CN117425727A

CN117425727A - Variants of C-glucosyltransferase and uses thereof

Info

Publication number: CN117425727A
Application number: CN202280016203.2A
Authority: CN
Inventors: 李相烨; 梁东秀; 张宇旲
Original assignee: Korea Advanced Institute of Science and Technology KAIST
Current assignee: Korea Advanced Institute of Science and Technology KAIST
Priority date: 2021-01-27
Filing date: 2022-01-27
Publication date: 2024-01-19

Abstract

The present invention relates to novel variants of C-glucosyltransferase and uses thereof. The variant of C-glucosyltransferase according to the present invention has improved glycosidic bond formation ability as compared with wild-type C-glucosyltransferase, and thus can increase the glycoside-producing effect of polyketides and pseudo-natural products, particularly type I, type II, type III polyketides, non-ribosomal peptides, phenylpropanoids and other aromatic natural products, and thus can be used for the preparation of pharmaceuticals, food additives, nutritional supplements and the like containing C-glycoside compounds as constituent components.

Description

Variants of C-glucosyltransferase and uses thereof

Technical Field

The present invention relates to novel variants of C-glucosyltransferase and their use, more particularly to variants of C-glucosyltransferase which enhance glycosylation of substrate carbons due to amino acid mutation at the active site of the C-glucosyltransferase, and their use in the production of polyketide (polyketide glycoside) and phenylpropanoid glycoside (polyketide glycoside).

Background

Polyketones are an attractive natural product with various biological effects and are widely used in foods, cosmetics, medicines, etc. in daily life. Enzymes that biosynthesize polyketides are collectively referred to as polyketide biosynthetic enzymes (polyketide synthases) (PKSs), and fall into three types, i.e., type I, type II, and type III, depending on the biosynthetic mechanism. Among them, macrolide-based polyketones are produced mainly by PKS type I, and aromatic polyketones are produced mainly by type II and type III.

For materials with pharmaceutical efficacy, such as pharmaceuticals or nutritional supplements, glycosides have greatly improved stability, hydrolysis resistance, bioavailability, etc. by forming glycosidic linkages, glycosides are generally more preferred than non-glycosides. In particular, stable C-glycosidic linkages are more chemically stable than O-glycosidic linkages. However, although examples of O-glycosylation of some natural products have been reported in E.coli (Escherichia coli) (Chen, D.; chen, R.; xie, K.; duan, Y.; dai, J.; production of acetophenone C-glucosides using an engineered C-glycosyltransferase in Escherichia coli. Tetrahedron Lett.2018,59 (19), 1875-1878), few examples of C-glycosylation have been reported. In nature and in E.coli, there have been many reports on O-glycosylation rather than C-glycosylation.

Typical C-glycoside natural products include carminic acid, aloesin (aloesin), and the like.

Carminic acid is a widely used red dye and is used in foods, cosmetics and pharmaceuticals. Carminic acid is extracted directly from scale insects such as cochineal (Dactylopius coccus) and added to foods such as tomato paste, strawberry milk, candies, etc. and cosmetics such as eye shadow, nail polish, lipstick, etc. However, cochineal growth is slow and the growing area is limited (may only grow in hot and dry areas), making it difficult to easily increase productivity, which limits commercial production. Especially, the extraction process efficiency is extremely low; for example, 70,000 female cochineal worms are required to produce one pound of carminic acid. In this case, it is necessary to develop a more sustainable process for producing carminic acid.

Aloin is extracted from Aloe (Aloe vera) and is widely used as a whitening agent in the cosmetic industry due to its anti-tyrosinase and anti-melanin production effects. In addition, since aloin exhibits anti-inflammatory and anti-radical effects, it can be used as a main ingredient of various medicines or cosmetics. However, the amount of aloin extracted from aloe plants is very small, and thus there is a need to develop a more efficient and more sustainable bio-based production process.

As described above, the demand for C-glycoside natural products is very high and the supply thereof is insufficient, but a method capable of producing the same has not been developed yet. In particular, even when the compound is produced by biological methods, enzymes thereof are not well known or efficient production from a microbial cell factory is impossible due to low enzymatic conversion efficiency.

In this technical background, the present inventors have made a great effort for developing a C-glycosyltransferase having an excellent C-glycosylation ability, thereby developing a C-glycosyltransferase variant exhibiting an excellent C-glycosylation ability by amino acid substitution, and confirming that in recombinant microorganisms into which C-glycosyltransferase variant genes are introduced, the ability of C-glycosyltransferase variants to produce glycosides is very high for type I, type II and type III polyketides, non-ribosomal peptides, phenylpropanoids and aromatic natural products, thereby finally forming the present invention.

The above information described in the background section is only for enhancement of understanding of the background of the invention and it does not contain information that forms the prior art that is already known to those of ordinary skill in the art to which the invention pertains.

[ quotation list ]

[ non-patent literature ]

(non-patent document 1) Chen, d.; chen, r.; xie, k.; duan, Y.; dai, J., production of acetophenone C-glucosides using an engineered C-glycosyltransferase in Escherichia colli.tetrahedron Lett.2018,59 (19), 1875-1878.

Disclosure of Invention

It is an object of the present invention to provide novel variants of C-glucosyltransferase and uses thereof.

In order to achieve the above-mentioned object,

the present invention provides a C-glucosyltransferase variant comprising a mutation of at least one amino acid of the group consisting of: f17, V93, V132, Y193, L164 and R322 in the C-glycosyltransferase shown in SEQ ID NO. 1.

Furthermore, the invention provides a nucleic acid encoding a C-glucosyltransferase variant.

Furthermore, the present invention provides a recombinant microorganism into which a nucleic acid is introduced.

In addition, the present invention provides a method of producing polyketide and/or phenylpropanoid glycosides, comprising:

(a) Producing polyketide and/or phenylpropanoid glycosides by culturing the recombinant microorganism of the present invention; and

(b) Recovering the polyketide and/or phenylpropanoid glycoside produced.

In addition, the present invention provides a method for producing polyketide and/or phenylpropanoid glycosides, comprising:

(a) Producing polyketide and/or phenylpropanoid glycosides by reacting a C-glucosyltransferase variant of the invention or a microorganism expressing the C-glucosyltransferase variant with polyketides and/or phenylpropanoids; and

(b) Recovering the polyketide and/or phenylpropanoid glycoside produced.

Drawings

FIG. 1 shows the carminic acid production pathway;

FIG. 2 shows the production of xanthone acid (flavokermesic acid) when different metabolic engineering strategies are introduced, wherein higher concentrations of FK are produced using type II polyketide synthases (AntDEFBG from P.luminescens) and ZhuiJ compared to when using type III polyketide synthases (AaPKS 5 from Aloe arborescens);

FIG. 3 shows that the production of nopaline acid is a function of the introduction of DnrF;

FIG. 4 shows the original enzymatic reactions of a candidate C-glucosyltransferase and a candidate enzyme for dcII production;

FIG. 5 shows the results of a comparison of dcII-producing ability of nine candidate enzymes;

FIG. 6 shows the results of homologous modeling and docking simulation to increase the production of KA and dcII: (a) the KA-producing ability of the selected variant by DnrF simulation, (b) the protein structure simulation result of the most potent DnrF variant (P217K), (c) the dcII-producing ability of the selected variant by GtCGT simulation, and (d) the protein structure of the most potent GtCGT variant (V93Q/Y193F);

FIG. 7 shows the production of carminic acid from glucose: (a) carminic acid production under different conditions, (b) carminic acid analysis by LC-MS/MS, the upper graph data showing the analysis results of commercially available carminic acid, the lower graph data showing the analysis results of samples containing carminic acid produced from glucose in e.coli, the left graph showing the Extracted Ion Chromatogram (EIC), the right graph showing the MS/MS fragmentation pattern, and (c) the fed-batch fermentation graph of the final strain, the red arrow representing the time point at which gene expression is initiated by IPTG, and DCW representing the dry cell weight;

FIG. 8 shows the aloin production pathway;

fig. 9 shows aloin production by e.coli: (a) constructing another RpALS-containing plasmid for increasing the test results of aloin production, (b) GtCGT and variants thereof for generating aloin production, and (c) analyzing aloin by LC-MS/MS, the upper graph data showing the analysis results of commercially available aloin, the lower graph data showing the analysis results of samples containing aloin produced from glucose in e.coli, the left graph showing the Extracted Ion Chromatogram (EIC), the right graph showing the MS/MS fragmentation pattern;

FIG. 10 shows the test results of additional variants of GtCGT for increasing aloin production, wherein the additional variants were predicted by analysis of the structural model of the GtCGT variant (V93Q/Y193F);

FIG. 11 shows test results of additional variants of GtCGT for increasing aloin production, wherein the additional variants were predicted by docking simulations based on the GtCGT variant (V93Q/Y193F);

FIG. 12 shows various phenylpropanoid C-glucosides (expressed as% conversion) produced by GtCGT variants (V93Q/Y193F); and

FIG. 13 shows K for calculation of GtCGT and GtCGT variants (V93Q/Y193F) _M And V _max Lineweaver-Burk plot of values.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is well known and typical in the art.

In the present invention, in order to find a variant of C-glucosyltransferase having an extremely high capacity to form glycosidic bonds as compared with a wild-type enzyme, the protein structure is predicted, and variant candidates having increased activity are obtained by protein structure analysis and computer simulation, wherein an effective variant having an increased capacity to bind to a substrate and capable of enhancing glycosylation reaction can be selected.

Accordingly, one aspect of the present invention relates to variants of C-glucosyltransferase having increased C-glycosylation capacity.

In the present invention, a C-glycosyltransferase that is a template (or wild-type) for a variant according to the present invention is an enzyme that induces C-glycosylation by forming a C-glycosidic bond with a carbon of a substrate (e.g., a compound, a protein, etc.).

In the present invention, the C-glucosyltransferase is represented by SEQ ID NO. 1, but is not limited thereto, and is understood as including a protein in which an amino acid residue is conservatively substituted at a certain amino acid residue position.

In the present invention, a "conservative substitution" is a modification of a C-glycosyltransferase that involves substitution of at least one amino acid with an amino acid having similar biochemical properties without resulting in loss of biological or biochemical function of the C-glycosyltransferase or variant thereof.

In the present invention, a "conservative amino acid substitution" is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues with similar side chains are defined and well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), uncharged amino acids with polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β -branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Thus, a C-glucosyltransferase as a template for a variant of the invention is understood as encompassing all C-glucosyltransferases, recombinant C-glucosyltransferases and fragments thereof, not only SEQ ID NO 1 but also having essentially the same function and/or effect and having 40% or more, 50% or more, 60% or more, 70% or more, preferably 80% or more or 85% or more, more preferably 90% or more or 95% or more, most preferably 99% or more amino acid sequence homology.

As used herein, the term "fragment" refers to a portion resulting from cleavage of a parent protein, and may be obtained by cleavage of the C '-end and/or the N' -end. The fragment of the present invention is a fragment having substantially the same function and/or effect as the deglycosylated C-glucosyltransferase of the present invention. For example, fragments may include fragments in which the signal sequence is cleaved from the full-length protein.

In the present invention, the C-glucosyltransferase may be derived from other strains or organisms in addition to the GtUF6CGT from gentian trislot (Gentiana triflora) shown in SEQ ID NO. 1. Examples thereof may include, but are not limited to, iroB (EnCGT) from escherichia coli Nissle, UGT708A6 (ZmCGT) from corn (Zea mays) as a dual C/O-glycosyltransferase, UGT708C2 (FeCGT) from buckwheat (Fagopyrum esculentum), miCGT from mango (Mangifera indica), osCGT 708D1 (GmCGT) from rice (Oryza sativa), UGT708D1 (GmCGT) from soybean (Glycine max), gtUF6CGT1 (GtCGT) from gentiana rigescens, and AvCGT from aloe, preferably GtUF6CGT1 (GtCGT) from gentiana rigescens or UGT708A6 (ZmCGT) from corn as a dual C/O-glycosyltransferase.

In an embodiment of the present invention, it has been confirmed that when variants are produced by substituting a part of amino acids of a wild-type C-glycosyltransferase, they exhibit extremely high ability to induce C-glycosylation, and it has also been confirmed that when the C-glycosyltransferase is introduced into a recombinant strain for polyketide synthesis, C-glycosylated polyketides can be produced in high yield.

In the present invention, the C-glycosyltransferase variant may comprise a mutation of at least one amino acid selected from the group consisting of F17, V93, V132, Y193, L164 and R322 in the C-glycosyltransferase shown in SEQ ID NO. 1, and preferably comprises a mutation in amino acid V93 and/or Y193.

In the present invention, the C-glycosyltransferase variant may comprise a mutation of one or more other amino acids in addition to a mutation of at least one amino acid selected from the group consisting of F17, V93, V132, Y193, L164 and R322 in the C-glycosyltransferase shown in SEQ ID NO. 1.

In the present invention, the C-glucosyltransferase variant may further comprise a mutation of at least one amino acid selected from the group consisting of C-glucosyltransferase shown in SEQ ID No. 1: f17, V405, P107, L208, L164, P45, I305, L316, F401, Y94, N57, Y187, C16, P319, F167, V132, N206, R406, Q386, V129, L125, L194, I95, S215, L184, Y158, L29, L27, F202, H159, S370, H365, V329, M301, V315, V190, C366, W80, L58, Q210, F312, D61, I207, L363, P196, L106, V93, A394, W314, S155, P88, D99, Y284, E189, G49, H328, E399, T392, F387, A44, P199E 46, R28, V285, I124, R419, L306, Y157, Y200, E373, P191, L214, S376, V15, E332, E51, I417, L98, I323, H161, T383, P127, E309, N84, L313, Q104, T371, N213, G79, L330, N307, K105, L128, A152, I18, N59, W147, S86, L293, E296, S377, L185, K216, F89, S286, F396, F211, Y303, D223, R415, N96, V22, S153, F154, D192, Y193, H195, P201, Y292, and R322.

In the present invention, the C-glycosyltransferase variant may further comprise a mutation of at least one amino acid selected from the group consisting of I18, Q20, T50, I95, V290, I323, V22, L29, E46, V48, E51, A55, S86, D99, R103, C151, L184, L194, E332 and P385 in the C-glycosyltransferase shown in SEQ ID NO. 1.

In the present invention, the C-glycosyltransferase variant preferably further comprises a mutation of at least one amino acid selected from the group consisting of I323, T50, I18, I95, Q20, P385, L194 and V48 in the C-glycosyltransferase variant shown in SEQ ID NO. 1.

As used herein, the term "variant" is used to cover all concepts that cover not only mutations, preferably substitutions, deletions and/or insertions of some amino acid residues, more preferably substitutions, but also deletions of some amino acid residues at the N-terminus or C-terminus, as well as substitutions, deletions and/or insertions of such amino acid residues in the amino acid sequence of a reference sequence (e.g., wild-type C-glucosyltransferase sequence, SEQ ID NO: 1). In an embodiment of the present invention, the variant is produced by substituting a part of the amino acid of SEQ ID NO. 1, but the present invention is not limited thereto.

In the present invention, a "mutation" may be an amino acid substitution.

In the present invention, the C-glycosyltransferase variant may comprise a substitution of at least one amino acid selected from the group consisting of F17G, V93Q, V132A, Y193F, L164G and R322D, more preferably a substitution of amino acids V93Q and/or Y193F, most preferably a substitution of amino acids V93Q and Y193F in the C-glycosyltransferase shown in SEQ ID NO. 1.

In the present invention, the C-glycosyltransferase variant may further comprise a substitution of at least one different amino acid in addition to the substitution of at least one amino acid selected from the group consisting of F17G, V93Q, V132A, Y193F, L164G and R322D in the C-glycosyltransferase shown in SEQ ID NO. 1.

In the present invention, the C-glycosyltransferase variant may further comprise a substitution of at least one different amino acid in addition to the substitutions of amino acids V93Q and Y193F in the C-glycosyltransferase shown in SEQ ID NO. 1.

In the present invention, the substitution of a different amino acid which may further be included in the variant C-glycosyltransferase may be a substitution of at least one amino acid selected from the group consisting of the C-glycosyltransferases shown in SEQ ID NO: 1: f17 405 107 208 45,107,316 45,401 94,57,187,57,187,187,319,132,132,206,386 129,194,194,29,202,159,370,329 301 315 190 366 80, 58, 312, 207, 363, 61, 196, 106, 93, 394, 155, 99, 284, 189, TH328, 399, 392, 44, 199, 46, 285, 124 419 306,157,373,373,201,191,214,376 15,332,417,98,323,383 127 309, 313, 104, 371, 213, 79, 330, 307, 105, 128, 152, 153, 59 147 86 293 296 377 185 216 89 286 396 211 223 96 93 93 93 93 154 193 195 195 292 292D and R322A.

In the present invention, the substitution of different amino acids which may be further included may be a substitution of at least one amino acid selected from the group consisting of the C-glycosyltransferases shown in SEQ ID NO: 1: I18P, Q20M, T50N, T50Q, T K, T50R, T95 5446 95M, I95T, V290G, V A, I79323S, I323A, I323L, V22A, L29A, E46G, V48G, E51C, A86 5299C, A103C, A151C, A184C, A194C, A332C, A a and P385A.

In the present invention, the substitution of different amino acids which may be further included is preferably a substitution of at least one amino acid selected from the group consisting of I323S, T50R, T50V, I18P, I95T, Q20M, I323A, P385A, L A and V48G in the C-glycosyltransferase shown in SEQ ID NO. 1.

In embodiments of the invention, it was demonstrated that the C-glucosyltransferase variants exhibit extremely excellent C-glycosylation when included below:

i) Substitutions of amino acids V93Q and Y193F in the C-glycosyltransferase shown in SEQ ID NO. 1;

ii) substitution of amino acids V93Q, Y193F and I323S in the C-glucosyltransferase of SEQ ID NO. 1; or (b)

iii) Substitutions of amino acids V93Q, Y193F and P385A in the C-glycosyltransferase shown in SEQ ID NO. 1, but the present invention is not limited thereto.

The amino acid residue positions in the amino acid mutations were numbered exactly using the amino acid sequence shown in SEQ ID NO. 1 as a reference. Here, "residue Xn" refers to a residue X at the n-th position in the amino acid sequence shown in SEQ ID NO. 1, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "residue V93" means the amino acid residue V at position 93 in the amino acid sequence shown by SEQ ID NO. 1.

In the present invention, an "amino acid mutation" may be an "amino acid substitution Xn", for example, refers to an amino acid substitution occurring from an amino acid residue X at the n-th position in the amino acid sequence shown in SEQ ID No. 1, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "amino acid substitution V93" means an amino acid substitution occurring in amino acid residue V at position 93 in the amino acid sequence shown in SEQ ID NO. 1.

In the present invention, when a C-glycosyltransferase having an amino acid sequence different from that of SEQ ID NO. 1 used as a reference sequence is used as the reference sequence, amino acid residues corresponding to the specific amino acid residues listed in the reference SEQ ID NO. 1 can be generally obtained by aligning the amino acid sequences under optimal conditions. Sequence alignment may be performed in a manner understood by those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or MegAlign (DNASTAR) software, and the like. One skilled in the art can determine the appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment over the full length sequences compared.

Amino acid substitutions of the invention may be non-conservative substitutions. Such non-conservative substitutions may include altering an amino acid residue of the target protein or polypeptide in a non-conservative manner, such as replacing an amino acid residue having a particular side chain size or a particular property (e.g., hydrophilicity) with an amino acid residue having a different side chain size or a different property (e.g., hydrophobicity).

Furthermore, amino acid substitutions may be conservative substitutions. Such conservative substitutions may include altering an amino acid residue of the target protein or polypeptide in a conservative manner, such as replacing an amino acid residue having a particular side chain size or a particular property (e.g., hydrophilicity) with an amino acid residue having the same or similar side chain size or the same or similar property (e.g., hydrophilicity). Conservative substitutions typically do not greatly affect the structure or function of the protein produced. In the present application, an amino acid sequence variant is a mutation of a fusion protein, a fragment thereof or a variant thereof in which one or more amino acids are replaced, and an amino acid sequence variant may include conservative amino acid substitutions that do not greatly alter the structure or function of the protein.

For example, mutual substitutions between amino acids in the following groups may be considered conservative substitutions in the present application:

Amino acid group with nonpolar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine;

uncharged amino acid groups with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;

negatively charged amino acid groups with polar side chains: aspartic acid and glutamic acid;

positively charged basic amino acid group: lysine, arginine, and histidine;

amino acid group having phenyl group: phenylalanine, tryptophan and tyrosine.

The protein, polypeptide and/or amino acid sequences encompassed by the present invention are also understood to include at least the following ranges: variants or homologues having the same or similar function as the protein or polypeptide.

In the present invention, a variant may be a protein or polypeptide produced by substitution, deletion or addition of at least one amino acid compared with the amino acid sequence of a wild-type C-glucosyltransferase. For example, a functional variant may include a protein or polypeptide having an amino acid change by substitution, deletion, and/or insertion of at least one amino acid (e.g., 1-30, 1-20, or 1-10 amino acids, or alternatively 1, 2, 3, 4, or 5 amino acids). Functional variants may substantially retain the biological properties of the protein or polypeptide prior to alteration (e.g., substitution, deletion, or addition). For example, a functional variant may retain at least 60%, 70%, 80%, 90% or 100% of the biological activity of a protein or polypeptide prior to alteration.

In the present invention, a homolog may be a protein or polypeptide having at least about 80% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence homology to the amino acid sequence of the protein and/or polypeptide.

In the present invention, homology generally means similarity, similarity or relatedness between two or more sequences. "percent sequence homology" can be calculated by comparing two aligned sequences in a comparison window that determines the number of positions at which the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) occurs, and the percent sequence homology can be obtained by dividing the number of matched positions by the total number of positions and then multiplying the result by 100 to provide the number of matched positions in the comparison window (i.e., window size). The alignment used to determine the percent sequence homology can be performed in a variety of ways known in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MegAlign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment within the compared full-length sequences or within the target sequence region. Homology can also be determined by the following method: FASTA and BLAST. The FASTA algorithm is described, for example, in w.r.pearson and d.j.lipman's "Improved Tool for Biological Sequence Comparison", proc.Natl.Acad.Sci.,85:2444-2448,1988; and D, J.Lipman and W.R. Pearson's "Fast and Sensitive Protein Similarity Search", science,227:1435-1441,1989, and BLAST algorithms are provided by S.Altschul, W.Gish, W.Miller, E.W.Myers and D.Lipman, "ABasic Local Alignment Search Tool", journal of Molecular Biology,215:403-410,1990.

In the present invention, the C-glucosyltransferase variant is characterized by an enhanced glycosylation reaction of the substrate carbon compared to the wild-type.

In the present invention, the mutated amino acid is located at the active site of the enzyme, and the ability of the variant to bind to the substrate can be increased by 10% or more, preferably 20% or more, more preferably 50% or more by amino acid mutation as compared with the wild type.

In an embodiment of the present invention, it has been demonstrated that the C-glucosyltransferase variants of the present invention highly promote C-glycosylation of various polyketides (xanthone acid, nopsurrounding acid, aloe-pine) or phenylpropanoid-based compounds (naringenin, apigenin or luteolin) used as substrates, regardless of the type of substrate, compared to the wild type. Thus, the C-glucosyltransferase variants of the invention may be used for C-glycosylation of various compounds or proteins as substrates. For example, the C-glucosyltransferase variants of the present invention may be used for C-glycosylation of polyketides or phenylpropanoid-based compounds, but the present invention is not limited thereto.

In the present invention, a polyketide or phenylpropanoid-based compound may be used as a substrate without limitation, and as demonstrated in the embodiments, preferred examples of the substrate include, but are not limited to, xanthonic acid, nopronic acid, aloe-pine, naringenin, apigenin, and luteolin.

The substrate is preferably a xanthogenic acid or a nopic acid, and the variant is capable of glycosylation at carbon number 2 of the xanthogenic acid, but the invention is not limited thereto.

The substrate is preferably aloe vera, and the variant is capable of glycosylation at carbon number 8 of aloe vera, but the invention is not limited thereto.

Another aspect of the invention relates to a nucleic acid encoding a variant C-glucosyltransferase.

A further aspect of the invention relates to a vector comprising the nucleic acid.

Yet another aspect of the invention relates to a recombinant microorganism into which the nucleic acid is introduced.

In the present invention, the recombinant microorganism may be configured such that the nucleic acid is introduced into the host microorganism in the form of a plasmid or inserted into the genome.

In the present invention, the recombinant microorganism may be used to produce polyketide and/or phenylpropanoid glycosides, but the present invention is not limited thereto.

In the present invention, the recombinant microorganism may have the ability to produce polyketides and/or phenylpropanoids as substrates for the C-glucosyltransferase of the present invention, and the polyketides and/or phenylpropanoids may be glycosylated by the C-glucosyltransferase expressed by the recombinant microorganism of the present invention and thus converted into polyketide and/or phenylpropanoid glycosides.

In the present invention, the polyketone may be selected from the group consisting of, but not limited to:

a type I polyketone selected from the group consisting of rapamycin (rapamycin), lovastatin, erythromycin (erythromycin), rifamycin (rifamycin), avermectin (avermectins), geldanamycin (geldanamycin), ivermectin (ivermectin), calicheamicin (calicheamicin), epothilone (epothilone), triacetin and 6-methyl salicylic acid;

a type II polyketone selected from the group consisting of actinorhodin (acteosin), doxorubicin (doxorubicin), daunorubicin (daunorubicin), oxytetracycline (oxytetracycline), SEK4b, SEK34, SEK15, SEK26, FK506, DMAC, aclacinone (aklavonone), aclacin (aklanonic acid), epsilon-Luo Duoxin quinone (epsilon-rhodomycinone), doxycycline, aflatoxin (anthramycin), teframycin (tetracyclic mycin), carminic acid, and Furanomycin (frenolicin); and

a type III polyketone selected from the group consisting of aloin, barbaloin, 5, 7-dihydroxy-2-methyl chromone, and aloeresin.

In the present invention, the phenylpropanoids may be selected from the group consisting of, but not limited to:

Non-ribosomal peptides including actinomycin (acteosin), bacitracin (bacitracin), daptomycin (daptomycin), vancomycin (vancomycin), thaumatin (teixobatin), gramicidin (tyrocidin), gramicidin (gramicidin), bivalve A (zwittermicin A), bleomycin (bleomycin), cyclosporin (ciclosporin), siderophilic (pyverine), enterobacterin (enterobacin), mucochelatin A (myxochelin A), natural blue pigment (indigoidin) and phycocyanin (cyanomycins), pinocembrin (pinocembrin), dihydrokaempferol (dihydrokaempferol), eriodictyol (dihydroquercetin), coniferol, silybin (silybin), isosilybin (isosilybin), silymarin (silychristin), silylide, 2, 3-dehydrosilybin (silymarin), silymarin (silydianin), soyabean extract (daidzein), genistein (genistein), apigenin (apigenin), luteolin (luteolin), kaempferol (kaempferol), quercetin (quercetin), catechin, pelargonin (pelargonidin), anthocyanin (cyanidin), alfucatechin (afzelechin), myricetin (myretin), non-sirtuin (fitin), galangin (galangin), hesperetin (hesperetin), naringenin (tangeritin), delphinidin (delphinidin), epicatechin (chrysin), resveratrol and naringenin (naringenin).

In the present invention, the host microorganism may have the ability to produce a precursor of the polyketide and/or phenylpropanoid glycoside of interest.

In the present invention, the precursor of polyketide and/or phenylpropanoid glycoside may be polyketide and/or phenylpropanoid, preferably non-glycosylated polyketide and/or phenylpropanoid.

In the present invention, the host microorganism is capable of naturally producing a precursor of polyketide and/or phenylpropanoid glycoside, or may be a recombinant microorganism producing a precursor of polyketide and/or phenylpropanoid glycoside by genetic manipulation.

In the present invention, the recombinant microorganism may be characterized by enhanced production of nucleotides, preferably NTP-sugars, to increase the glycosidic conversion efficiency of the introduced C-glucosyltransferase. For example, the recombinant microorganism of the present invention is further characterized by enhanced expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase and/or nucleoside diphosphate kinase, but the present invention is not limited thereto.

In the present invention, UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase and/or nucleoside diphosphate kinase may be derived from E.coli, but the present invention is not limited thereto, and the expression of genes involved in NTP-sugar production may be enhanced depending on the host strain.

In the embodiment of the present invention, flavonone acid, nopal acid, aloe-pine, naringenin, apigenin or luteolin is used as a precursor of polyketide and/or phenylpropanoid glycoside, but the present invention is not limited thereto, and various precursors of the above polyketide and/or phenylpropanoid glycoside are well known in the art and may be easily selected therefrom.

As used herein, the term "nucleic acid" refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide, or an analog of any length thereof, typically isolated from a natural environment or synthesized artificially. The nucleic acids of the invention may be isolated. For example, a nucleic acid may be produced or synthesized by: (i) in vitro amplification such as Polymerase Chain Reaction (PCR) amplification, (ii) clonal recombination, (iii) purification such as fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesis such as chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA techniques. In the present invention, nucleic acids encoding variants can be produced by various methods known in the art. Such methods include, but are not limited to, restriction fragment processing or overlap extension PCR using synthetic oligonucleotides. Methods and principles of production are described in Sambrook et al, molecular Cloning, A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY,1989; and Auube et al Current Protocols in Molecular Biology, greene Publishing and Wiley-Interscience, new York N Y,1993.

As used herein, the term "plasmid" is used interchangeably with vector and generally refers to a nucleic acid molecule capable of self-replication in a host cell or microorganism by delivering an inserted nucleic acid to the host cell (including the host microorganism). Examples of the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for DNA or RNA replication, and a vector mainly used for transcription and/or translation expression of DNA or RNA. Examples of the carrier may also include a carrier having various functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector is capable of producing the desired expression product by culturing a suitable host cell containing the vector. In the present invention, the vector may include at least one nucleic acid. For example, a vector may include all nucleic acid molecules required to encode a variant. Thus, only one vector is required to obtain the fusion proteins of the present application. In some embodiments, the vector may include a nucleic acid molecule encoding a portion of the variant. Alternatively, the vector may comprise a nucleic acid molecule, e.g. for modulating gene expression in a recombinant microorganism. Here, two or more different vectors may be required to obtain the recombinant microorganism of the present invention.

In addition, the vector may include other genes, such as marker genes, which select the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may include expression control elements to properly express the coding region in a suitable host. These control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatory element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically includes 5' and 3' untranslated sequences as well as 5' untranslated sequences involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may include a promoter region and a promoter region may include a promoter sequence for transcriptional control of a functionally linked nucleic acid. In the present invention, the vector may be selected from the group consisting of pET-30a-c (+), pET-22b (+), pCDFDuet-1, pACYCDuet-1, pRSFDuet-1, pBBR1MCS, pSC101, pTac15K, pTrc99A, pCOLADuet-1 and pBR322, but is not limited thereto, and a person skilled in the art will be able to appropriately select and use a vector commonly used in the art in addition to the above vectors.

In the present invention, the terms "host cell", "host microorganism" and "host" are used interchangeably and generally refer to an individual cell, cell line, microorganism or cell culture capable of expressing a plasmid or vector comprising or capable of comprising a nucleic acid of the invention, a variant of the invention, or a protein or polypeptide whose expression is modulated. The host cell may comprise a progeny of a single host cell. The progeny cells and the original parent cells need not be identical in morphology or genome due to natural, accidental or deliberate mutation, so long as they are capable of expressing the target protein or polypeptide of the invention. Host cells can be obtained by transfecting cells in vitro with the vectors of the invention. The host cell is preferably a microorganism and may be selected from the group consisting of: for example, escherichia coli, rhizobium (Rhizobium), bifidobacterium (Bifidobacterium), candida (Candida), erwinia (Erwinia), enterobacter (Enterobacter), pasteurella (Pastellella), mannheimia (Mannheimia), actinobacillus (Actinobacillus), aggregaria (Aggregaria), xanthomonas (Xanthomonas), vibrio (Vibrio), azotobacter (Azotobacter), acinetobacter (Azotobacter), rhodobacter (Rhodobacter), zymomonas (Zymomonas), bacillus (Bacillus), staphylococcus (Staphylococcus), lactobacillus (Lactobacillus), streptococcus (Lactobacillus), lactobacillus (Lactobacillus), corynebacterium (Lactobacillus), and Corynebacterium (Lactobacillus), but not belonging to the genus Corynebacterium (Lactobacillus).

Furthermore, in the present invention, it was confirmed that various polyketide or phenylpropanoid glycosides can be efficiently produced using a recombinant microorganism capable of expressing a variant of C-glucosyltransferase.

Thus, in a further aspect the invention relates to a recombinant microorganism for producing polyketide or phenylpropanoid glycosides, into which a nucleic acid encoding a C-glucosyltransferase variant of the invention has been introduced.

In the present invention, the polyketide may be a type I polyketide, a type II polyketide, or a type III polyketide.

a polyketone of type I selected from the group consisting of rapamycin, lovastatin, erythromycin, rifamycin, avermectin, geldanamycin, ivermectin, calicheamicin, epothilone, triacetin and 6-methyl salicylic acid;

a type II polyketone selected from the group consisting of actinomycin, doxorubicin, daunorubicin, terramycin, SEK4b, SEK34, SEK15, SEK26, FK506, DMAC, aclacinone, aclacin, epsilon-Luo Duoxin quinone, doxycycline, aflatoxin, carminic acid, and frenolicin; and

a type III polyketone selected from the group consisting of aloin, 5, 7-dihydroxy-2-methyl chromone, and aloeresin.

In the present invention, the recombinant microorganism used for producing polyketide or phenylpropanoid glycosides is capable of producing precursors of each glycoside. For example, recombinant microorganisms are capable of producing polyketides or phenylpropanoids, which are precursors for each glycoside.

In the present invention, the recombinant microorganism for producing polyketide or phenylpropanoid glycoside can produce polyketides or phenylpropanoids by additional gene transfer. Recombinant microorganisms capable of synthesizing polyketides by gene transfer can be prepared using the genes and methods described in the articles disclosed by the present inventors, for example, yang, d., kim, w.j., yoo, s.m., choi, j.h., ha, s.h., lee, m.h., and Lee, s.y., "Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria", proc.Natl.Acad.Sci (PNAS), 115 (40) 9835-9844 (https:// doi.org/10.1073/pnas.1808567115) (2018.10.2) and korean patent No. 10-2187682, but the present invention is not limited thereto. Recombinant microorganisms capable of synthesizing polyketides or phenylpropanoids can be produced by one skilled in the art by using the various polyketide or phenylpropanoid synthetic pathways described in the art and genes involved therein introduced into various host microorganisms.

In the present invention, the recombinant microorganism for producing polyketide or phenylpropanoid glycoside may be further characterized by introducing therein polyketide synthase or phenylpropanoid synthase.

In the present invention, examples of polyketide synthases may include, but are not limited to, type I polyketide synthases, type II polyketide synthases, and type III polyketide synthases.

In the present invention, when the recombinant microorganism for producing polyketide or phenylpropanoid glycoside does not produce a precursor of each glycoside, the precursor of each glycoside may be added to a medium to obtain polyketide or phenylpropanoid glycoside.

In the present invention, recombinant microorganisms can be used to produce polyketide type I.

In the present invention, the recombinant microorganism used to produce the type I polyketide is capable of producing a precursor of the type I polyketide. Examples of precursors of type I polyketide may include, but are not limited to, rapamycin, lovastatin, erythromycin, rifamycin, and the like.

In the present invention, the recombinant microorganism for producing a type I polyketide can be introduced with an additional gene to produce a precursor of a type I polyketide.

In the present invention, the recombinant microorganism used for producing the type I polyketide may be characterized by:

(i) In addition, a gene encoding type I polyketide synthase is introduced therein.

In the present invention, type I polyketide synthases can be readily selected from a variety of protein and gene databases.

Thus, a host microorganism into which a nucleic acid encoding a C-glycosyltransferase variant of the invention and a polyketide synthase type I gene are introduced may have the ability to produce coenzyme A, preferably malonyl-CoA or acetyl-CoA.

Thus, in the present invention, the recombinant microorganism may be characterized by enhanced production of coenzyme A. For example, in the present invention, the recombinant microorganism is further characterized in that (ii) the expression of the pabA gene is inhibited or attenuated, but the present invention is not limited thereto, and the recombinant microorganism having enhanced production of CoA can be prepared using various strategies known in the art for large-scale production of CoA.

In the present invention, the recombinant microorganism may be characterized by enhanced production of nucleotides, preferably NTP-sugars, to increase the efficiency of glycoside conversion by the introduced C-glucosyltransferase. For example, in the present invention, the recombinant microorganism may be further characterized by (iii) enhanced expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase and/or nucleoside diphosphate kinase, but the present invention is not limited thereto.

In the present invention, recombinant microorganisms can be used to produce type II polyketide. For example, the type II polyketide may be carminic acid, but is not limited thereto.

In the present invention, the recombinant microorganism used to produce the type II polyketide is capable of producing a precursor of the type II polyketide. For example, the precursor of the type II polyketide may be, but not limited to, xanthonic acid or nopic acid.

In the present invention, the recombinant microorganism for producing type II polyketide can be introduced with another gene to produce a precursor of type II polyketide.

In the present invention, the recombinant microorganism for producing a type II polyketide may be characterized in that (i) a gene encoding a type II polyketide synthase is additionally introduced thereto.

In the present invention, the recombinant microorganism for producing a polyketide type II, preferably carminic acid, may be characterized in that any one or more genes selected from the group consisting of:

(i) A gene encoding a type II polyketide synthase;

(ii) A gene encoding 4'-phosphopantetheinyl transferase (4' -phosphopantetheinyl transferase);

(iii) A gene encoding a cyclase;

(iv) A gene encoding acetyl-CoA carboxylase; and

(v) A gene encoding an aclacinone 12-hydroxylase.

As shown in FIG. 1, for type II polyketides, which are substrates for the C-glucosyltransferase of the present invention, for example, coenzyme A (CoA) such as malonyl-CoA or acetyl-CoA may be converted into type II polyketides, which are substrates for the C-glucosyltransferase of the present invention, produced by an enzyme encoded by the introduced gene. Thus, a host microorganism into which nucleic acids encoding a C-glucosyltransferase variant and a type II polyketide synthase gene or genes of (i) to (v) above are introduced may have the ability to produce coenzyme A, preferably malonyl-CoA or acetyl-CoA.

In an embodiment of the present invention, it was confirmed that coenzyme A is accumulated by inhibiting or attenuating the expression of the pabA gene, thereby improving the synthesis of polyketide which is a precursor of the C-glucosyltransferase of the present invention.

In the present invention, the gene encoding type II polyketide synthase may be at least one selected from the group consisting of antD (ketosynthase), antE (chain length factor), antF (ACP), antB (phosphopantetheinyl transferase), and antG (malonyl-CoA: ACP malonyl transferase), or a combination thereof, but is not limited thereto.

In the present invention, the aclacinone 12-hydroxylase may include a proline to lysine mutation (P217K) at amino acid position 217 in the amino acid sequence shown in SEQ ID NO. 2, but is not limited thereto.

In the present invention, the type II polyketide synthase may be derived from a photorhabdus luminophorus;

the 4' -phosphopantetheinyl transferase may be derived from bacillus subtilis (Bacillus subtilis) or photorhabdus luminescens;

the cyclase may be derived from Streptomyces sp;

acetyl-CoA carboxylase may be derived from corynebacterium glutamicum (Corynebacterium glutamicum); and/or

The aclacinone 12-hydroxylase may be derived from Streptomyces wave (Streptomyces peucetius), but the present invention is not limited thereto.

In the present invention, recombinant microorganisms can be used to produce polyketide type III. For example, in the present invention, the type III polyketide may be aloin, but is not limited thereto.

In the present invention, the recombinant microorganism used to produce a type III polyketide is capable of producing a precursor of a type III polyketide. For example, the precursor of the type III polyketide may be aloe vera, but is not limited thereto.

In the present invention, the recombinant microorganism for producing a type III polyketide can be introduced with an additional gene to produce a precursor of a type III polyketide.

In the present invention, the recombinant microorganism for producing a type III polyketide may be characterized by:

(i) Into which a gene encoding type III polyketide synthase is introduced. For example, the type III polyketide synthase may be aloe vera synthase, but is not limited thereto.

In the present invention, aloe-pine synthase may be derived from rheum palmatum (r.palmatum), but the present invention is not limited thereto.

As shown in FIG. 8, for a type III polyketone (e.g., aloe vera), which is a substrate for the C-glucosyltransferase of the present invention, for example, coenzyme A (CoA) such as malonyl-CoA or acetyl-CoA may be converted to a type III polyketone, which is a substrate for the C-glucosyltransferase of the present invention, produced by an enzyme encoded by the introduced gene. Thus, a host microorganism into which nucleic acid encoding a C-glucosyltransferase variant and a polyketide synthase type III gene has been introduced may have the ability to produce coenzyme A, preferably malonyl-CoA or acetyl-CoA.

In the present invention, recombinant microorganisms can be used to produce phenylpropanoid glycosides. For example, the phenylpropanoid glycoside may be, but is not limited to, vitexin (vitexin), naringenin-6-C-glucoside, or isoorientin (isoorinentin).

In the present invention, the recombinant microorganism for producing a phenylpropanoid glycoside is capable of producing a precursor of a phenylpropanoid glycoside. For example, the precursors of phenylpropanoid glycosides may be, but are not limited to, apigenin, naringenin, or luteolin.

In the present invention, the recombinant microorganism for producing phenylpropanoid glycosides can be introduced with an additional gene to produce a precursor of phenylpropanoid glycosides.

In the present invention, the recombinant microorganism for producing phenylpropionic acid glycoside may be characterized by:

(i) In addition, a gene encoding phenylpropanoid synthase was introduced therein.

For phenylpropanoids, coenzyme a (CoA), such as malonyl-CoA or aromatic-CoA (e.g., coumaroyl-CoA), can be converted to phenylpropanoids by an enzyme encoded by the introduced gene, which is a substrate for the C-glucosyltransferase of the present invention. Thus, a host microorganism into which nucleic acid encoding a C-glucosyltransferase variant and a phenylpropanoid synthase gene has been introduced may have the ability to produce coenzyme A, preferably malonyl-CoA or coumaroyl-CoA.

In an exemplary embodiment, the recombinant microorganism of the present invention may be a recombinant microorganism for producing carminic acid, wherein in the recombinant microorganism into which a nucleic acid encoding the C-glucosyltransferase of the present invention is introduced, at least one gene selected from the group consisting of:

(i) Introducing a gene encoding a type II polyketide synthase;

(ii) Introducing a gene encoding 4' -phosphopantetheinyl transferase;

(iii) Introducing a gene encoding a cyclase;

(iv) Introducing a gene encoding acetyl-CoA carboxylase;

(v) Introducing a gene encoding an aclacinone 12-hydroxylase;

(vi) Enhancing expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase and/or nucleoside diphosphate kinase; and

(vii) Attenuating the expression of the pabA gene.

In another exemplary embodiment, the recombinant microorganism of the present invention may be a recombinant microorganism for producing aloin, wherein in the recombinant microorganism into which a nucleic acid encoding a C-glucosyltransferase is introduced, at least one gene selected from the group consisting of:

(i) Introducing a gene encoding aloe vera pine synthase;

(ii) Attenuating expression of the pabA gene; and

(iii) The expression of the gene encoding glucose 6-phosphate 1-dehydrogenase is enhanced.

In another exemplary embodiment, the recombinant microorganism of the present invention may be:

recombinant microorganism for the production of polyketide or phenylpropanoid glycosides, wherein in the recombinant microorganism into which a nucleic acid encoding a C-glucosyltransferase of the invention has been introduced, the expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase and/or nucleoside diphosphate kinase is enhanced.

In the present invention, the introduction of a gene refers to the introduction of a foreign gene into a host microorganism through a vector such as a vector, or the direct insertion into the genome of the host microorganism.

In the present invention, the enhancement of expression of a gene means that when a peptide or protein produced by the gene is not present in a host microorganism, the activity or function of the peptide or protein is imparted by artificial expression in the host microorganism, and also means that when the gene is already present in the host microorganism, an endogenous promoter controlling the expression of the gene is replaced with a strong constitutive expression promoter, or the gene is additionally introduced from the outside using a vector or the like having a strong replication ability to increase the copy number of the gene, thereby inducing overexpression of the gene or modifying the activity or function of the peptide or protein produced by the gene to be enhanced as compared with the intrinsic activity or function.

In the present invention, the expression attenuation of a gene means that the gene concerned is not expressed by mutating, substituting or deleting some or all bases of the gene concerned, or by introducing an inhibitor capable of inhibiting the expression of the gene (for example, sRNA or the like), or does not exhibit activity or function even when expressed, and is a concept covering modification such that the activity or function of a peptide or protein produced by the gene is attenuated as compared with the intrinsic activity or function.

As used herein, the term "intrinsic activity or function" refers to the activity or function of an enzyme, peptide, protein, etc. that the microorganism originally had in an unmodified state.

In the present invention, "modified to be enhanced as compared with the intrinsic activity or function" means that the activity or function of the microorganism is newly produced or increased after the manipulation as compared with the activity of the microorganism before the manipulation, such as an increase in the number of genes or copies of genes into which the genes are introduced to exhibit the activity or function (for example, expression using a plasmid into which the genes are introduced), deletion of a gene expression inhibitor, or modification of an expression control sequence, for example, using an improved promoter, etc.

In the present invention, "modified to be reduced in comparison with intrinsic activity or function" means that the function of a microorganism is reduced or lost after an operation as compared with the function of a microorganism before an operation, such as deletion of a gene exhibiting activity or function or inactivation of a gene (e.g., substitution with a mutant gene), attenuation of gene expression (e.g., substitution with a weak promoter, introduction of siRNA, gRNA, sRNA, etc., substitution of an initiation codon from ATG to GTG, etc.), inhibition of the function of a peptide expressed by a gene (e.g., addition of a non-competitive or competitive repressor), etc.

In the present invention, "substitution" of a gene or a promoter means that a conventional gene or a promoter is removed and a different gene (e.g., a mutant gene or the like) or a promoter having a different intensity is newly introduced, and the concept of removing a conventional gene or a promoter includes not only deletion of the corresponding gene or promoter but also inhibition or reduction of the function thereof.

As used herein, the term "overexpression" refers to a higher level of expression than the level of expression of the relevant gene in a cell under normal conditions, and the concept includes increasing the level of expression by replacing the promoter of the gene present in the genome with a strong promoter or by cloning the relevant gene into an expression vector and transforming the cell with it.

As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particle, or simply a potential genomic insert. Once transformed into an appropriate host, the vector may replicate and function independently of the host genome, or in some cases may integrate into the genome itself. Since plasmids are the most commonly used form of vector at present, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, plasmid vectors are preferably used. Typical plasmid vectors useful for this purpose may be configured to include (a) an origin of replication that achieves efficient replication, such that each host cell includes one to hundreds of plasmid vectors, (b) an antibiotic resistance gene that achieves selection of host cells transformed with the plasmid vectors, and (c) a restriction enzyme cleavage site into which an exogenous DNA fragment may be inserted. The vector and the foreign DNA can be easily ligated using synthetic oligonucleotide adaptors or linkers according to typical methods even in the absence of appropriate restriction enzyme cleavage sites. After ligation, the vector must be transformed into an appropriate host cell. Transformation can be readily achieved using the calcium chloride method or electroporation (Neumann et al, EMBO J.,1:841, 1982).

The promoter of the vector may be constitutive or inducible, and may be additionally modified to achieve the effects of the present invention. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector, and in the case of replicable expression vectors, an origin of replication (Ori). The vector may replicate autonomously or may be integrated in the host genomic DNA. Preferably, the gene inserted and delivered into the vector is irreversibly fused into the genome of the host cell, such that gene expression is stably maintained in the cell for a long period of time.

A base sequence is "operably linked" when it is in a functional relationship with another nucleic acid sequence. This may be a gene and one or more regulatory sequences linked in such a way: the gene is enabled to be expressed when an appropriate molecule (e.g., a transcriptional activator) is associated with one or more regulatory sequences. For example, when expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of the preprotein or secretion leader is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; the ribosome binding site is operably linked to a coding sequence when it affects the transcription of the sequence; or operably linked to a coding sequence when the ribosome binding site is arranged so as to facilitate translation. Typically, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, the enhancers do not have to be contiguous. Ligation of these sequences is accomplished by ligation at convenient restriction enzyme sites. When such a site is not present, synthetic oligonucleotide adaptors or linkers according to typical methods are used.

As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene must be operably linked to transcriptional and/or translational expression control sequences that function in the selected expression host. Preferably, the expression control sequences and/or the corresponding genes are contained in a single recombinant vector comprising the bacterial selection marker and the origin of replication. When the host cell is a eukaryotic cell, the recombinant vector must further include an expression marker that can be used in a eukaryotic expression host.

A further aspect of the invention relates to a host cell transformed with the recombinant vector described above. As used herein, the term "transformation" means introducing DNA into a host such that the DNA becomes replicable as an extrachromosomal element or by completing chromosomal integration.

It will be appreciated that not all vectors function equally well in expressing the DNA sequences of the present invention. Also, not all hosts will function equally well with the same expression system. However, one skilled in the art will be able to make appropriate selections among various vectors, expression control sequences, and hosts without departing from the scope of the invention and without undue experimentation. For example, in selecting a vector, the host must be considered, as the vector must replicate therein.

Yet another aspect of the invention relates to a method of producing a polyketide or a phenylpropanoid glycoside, comprising:

(a) Producing polyketide or phenylpropanoid glycoside by culturing a recombinant microorganism into which a nucleic acid encoding a C-glucosyltransferase variant of the invention has been introduced; and

(b) Recovering the polyketide or phenylpropanoid glycoside produced.

In the present invention, step (a) may be performed by culturing a recombinant microorganism into which a nucleic acid encoding a C-glucosyltransferase variant has been introduced by adding a precursor of polyketide or phenylpropionate.

In the present invention, in the step (a), the recombinant microorganism into which the nucleic acid encoding the C-glucosyltransferase variant is introduced may be configured such that the nucleic acid encoding the C-glucosyltransferase variant is introduced into a host microorganism capable of producing polyketide or a precursor of phenylpropanoid glycoside, and the host microorganism may be a recombinant microorganism into which a foreign gene is introduced or in which gene expression is controlled.

In the present invention, the recombinant microorganism into which the nucleic acid encoding the C-glucosyltransferase variant is introduced may have the same characteristics as those described in the recombinant microorganism for producing polyketide and/or phenylpropionic acid glycoside according to the present invention.

In the present invention, the step (a) may be performed in such a manner that the microorganism is cultivated by adding ascorbic acid to the medium during the cultivation. Here, the microorganism may be cultured by adding ascorbic acid preferably 0.1 to 1.5g/L, more preferably 0.2 to 1.0g/L, but the present invention is not limited thereto.

Unless otherwise indicated, the preparation method of the present invention may have the same features as those described in other aspects within the scope that one skilled in the art can understand.

Yet another aspect of the invention relates to a method of producing polyketide and/or phenylpropanoid glycosides, comprising:

(b) Recovering the polyketide and/or phenylpropanoid glycoside produced.

Although specific gene names are described in the present invention, it will be clear to those skilled in the art that the present invention is not limited to the corresponding genes.

Furthermore, even in the genes introduced in the present invention, the names of genes derived from specific microorganisms are described, but the scope of protection of the present invention is not limited to the corresponding names of genes, and is within the following ranges: it will be appreciated by those skilled in the art that the same functions as the corresponding genes are exhibited, and that when genes derived from another microorganism having a different gene name are introduced according to the technical features of the present invention, it is apparent that the corresponding recombinant microorganism also falls within the scope of the present invention.

Examples

The invention will be better understood by the following examples. However, these examples should not be construed as limiting the present invention and various changes or modifications can be made within the spirit and scope of the present invention as will be apparent to those skilled in the art.

Example 1 Experimental methods

1-1 flask culture

Flask culture was performed under the following conditions. Colonies were inoculated into 10mL of LB medium supplemented with the appropriate concentration of antibiotic, followed by overnight incubation at 37 ℃. The culture broth thus prepared was passed into a 250mL baffled flask containing 50mL of R/2 medium supplemented with 3g/L of yeast extract and 20g/L of glucose (containing 0.45g/L of ascorbic acid, if necessary), followed by culturing at 30℃and 200 rpm. The composition of the R/2 medium (pH 6.8) is as follows (per liter): 2g (NH) ₄ ) ₂ HPO ₄ KH of 6.75g ₂ PO ₄ 0.85g of citric acid, 0.7g of MgSO ₄ ·7H ₂ O and 5ml Trace Metal Solution (TMS) [ per liter 5M HCl,10g FeSO ] ₄ ·7H ₂ O, 2.25g ZnSO ₄ ·7H ₂ O, 1g of CuSO ₄ ·5H ₂ O, 0.5g MnSO ₄ ·5H ₂ O, 0.23g of Na ₂ B ₄ O ₇ ·10H ₂ O, 2g CaCl ₂ ·2H ₂ O and 0.1g (NH) ₄ ) ₆ Mo ₇ O ₂₄ ]. When the OD600 of the culture solution reached 0.6-0.8, exogenous gene expression was induced by adding 1mM isopropyl β -D-1-thiogalactoside (IPTG), followed by culturing for 48 hours.

1-2 fed batch fermentation

Fed batch dispensingThe fermentation was performed in R/2 medium (pH 6.8) containing 20g/L glucose, 3g/L yeast extract, 0.45g/L ascorbic acid and the appropriate antibiotics using a 6.6L BioFlo 320 fermenter (Eppendorf). Colonies were inoculated into 10mL of LB medium supplemented with the appropriate concentration of antibiotic, followed by overnight incubation at 37 ℃. The culture broth thus prepared was passed into a 250mL baffled flask containing 50mL of R/2 medium supplemented with 3g/L yeast extract, 20g/L glucose and 0.45g/L ascorbic acid, followed by cultivation at 30℃and 200rpm until OD600 reached about 4. After inoculation with the fermenter, the pH was maintained at 6.8 by automatic addition of ammonia solution and the temperature was maintained at 30 ℃. Oxygen saturation (DO) was maintained at 40% of air saturation, and 1vvm [ (air volume) ] was continuously blown in (workload) ^-1 (minutes) ^-1 ]Provided DO is maintained by increasing the stirring speed or adding pure oxygen concentration. IPTG (0.5 mM) was added when the OD600 reached about 20 and the depleted carbon source and other nutrients were automatically added to the fermentor by pH-stat strategy. Thus, the addition liquid per liter contains the following components: 650g glucose, 5g ascorbic acid, 6mL TMS and 8g MgSO ₄ ·7H ₂ O. When the pH is higher than 6.85, the additive liquid is automatically added into the fermentation tank.

1-3 production analysis

After the cultivation, production analysis was performed under the following conditions. After flask incubation, 50mL of the culture broth was centrifuged at 4,000g for 30 min, and the supernatant was desalted and concentrated. Here, oasis HLB cartridges (Water) is used. The supernatant was concentrated 1-fold for FK, 30-fold for KA, 45-fold for dcII and 200-fold for carminic acid. The concentrated sample was redissolved in an appropriate volume of DMSO and then the impurities were removed therefrom using a 0.22 μm PTFE filter. The prepared samples were analyzed by MS (LC/MSD VL; agilent) in combination with HPLC (1100 series; agilent). Eclipse XDB-C18 column was used, 0.1% formic acid was used as buffer A, and methanol was used as buffer B. Analysis was performed in ESI negative mode. For more accurate analysis of carminic acid, LC-MS/MS was performed using an HPLC triple quadrupole mass spectrometer (LCMS-8050, shimadzu) (MRM mode).

In addition, MS (Agilent 6550iFunnel Q-TOF LC/MS system) was used in combination with super HPLC (UHPLC; 1290 information II LC system; agilent) in LC-MS/MS analysis of aloin. Here, eclipse-plus C18 column was used, with 0.1% formic acid as buffer A and acetonitrile with 0.1% formic acid added as buffer B.

Example 2: identification of C-glucosyltransferase for producing carminic acid

Although the pathway of carminic acid production has not been specifically determined, PKS was used to induce carminic acid production because the carbon backbone of carminic acid has an anthraquinone-based structure (FIG. 1).

Thus, a production method of E.coli BAP1 strain (E.coli BL21 (DE 3) (Invitrogen)) was used, in which Sfp from Bacillus subtilis was introduced into the genome to exert the activity of an exogenous Acyl Carrier Protein (ACP), and this production method was carried out with reference to B.Pfeifer et al, science 2001,291 (5509), 1790-1792/D.Yang et al, PNAS2018,115 (40) 9835-9844. Thereafter, in order to apply type II PKS from Photorhabdus luminescens (Photorhabdus luminescens), pDS00-antDEFBG was constructed to introduce antD (ketosynthase), antE (chain length factor), antF (ACP), antB (phosphopantetheinyl transferase) and antG (malonyl-CoA: ACP malonyl transferase) derived from Photorhabdus luminescens. Specifically, the antDEF gene was amplified by PCR from the genome DNA of Photorhabdus luminescens using the antDE_F primer and the antDEF_R primer, and then inserted into NdeI and EcoRI restriction enzyme sites of pDS00 (a platform plasmid identical to pET-30a (+) primer except for the restriction enzyme sequence, novagen). The pDS00 plasmid was constructed as follows. Gene fragments containing the T7 promoter, multiple Cloning Site (MCS) and T7 terminator were amplified from pET-30a (+) using pET_NheI_DraIII and pET_SpeI_SphI primers, treated with SphI and DraIII restriction enzymes, and inserted into the SphI and DraIII sites of the pET-30a (+) plasmid to construct the pDS00 plasmid. Thereafter, antB was amplified from the genome DNA of P.photoperiod using the antB_F primer and the antB_R primer, and inserted into the HindIII site of pDS00 to construct the pDS00-antB plasmid. Subsequently, after digestion with NdeI and EcoRI restriction enzymes, the pDS00-antDEF plasmid was also digested with NdeI and EcoRI restriction enzymes to obtain two antDEF fragments, which were then combined using Gibson assembly to produce the pDS00-antDEFB plasmid. In addition, anti-G was amplified from the genome DNA of P.photoperiod using the anti-G_F primer and the anti-G_R primer, and inserted into the NdeI and EcoRI sites of pDS00 to construct a pDS 00-anti-G plasmid. The constructed plasmid was digested with NheI and SpeI restriction enzymes to obtain an antG fragment, which was then inserted into the NheI site of the pDS00-antDEFB plasmid to construct the pDS 00-antDEFBS plasmid.

TABLE 1

To produce xanthogenic acid (FK), zhuI and ZhuJ, which are cyclases derived from Streptomyces, were introduced. For this purpose, a pFK (pDS 00-antDEFBG-zhuiJ) plasmid was constructed. Specifically, based on zhuIJ DNA codon optimized for expression in escherichia coli, zhuIJ fragments were amplified by PCR using zhuij_f primers and zhuj_r primers and inserted into NdeI and EcoRI sites of pDS 00. The thus constructed pDS00-zhuiJ was digested with NheI and SpeI restriction enzymes to obtain zhuiJ fragment, which was then inserted into the NheI site of pDS00-antDEFBG to construct pFK.

The strain that transformed BAP1 with pDS00-antDEFBG-zhuiJ produced 88mg/L FK from glucose. The color of the culture broth was observed to be bright red at the beginning of the culture and turned to a cloudy brown color over time. This is thought to be due to the conversion of FK to melanin analogues and the like, and therefore, 0.45g/L ascorbic acid was added to the medium to prevent FK blackening, thereby increasing the FK yield to 154.9mg/L.

TABLE 2

Increasing intracellular malonyl-CoA concentration is also predicted to increase the manner of FK production, and acetyl-CoA carboxylase from corynebacterium glutamicum (encoded by accBCD1 gene) is over-expressed or the pabA gene is knockdown. Thus, in the accBCD1 overexpressing strain, FK production increased to 180.3mg/L (fig. 2).

Flask culture was started by inoculating colonies on LB agar plates into tubes containing 10mL of LB. Thus, an appropriate concentration of antibiotic was added, followed by incubation at 37℃and 220rpm overnight. 1mL of the seed culture thus prepared was inoculated into a 250mL baffle flask containing 50mL of R/2 medium (supplemented with 3g/L yeast extract and 20g/L glucose), followed by cultivation at 30℃and 200 rpm. The composition of the R/2 medium was as follows (pH 6.8, per 1L): 2g (NH) ₄ ) ₂ HPO ₄ KH of 6.75g ₂ PO ₄ 0.85g of citric acid, 0.8g of MgSO ₄ ·7H ₂ O and 5ml of Trace Metal Solution (TMS). The composition of TMS is as follows (based on 0.1M HCl, per 1L): 10g of FeSO ₄ ·7H ₂ O, 2.25g ZnSO ₄ ·7H ₂ O, 1g of CuSO ₄ ·5H ₂ O, 0.58g MnSO ₄ ·5H ₂ O, 0.02g of Na ₂ B ₄ O ₇ ·10H ₂ O, 2g CaCl ₂ ·2H ₂ O and 0.1g (NH) ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O. When the OD600 of the culture broth reached 0.6-0.8, exogenous enzyme expression was induced by the addition of 0.5mM IPTG, followed by 48 hours of culture. To all experiments for the production of xanthonic acid, nopic acid, dcII and carminic acid, 0.45g/L of ascorbic acid was further added.

Based on the results of flask culture, a small amount of nopaline acid (KA) (0.14 mg FK equivalent/L; i.e., mg FK eq/L) was also observed. This is thought to be caused by E.coli endogenous oxidase or ZhuiJ, but the conversion efficiency is too low and the enzyme responsible for the relevant reaction has not been determined. In the present invention, biochemical reaction analysis was performed using previously reported literature and compound databases.

Thus, the gene encoding the aclacinone 12-hydroxylase (DnrF) from streptomyces was predicted to be an enzyme that underwent the relevant reaction, and amplified using the dnrf_f primer and the dnrf_r primer, and then inserted into the NdeI and EcoRI sites of pDS00 to construct pDS00-DnrF. The dnrF gene was amplified by PCR using pET30a_frag_F primer and pET30a_frag_R primer based on the corresponding plasmids, and the pBBR1-T7 plasmid was amplified by inverse PCR using pET30a_IV_R primer and rrnB_IV_F primer (constructed by the method of S.Y. park et al, bioRxiv, DOI:10.1101/2020.11.27.401000from Kovach,M.E; phillips, R.W., elzer, P.H., roop, R.M., II; peterson, K.M., pBBR1MCS: a broad-host-range cloning vector.Biotechniques 1994,16 (5), 800-802), and the two DNA fragments were ligated using Gibson assembly to construct pBBR1-dnrF. After pBBR1-dnrF was introduced into FK-producing strain, flask culture was performed. Thus, KA (FIG. 3 a) was produced at 1.20mg FK eq/L.

TABLE 3

The production of Carminic Acid (CA) from FK proceeds through two biosynthetic pathways, both of which require monooxygenase and C-glucosyltransferase. FK may be oxidized to KA or may be C-glycosylated to dcII. DcUGT2 from cochineal has been shown to catalyze the conversion from FK to dcII (or from KA to CA) and its activity has been demonstrated in Saccharomyces cerevisiae (S. Cerevisiae) (Kannengara et al, nat Commun 2017,8). However, in order for the enzyme to be active, it must be glycosylated, and since it also has a transmembrane helix and a signal peptide, successful expression in cells such as E.coli is considered to be difficult. In fact, dcUGT2 cannot produce dcII when it is introduced into FK-producing escherichia coli. To solve the problems associated with DcUGT2 expression, ntr-DcUGT2 with the N-terminal signal peptide removed, ctr-DcUGT2 with the C-terminal transmembrane helix removed, and Ntr-Ctr-DcUGT2 with the N-terminal signal peptide and the C-terminal transmembrane helix removed were constructed, and plasmids with E.coli OmpA signal peptide attached to the N-terminal ends of Ntr-DcUGT2 and Ntr-Ctr-DcUGT2 were also constructed, but none produced dcII. Thus, it was concluded that DcUGT2 was not active in e.

Some examples of O-glycosylation of natural products have been reported in E.coli, but few examples of C-glycosylation have been reported. Thus, in the present invention, UDP-glycosyltransferase was selected which was found to undergo C-glycosylation in E.coli by biochemical analysis. Eight enzyme candidates were selected as follows: iroB (EnCGT) from escherichia coli Nissle; UGT708A6 (ZmCGT) from corn as a dual C/O-glycosyltransferase; UGT708C2 (FeCGT) from buckwheat; miCGT from mango; osCGT from rice; UGT708D1 (GmCGT) from soybean; gtUF6CGT1 (GtCGT) from gentiana rigescens; and AvCGT from aloe vera (fig. 4).

For the enzymes selected above, all genes except for the iroB gene amplified from the genomic DNA of escherichia coli Nissle using irob_gib_f primer and irob_gib_r primer were constructed and inserted into the NdeI site of the pCDFDuet-1 plasmid using Gibson assembly.

TABLE 4

Only GtCGT and ZmCGT were able to successfully convert FK to dcII. For ZmCGT, the main product is O-glycosylated FK and little dcII is produced. For GtCGT, 0.13mg CA equivalent/L (mg CA eq/L) of dcII was produced (FIG. 5). Since C-glycosylation requires large amounts of UDP-glucose, galU (encoding UTP-glucose-1-phosphate uridyltransferase), pgm (encoding phosphoglucomutase) and ndk (encoding nucleoside diphosphate kinase) are overexpressed, resulting in an increase in dcII yield to 0.30mg CA eq/L (FIG. 5). To construct the pBBR1-galU-pgm-ndk plasmid (all three genes were amplified from E.coli BL21 (DE 3) strain), the galU gene was amplified from the galU_gib_F primer and galU_gib_R primer and inserted into the EcoRI site of the pBBR1TaC plasmid by Gibson assembly. The pgm gene was amplified from the pgm_gib_f and pgm_gib_r primers and inserted into the kpnl site of the pBBR1TaC-galU plasmid, and the ndk gene was amplified from the ndk_gib_f and ndk_gib_r primers and inserted into the SphI site of the pBBR1-galU-pgm plasmid, thereby constructing pBBR1-galU-pgm-ndk.

TABLE 5

In order to successfully produce carminic acid by increasing the activity of GtCGT and DnrF, the present inventors tried to create mutants with increased activity by computer simulation. However, since the structure of the relevant enzyme has not been revealed, MODELLER was used to analyze protein structure (Webb, B.; sali, A.; comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 2016,54,5.6.1-5.6.37). Thereafter, mutants with increased activity were screened by PyRosetta using a docking simulation (Chaudhury, S.; lyskov, S.; gray, J.J., pyRosetta: script-based interface for implementing molecular modeling algorithms using Rosetta. Bioinformatics 2010,26 (5), 689-691). In addition to predictions based on computer simulations, mutants with expected increased activity were selected based on structural analysis results.

The homology modeling was performed on GtCGT (C-glucosyltransferase from trollius chinensis (Trollius chinensis); PDB ID 6JTD; protein sequence similarity 35.1%) as a control. Computer-based docking simulations (SW: autoDock Vina) using FK as ligand were performed using the resulting GtCGT structural model. Thus, 239 mutants were generated, 122 of which showed higher docking scores than the wild-type enzyme (table 6). Experiments were performed with the first 20 mutants and additional 14 mutants were tested for which an increase in activity was expected based on the predicted structural analysis results of GtCGT. Six mutants with higher KA yield than the wild type GtCGT were selected based on the results of flask culture after transformation of 34 mutants into FK strain (V93Q, Y193F, L164G, F G, R322D and V132A). Among them, the mutant showing the highest dcII production was identified as GtCGT ^V93Q And the yield increased by about 2.9 times (fig. 6 c). In this mutant, the Gln93 amino acid was judged to be located at the active site and thus bound directly to FK. Gln93 amino acid forms hydrogen bond with the hydroxyl group of C6, which would be expected to orient FK ligand correctly for C-glycosylation at C2. The Y193F mutant showed the second highest dcII concentration. To evaluate the synergistic effect between these two mutants, a double mutant (GtCGT was constructed ^V93Q/Y193F ) And then guide itInto FK strains. The double mutant produced 0.74mg CA eq/L dcII, 5.3-fold increase compared to wild type GtCGT (FIG. 6 c). In the V93Q mutant, the Tyr193 amino acid forms hydrogen bonds with the carbonyl group of C10 and blocks Gln93 from forming hydrogen bonds with the hydroxyl group of C6. Thus, it was hypothesized that FK ligand binding was improved by replacing Tyr193 with Phe193 and inhibiting hydrogen bonding at C10 (fig. 6 d).

TABLE 6

* The 20 highest docking scores are indicated in bold; mutants additionally selected according to structure are represented in blue;wild type GtCGT.

Constructing a mutant library by adopting the same method as DnrF, and determining the mutant with the highest KA yield as DnrF based on the mutant library ^P217K And KA production increased by about 2.2-fold (2.68 mg FK eq/L) (FIGS. 6a and 6 b).

Mutagenesis of specific sequences was performed as reported in the prior art (Zheng, l.; baumann, u.; reymond, j.l.; an effector one-step site-directed and site-saturation mutagenesis protocols.nucleic Acids Res 2004,32 (14), e 115). Here, dnrF is introduced therein ^P217K The plasmid pBBR1-T7 was designated as pKA, into which GtCGT was introduced ^V93Q/Y193F The plasmid pCDFDuet-1 of (E) was designated as pdcII. Construction of a P217K mutant of DnrF using DnrF_P217K_F and DnrF_P217K_R primers, construction of a V93Q mutant of GtCGT using GtCGT_V93Q_F and GtCGT_V93Q_R primers, and construction of a Y193F mutant of GtCGT using GtCGT_Y193F and GtCGT_Y193F_R primers.

TABLE 7

Name of the name	Sequence(s)	SEQ ID NO:
			DnrF_P217K_F primer (Forward)	5’-gtcgtaccgAAGgggtccaccggctggtac-3’	27
DnrF_P217K_R primer (reverse)	5’-ggtggacccCTTcggtacgacggcggtgag-3’	28
			GtCGT_V93Q_F primer (Forward)	5’-gacgtatggCAAtacatcaatcacttagac-3’	29
GtCGT_V93Q_R primer (reverse)	5’-gattgatgtaTTGccatacgtcaaacggtt-3’	30
			GtCGT_Y193F_F primer (Forward)	5’-gtgcccgacTTCctgcatccgcgcacaccc-3’	31
GtCGT_Y193F_R primer (reverse)	5’-cggatgcagGAAgtcgggcacttcatcata-3’	32

The protein sequence of the GtCGTV93Q/Y193F (GtUF 6CGT1V 93Q/Y193F) mutant constructed by the invention is described as follows.

MGSLTNNDNLHIFLVCFIGQGVVNPMLRLGKAFASKGLLVTLSAPEIVGTEIRKANNLNDDQPIKVGSGMIRFEFFDDGWESVNGSKPFDVWQYINHLDQTGRQKLPIMLKKHEETGTPVSCLILNPLVPWVADVADSLQIPCATLWVQSCASFSAYYHYHHGLVPFPTESEPEIDVQLPGMPLLKYDEVPDFLHPRTPYPFFGTNILGQFKNLSKNFCILMDTFYELEHEIIDNMCKLCPIKPIGPLFKIPKDPSSNGITGNFMKVDDCKEWLDSRPTSTVVYVSVGSVVYLKQEQVTEMAYGILNSEVSFLWVLRPPSKRIGTEPHVLPEEFWEKAGDRGKVVQWSPQEQVLAHPATVGFLTHCGWNSTQEAISSGVPVITFPQFGDQVTNAKFLVEEFKVGVRLGRGELENRIITRDEVERALREITSGPKAEEVKENALKWKKKAEETVAKGGYSERNLVGFIEEVARKTGTK

After constructing DnrF and GtCGT mutants with increased activity as described above, CA strains were constructed by combining these two mutant enzymes. By combining pFK with pCA (pCDF-dnrF ^P217K -GtCGT ^V93Q/Y193F ) Transformation of the plasmid into BAP1 strain CA strain was constructed. Thus, in order to insert two genes into one plasmid, dnrF was amplified from pKA by PCR amplification using the dnrF_NcoI_F primer and the dnrF_BamHI_R primer ^P217K And then inserted into the NcoI and BamHI sites of pdiii to construct pCA. Based on the flask culture result of the CA strain thus constructed, 22.2. Mu.g/L carminic acid was produced (FIG. 7). The carminic acid produced by glucose was identified by LC-MS/MS as shown in fig. 7.

To increase carminic acid production capacity, corynebacterium glutamicum (c.glutamicum) accBCD1 overexpression, pabA knockdown and galU-pgm-ndk overexpression were tested alone or in combination, and the yields in each strain were as follows (fig. 7 a): pabA KD,25.9 μg/L; accBCD1 OE,74.9 μg/L; galU-pgm-ndk OE,41.0 μg/L; accBCD1 OE-galU-pgm-ndk OE,49.9 μg/L; pabAKD-galU-pgm-ndk OE, 57.7. Mu.g/L; pabAKD-accBCD1 OE, 57.2. Mu.g/L; and pabA KD-accBCD1 OE-galU-pgm-ndk OE, 25.2. Mu.g/L, confirming that carminic acid was produced at the highest concentration of 74.9. Mu.g/L in the strain carrying pFK, pCA, pACC overexpressing accBCD1 (BL 21 (DE 3)). pFK: pDS00 derivative (P) containing anti-DEFBG derived from Photorhabdus luminescens and codon-optimized zhuiJ derived from Streptomyces R1128 _T7 -antDEFBG-T7 _T -P _T7 -zhuIJ-T7 _T ) The method comprises the steps of carrying out a first treatment on the surface of the pCA: containing dnrF in different operons ^P217K And GtCGT ^V93Q/Y193F pCDFDuet-1 of (C)Derivatives (P) _T7 -dnrF ^P217K -T7 _T -P _T7 -GtCGT ^V93Q/Y193F -T7 _T ) The method comprises the steps of carrying out a first treatment on the surface of the pACC: pBBR1TaC derivatives containing accBC and accD1 from Corynebacterium glutamicum (ATCC 13032). In addition, the strain was subjected to fed-batch fermentation, thereby producing 0.65mg/L carminic acid.

TABLE 8

Example 3: by GtCGT ^V93Q/Y193F Production of aloin

Aloin is a representative cosmetic additive extracted from aloe. Aloin is used as a whitening agent in the cosmetic industry due to its anti-tyrosinase and anti-melanogenesis effects, and is attracting attention as a potential pharmaceutical or cosmetic raw material due to its anti-inflammatory and anti-radical properties. However, the aloin content in plants is low and more efficient preparation methods are required. The production of aloin has been reported (D Yang et al, proc. Natl. Acad. Sci. U.S. A.2018,115 (40), 9835-9844). However, no C-glucosyltransferase has been reported to convert aloe pine into aloin (fig. 8), and thus, in the present invention, whether the GtCGT mutant developed in example 2 was effective in producing aloin was tested.

In the present invention, in order to produce aloe-pine, E.coli BL21 (DE 3) strain was transformed with the following plasmid: pCDF-RpALS, pWAS-anti-pabA and pBBR1-zwf. Thus, the strain expresses the following genes: rpALS (encoding aloe vera pine synthase), anti-pabA synthesis modulating sRNA, and zwf (encoding e.coli glucose 6-phosphate 1-dehydrogenase) (Yang, d.; kim, w.j.; yoo, s.m.; choi, j.h.; ha, s.h.; lee, m.h.; lee, s.y.; repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proc.Natl. Acad. Sci.u.s.a.2018,115 (40), 9835-9844).

The strain produced 30.9mg/L of aloe vera pine from glucose. Prior to aloin production, rpALS was additionally introduced into a compatible plasmid to increase aloe-pine production. For this purpose, rpALS was introduced into the prsduet-1 plasmid with a high copy number RSF replication origin. RpALS was amplified from previously constructed pCDF-RpALS using als_ndei_f primer and als_ndei_r primer, and then inserted into the NdeI site of prsduet-1 using Gibson assembly to construct the relevant plasmid. Thereafter, pCDF-RpALS and pRSF-RpALS were simultaneously transformed into the E.coli BL21 (DE 3) strain which already contained the pWAS-anti-pabA plasmid and the pBBR1-zwf plasmid, and flask culture of the strain was performed. Thus, 102.1mg/L of aloe vera was produced, demonstrating a significant increase in aloe vera production compared to prior to modification.

TABLE 9

Thereafter, to test for aloin production, pCDF-GtCGT or pCDF-GtCGT was used ^V93Q/Y193F The plasmid was transformed into BL21 (DE 3) strain containing pWAS-anti-pabA, pRSF-RpALS and pBBR1-zwf plasmids, followed by flask culture. Thus, it contains GtCGT ^V93Q/Y193F 0.06 μg/L of aloin was produced and a greater amount of aloin was successfully produced than for the strain containing GtCGT (fig. 9).

Similar to the test to increase aloe-pine production, rpALS was additionally introduced to increase aloin production. To this end, pCDF-RpALS-GtCGT was constructed ^V93Q/Y193F Rather than introducing pCDF-GtCG ^TV93Q/Y193F . Specifically, rpALS was amplified from pCDF-RpALS using pcdfduret_f primer and pcdfduret_r primer, and inserted into the NcoI and BamHI sites of pCA plasmid by Gibson assembly. Thereafter, four plasmids, pRSF-RpALS, pCDF-RpALS-GtCGT ^V93Q/Y193 pWAS-anti-pabA and pBBR1-zwf were transformed into E.coli BL21 (DE 3), which was designated ALS strain.

ALS strains successfully produced 0.3 μg/L aloin from glucose. The aloin produced was identified by LC-MS/MS as shown in figure 9.

Thus, the present inventors succeeded in producing aloin in a state of unknown enzyme by introducing GtCGT, and were able to pass the introduction of GtCGT ^V93Q/Y193F Greatly improves aloin production Ability to develop. Here, gtCGT ^V93Q/Y193F The enzyme mutant can be said to be an enzyme capable of producing a glycoside of polyketide, and the present inventors are based on GtCGT ^V93Q/Y193F Additional mutants were further created to construct mutants capable of further enhancing aloin production.

Example 4: by introducing additional mutants into the GtCGT ^V93Q/Y193F To increase aloin production

The present inventors have attempted to develop GtCGT exhibiting activity against aromatic polyketones by further improving the above-developed GtCGT ^V93Q ^/Y193F Mutants to increase the conversion efficiency from aloe vera to aloin. For this purpose, aloe-pine as a new substrate was docked to the GtCGT produced in example 2 ^V93Q/Y193F And (5) a structural model. Thus, mutants expected to bind more stably to aloe-pine and increase enzyme activity were selected as shown in table 10 below.

TABLE 10

Mutagenesis of the specific sequence was performed in the same manner as in example 2. Here, pCDF-RpALS-GtCGT was used ^V93Q/Y193F Plasmids containing the gene mutants were constructed as templates and using the primer pairs shown in table 11 below. The thus constructed plasmids were transformed into BL21 (DE 3) strain together with three plasmids (i.e., pWAS-anti-pabA, pRSF-RpALS and pBBR 1-zwf), and then flask culture was performed under the same conditions as in example 3 using the thus constructed strain. The concentration of aloin produced was measured in MRM mode of HPLC triple quadrupole mass spectrometer (LCMS-8050, shimadzu) of KAIST Biocore Center.

TABLE 11

According to the results of flask culture, the introduction of additional mutants such as I323S, T50R, T50V, I18P, I95T, Q M and I323A increased aloin production by 10-fold or more, particularly 7.75 μg/L aloin by GtCGT V93Q/Y193F/I323S mutant (FIG. 10), which increased 25.8-fold or more compared to the conventional concentration of 0.3 μg/L.

In addition to structure-based search for additional mutants, computer-based docking simulations (SW: autoDock Vina) using aloe-pine as ligand were performed using the GtCGT structural model to further increase enzyme activity. Thus, 15 mutants showed higher docking scores than the wild-type enzyme (table 12).

TABLE 12

Numbering device	Mutant
		1	V22A
2	L29A
		3	E46G
4	V48G
		5	E51C
6	A55S
		7	S86V
8	D99G
		9	R103V
10	C151G
		11	L184G
12	L194A
		13	E332P
14	I18A
		15	P385A

Mutagenesis of the specific sequence was performed in the same manner as in example 2. Here, pCDF-RpALS-GtCGT was used ^V93Q/Y193F Plasmids containing the gene mutants were constructed as templates and using the primer pairs shown in table 13 below. The thus constructed plasmids were transformed into BL21 (DE 3) strain together with three plasmids (i.e., pWAS-anti-pabA, pRSF-RpALS and pBBR 1-zwf), and then flask culture was performed under the same conditions as in example 3 using the thus constructed strain. The concentration of aloin produced was measured in MRM mode of HPLC triple quadrupole mass spectrometer (LCMS-8050, shimadzu) of KAIST Biocore Center.

According to the results of flask culture, the introduction of additional mutants such as P385A, L194A and V48G increased aloin production by 5-fold or more, and in particular, 4.23. Mu.g/L aloin production by GtCGT V93Q/Y193F/P385A mutant (FIG. 11) by 14.1-fold or more compared to the conventional concentration of 0.3. Mu.g/L.

TABLE 13

Example 5: by GtCGT ^V93Q/Y193F Production of phenylpropanoid glycosides

To test the applicability of the C-glucosyltransferase developed in the present invention to phenylpropanoid-based natural products, the present inventors conducted the following experiments.

To confirm intracellular expression of GtCGT ^V93Q/Y193F Is expressed by pCDF-GtCGT ^V93Q/Y193F And pBBR1-galU-pgm-ndk into E.coli BL21 (DE 3), and 1mM IPTG was administered when cell growth reached an OD600 of 0.6-0.8. Thus, 70. Mu.M luteolin, 0.5mM naringenin, or 185.2. Mu.M apigenin was co-administered followed by incubation for an additional 36 hours. The amounts of substrate and product were analyzed by LC-MS. Flask culture was performed in a 250mL baffled flask containing 50mL of R/2 medium supplemented with 3g/L yeast extract and 20g/L glucose, and culture was performed at 30℃and 200 rpm.

According to the result of the culture, 15.0. Mu.M vitexin was produced from 185.2. Mu.M apigenin, 51.6. Mu.M naringenin-6-C-glucoside was produced from 0.5mM naringenin, and 27.9. Mu.M isoorientin was produced from 70. Mu.M luteolin. These values correspond to conversions of 8.1%, 10.3% and 27.9%, respectively (fig. 10).

As described above, the glycosyltransferases of the invention also exhibit activities for producing various phenylpropanoid C-glucosides, indicating that the enzymes of the invention are versatile enzymes capable of producing various polyketides and phenylpropanoid C-glucosides.

Example 6: gtCGT ^V93Q/Y193F Is purified of K _M And V _max Is measured by (a)

To investigate in more detail the C-glucosyltransferase GtCGT developed in the present invention ^V93Q/Y193F Purifying the enzyme and measuring the kinetic parameters of the enzyme. For enzyme purification using His-tag, expression GtCGT and GtCGT with 6 XHis-tag linked to N-terminal were constructed ^V93Q/Y193F pCDF-NHis-GtCGT and pCDF-NHis-GtCGTmut plasmids of (C). Each plasmid was constructed by PCR amplification of pCDF-GtCGT and pCDF-GtCGTmut using the GtCGT_N_His_IV_F primer and the GtCGT_N_His_IV_R primer, followed by blunt-end ligation by DpnI treatment and T4 PNK and T4 ligase treatment. Each of the two plasmids thus constructed was transformed into E.coli BL21 (DE 3) strain, followed by seed culture in a tube containing 10mL of LB, and then culture in a flask containing 500mL of LB at 37℃until OD ₆₀₀ Reaching 0.8. After enzyme expression was treated with 1mM IPTG, the cells were harvested by centrifugation and cultured at 20℃for an additional 16 hours, and then resuspended in 30mL of lysis buffer (50 mM NaH ₂ PO ₄ 0.3MNaCl, 10mM imidazole, pH 7.5). The cells were sonicated and then centrifuged at 10,000rpm at 4℃for 40 minutes to obtain a supernatant containing the water-soluble protein. The supernatant was passed through a TALON resin (Clontech) to purify only the His-tagged protein bound thereto. Washing buffer (50 mM NaH was used ₂ PO ₄ After removal of impurities, 0.3M NaCl, 20mM imidazole, pH 7.5), the enzyme was purified using elution buffer and 90, 160, 230 or 300mM imidazole was added to the lysis buffer.

TABLE 14

Purified enzyme was buffer exchanged with enzyme storage solution (50 mM HEPES,20% glycerol, pH 7.5) using Amicon Ultra-15 centrifugal filter (regenerated cellulose, 50,000NMWL; merck) and K was calculated _M And V _max Value, FK was converted to dcII using purified enzyme. Here, the extent of the reaction was determined using the UDP-Glo glycosyltransferase assay kit (Promega). Since the kit is capable of measuring free UDP generated as a reaction byproduct by luminescence,thus 200. Mu.L of enzyme reaction solution containing 0.1. Mu.M enzyme and FK (50 mM HEPES, 0.1mM UDP-glucose, 5mM MgCl) ₂ pH 7.5), at 25℃for 1 hour, 25. Mu.L was removed therefrom, and the amount of luminescence was measured using a kit. The reaction rate and substrate concentration were substituted into Michaelis-Menten equation and analyzed using OriginPro 2019 software to calculate GtCGT and GtCGT ^V93Q/Y193F K of (2) _M And V _max Values. Thus, compared to the GtCGT, the GtCGT ^V93Q / ^Y193F K of (2) _M The value was reduced by 19.5%, compared with the GtCGT ^V93Q / ^Y193F V of (2) _max The value increased by 18.2% (FIG. 13; table 15). Thus, compared to the GtCGT, the GtCGT ^V93Q ^/Y193F V of (2) _max /K _M The value increased by 46.8%, indicating that the GtCGT ^V93Q / ^Y193F The catalytic efficiency of the mutant is increased.

TABLE 15

^a Mean ± standard deviation (SD; n=3)

EXAMPLE 7 genetic information

1. ravC (SEQ ID NO: 37) from Streptomyces griseogray (Streptomyces ravidus)

atgtccagtttcagtattgatgatctgaagcgtatcttgcgcgaaggggcaggggcaacggctgagttagacggtgacattttagacgcctcctttgatgatttggggtatgattctttggctcttcttgaaacgggttcgcgcatcggacgtgaatacggtttggaatttgaggatacagctttcgccgacgtggaaacacctcgtgacttggtcggcgtagttaatgctcagttatcggccccggctccgcgtgggtaa

2. ZhuiJ (SEQ ID NO: 38) from Streptomyces R1128

atgcgtcatgtagagcatacagtcaccgttgcggccccagcagacttggtttgggaggtacttgccgatgtcttaggctatgctgacatcttcccaccgacggaaaaagttgaaattcttgaggaggggcaaggataccaggtagtgcgccttcacgtcgatgttgcgggtgagattaatacatggaccagtcgtcgcgatttagaccctgcgcgccgcgtaattgcttaccgccaacttgagacggctccgatcgtgggccacatgagcggggaatggcgtgctttcacactggatgccgaacgtacccaattagtcctgactcacgatttcgtaacccgtgcagccggggatgacggtttagtcgccggaaaattgaccccagatgaggcgcgcgaaatgttagaagcggtggtagaacgtaactctgtcgccgacttaaacgcggtccttggagaagctgagcgtcgcgtccgcgcagccggtggagttggtaccgtaactgcgtaataataattttgtttaactttaagaaggagatatatccatgtcagggcgcaaaacctttttagacttaagttttgctacccgcgacacaccgtcggaggcgactccggtggtggtagatttgctggaccacgtaactggagccaccgtattaggattatcacctgaggatttccccgatggtatggctatttccaatgagaccgttacgttgacgacccacactggcacgcacatggatgcgccactgcactatggtcccttaagtgggggagttccggcaaagtcgattgaccaagtgcccttggaatggtgctatggacctggagttcgtttggatgttcgccacgtgccggcaggagatggtattactgtcgatcatttgaacgccgcgttggatgcagcagagcacgatttggcccccggtgacattgtgatgctgtggaccggcgcggacgctctgtggggaacccgcgaatacttgagcacgtttccggggttaactgggaaggggacacaatttttggtcgaggcgggtgttaaagtcattggcattgatgcatggggactggatcgcccgatggcagctatgatcgaagaataccgtcgtacgggcgataaaggagcattatggccggctcacgtctatggacgcacacgcgaatacctgcaattagagaagcttaataatttgggcgctttaccaggagctacagggtatgacatttcatgctttccggttgcggttgcaggcactggagctgggtggactcgtgtggtcgccgttttcgagcaagaggaagaggattaataa

3. trTT7 from Arabidopsis thaliana (Arabidopsis thaliana) (SEQ ID NO: 39)

atgtcccatcgccgcaaccgtagccacaacaatcgtttacccccgggacctaatccctggcccattatcggaaacctgccgcacatgggtacgaagccccaccgtacgttgtcggctatggttacgacctatggcccaattctgcacctgcgtctgggttttgtagacgtggtcgtagcggcgtccaagtcggtcgccgagcagttcttgaagattcacgacgctaacttcgcttcacgtccccccaactcaggagccaaacacatggcgtataattatcaagacttggtattcgccccctatggacatcgctggcgcttattgcgcaagatcagctcggtgcatctgttctcggcaaaagcgttggaggactttaaacacgtgcgtcaggaagaggtcggaacactgactcgcgaattagtacgtgtcggcaccaaaccagtgaaccttgggcaactggtgaatatgtgcgttgtcaacgccttaggacgcgaaatgatcggacgccgcttattcggggccgatgccgatcataaggcggatgagttccgctcgatggtcacggagatgatggcgttagcgggggtctttaatatcggcgactttgtaccgtcattagactggcttgatctgcaaggggtcgctggaaagatgaagcgtttacataagcgttttgatgcgttcttaagttcgattttaaaagaacatgaaatgaatgggcaagaccaaaagcataccgatatgttatcgaccttaatcagccttaagggtacagatctggatggggatggcggctccttaacggatactgaaattaaggcgcttttattaaacatgttcacagccggaaccgacacatcagccagtacagtagattgggcaatcgctgaattgatccgccaccccgatatcatggtgaaggctcaggaagaattagatattgttgtaggtcgcgaccgccctgtgaatgagtctgatatcgcccaactgccgtacttacaggcggtaattaaggaaaattttcgtctgcatccacctactcccctgtctttgccgcacattgcgagtgaatcctgtgagattaacggttaccatattcccaaaggttcaacattacttaccaacatctgggctatcgcccgtgatccggaccagtggagcgatccgttagcttttaaaccagaacgttttctgccaggaggagaaaaatctggggttgatgtaaaaggtagtgatttcgagctgattccgttcggtgcaggccgtcgcatttgtgcaggcctgtctctgggtcttcgcacgatccagttcttaacagcgactttagtacaagggtttgattgggagttagctgggggagtgacgcccgaaaaactgaacatggaagaatcgtacgggttaactttgcaacgcgctgtccctctggtagtacacccgaaacctcgtttggccccgaatgtgtacgggctgggcagtggctaa

4. ATR2 from Arabidopsis thaliana (SEQ ID NO: 40)

atgtcttcttcttcttcttcttctacctctatgatcgacctgatggctgctatcatcaaaggtgaaccggttatcgtttctgacccggctaacgcttctgcttacgaatctgttgctgctgaactgtcttctatgttaattgagaatcgtcagtttgctatgatcgttacaacatccatcgcggtccttattggttgtatcgttatgttggtctggcgccgctctggttccggtaactctaaacgtgtggaaccgcttaaaccgctggtgatcaaacctcgtgaggaggaaatcgacgatggacgtaaaaaagtaacaatctttttcggaacgcagactggcactgcggaaggttttgccaaggcattaggtgaggaagctaaagctcgttatgaaaagacgcgcttcaagattgttgatctggacgattacgctgcagacgatgatgaatacgaggaaaaattaaaaaaagaggatgtagctttcttcttcttagctacgtatggcgatggtgaaccgacagataatgccgctcgtttttataagtggtttaccgaaggcaatgatcgtggtgagtggttgaaaaacttaaaatatggggttttcgggctgggcaatcgtcaatacgagcactttaacaaggtcgcgaaagtggtcgatgacattctggttgagcaaggcgcacagcgtctggtacaagtagggttaggggatgatgaccagtgtatcgaagatgatttcacagcttggcgcgaagcattgtggcccgagttggatacgattctgcgcgaagagggcgatacggctgttgccacaccctacacagccgcagtattagagtatcgcgtaagcatccatgatagcgaggatgccaaattcaatgatattaaccttgctaacggaaacgggtatacagtttttgacgctcaacatccgtataaggccaacgttgcggtcaaacgtgaattgcacaccccggagtccgaccgttcctgtatccatctggaatttgatattgcgggatcaggtttaacatacgaaactggagatcacgttggtgttctgtgcgataacttatccgagacggtggatgaggcactgcgccttttagacatgtcccctgacacgtattttagcttgcatgctgaaaaagaggacggtactccgatcagtagctcgctgccaccgccgtttccaccgtgcaatttacgcacggctttaacacgttacgcgtgcctgttgtcatctcctaagaaatccgccttagtggctttggctgcacacgctagtgatcccactgaggccgagcgcttgaaacacttagcaagccctgcaggtaaagacgagtactccaagtgggtagtagagtcacagcgtagtttattggaggtgatggccgagtttcctagtgcgaagccaccgttgggagttttctttgccggggtggctccgcgtttgcaaccacgtttttatagcatcagtagttctccaaaaatcgccgagactcgcattcacgttacatgtgccctggtctacgaaaaaatgccgactgggcgcatccacaagggtgtatgctcgacttggatgaagaacgccgtaccctacgaaaagtctgaaaactgcagctcggcgccaatcttcgtacgccagtccaatttcaagttgccgtcagattcaaaggtaccgatcattatgatcggtccaggaacggggttagctccgttccgtgggttcttacaggaacgcttagcactggtcgagtcgggggtagaattgggcccctccgtcttgtttttcgggtgtcgtaaccgtcgcatggacttcatctatgaagaagagctgcaacgtttcgtggaaagtggggcgcttgctgaactgtcggtggcgttttcccgcgaaggacccacgaaagaatatgttcaacacaaaatgatggacaaagcgtcggatatctggaacatgatttcacagggcgcttatttatatgtatgtggcgatgcgaaaggcatggcgcgtgacgtccaccgttctctgcacaccattgcgcaagagcaaggtagcatggattcaacgaaagcagaaggcttcgtgaagaatttacaaacctctgggcgctatcttcgtgatgtgtggtaa

5. UrdGT2 (SfCGT) from Streptomyces fradiae (Streptomyces fradiae) TU2717 (SEQ ID NO: 41)

atgtttgccctggctccgctggccacagcagctcgtaatgcaggtcatcaggtagtaatggcagcaaaccaggacatgggacctgtcgtaaccggggttggccttccagccgtagcaaccactgatcttccgatccgtcatttcatcactaccgatcgtgaaggacgtcccgaggccattccttctgacccggtcgcgcaggcccgtttcactggtcgctggttcgcccgtatggctgccagttccttgccccgtatgcttgatttttcacgtgcatggcgcccagacttaatcgtcggtggtactatgagctatgtcgctccgctgttagctcttcacctgggagtcccgcacgcccgtcagacttgggatgcggtagacgctgatggaatccacccaggtgctgatgctgagcttcgcccagagttaagcgaattgggattggagcgccttcccgcacccgatttgttcatcgacatttgcccgccctcgttacgtcctgccaacgcagcaccagctcgcatgatgcgccacgtagccacgagccgccaatgcccgttagagccgtggatgtatacacgtgacactcgccagcgtgttttagtgacgtcgggatcgcgtgttgcaaaagaatcttacgatcgtaatttcgattttttacgtggattagcgaaggatttggtgcgctgggatgttgaattaattgtggctgctcctgacaccgtggctgaggctcttcgtgccgaggtgccacaagctcgcgtagggtggacccctttagacgtcgtggcccctacatgcgatttattggtgcatcacgccggcggagtctctacgctgactggtttatcggctggcgagccccaattattaatcccaaagggcagtgtattggaagctcctgcgcgccgcgtagcagattacggcgcggcgattgcactgttgcctggtgaggactcgacggaagctatcgccgatagttgtcaggagttgcacgccaaggacacttatgcccgccgcgctcaagacttaagccgcgaaatttcagggatgcctctgccggccacagtggtgactgcactggaacagttagcctaa

6. DcUGT2 (DcCGT) from cochineal (SEQ ID NO: 42)

atggagttccgcttattgattctggcactgtttagtgtcttaatgagtacgtcaaatggtgccgagattcttgccctgtttccaattcacggaatttccaactacaacgtagcagaggcacttcttaaaacgcttgccaaccgtggacataacgtcactgtggtcacgtcattcccccagaagaaacccgtgccaaacttgtatgaaatcgacgtatctggggccaaaggtcttgctaccaactcaattcactttgagcgtcttcagactatcattcaagacgtgaagtccaacttcaagaatatggtgcgtctttcacgcacttactgcgagattatgttctccgatcctcgcgtacttaacatccgcgacaaaaagtttgatttggtaatcaacgcggtgttcggatcggattgtgacgctggctttgcgtggaaaagccaagcacccctgatctcaattttaaatgcacgccacaccccgtgggcgcttcaccgtatgggaaacccttctaaccctgcgtacatgccagtaatccactcacgcttcccagtaaaaatgaatttcttccagcgtatgatcaatacaggctggcatttatactttttgtatatgtacttttattatggcaacggagaagacgcaaataaaatggcgcgtaagtttttcggtaacgacatgcccgacattaacgaaatggtctttaacacatcgttgctttttgttaatactcactttagtgttgatatgccctaccctcttgttcctaactgcattgaaattggtgggattcacgtcaaagagccccaaccgcttcccctggagattcaaaaattcatggatgaagcagagcatggtgtaatctttttcactttgggctcgatggtccgcactagcacctttcccaatcagactatccaagcgtttaaagaggcgttcgcagaacttccacagcgtgttctttggaagtttgaaaacgagaatgaagacatgccttctaacgttttaatccgcaaatggtttccacagaacgatatctttggacataagaacattaaggcgtttatctcgcatggcggtaactcaggggcccttgaagccgtgcatttcggcgtgcccatcatcggcattcctctgttctatgatcagtatcgtaatatcttgagcttcgtgaaagaaggggttgcggtactgctggacgttaatgatttgacaaaggataacattctgtcgtctgttcgtacagtagtgaacgataaatcttacagcgaacgcatgaaagctctgtcccagctgtttcgcgatcgtccaatgagtcccctggacacggctgtctattggacggaatatgtaatccgtcaccgcggagcacatcatctgaagactgctggcgcttttttgcactggtatcagtatttgttgctggatgtgattactttccttttggtaacattctgtgccttttgcttcattgtcaagtacatctgtaaggcgctgattcaccattattggtcgagttccaagagtgaaaaattaaagaaaaactaa

7. UGT708A6 (ZmCGT) from maize (SEQ ID NO: 43)

atggctgctaatgggggggatcatacctccgcgcgcccacatgtggtgttgcttccatccgctggcatgggacatcttgtccctttcgcccgcttagctgtggctttatctgagggacacggctgcaacgtaagtgtagctgcagttcaaccaacggtttcctctgcggagtcgcgtctgttagacgcacttttcgtcgccgccgccccagccgtccgccgtcttgatttccgcctggcccccttcgatgaatccgagttccccggtgcagacccttttttcttacgcttcgaggcgacacgtcgctcggcaccgcttctggggccgttattagatgcggcggaagcctccgcacttgtgactgatattgtccttgcttcggtagcgttgccagtggcgcgcgagcgtggagttccctgctatgtgctttttacgtcatcggccgcaatgctgtcgttgtgtgcgtattttccagcttatttagatgcacatgcagcggccggctcggtcggggtcggagtagggaacgtcgacattccaggggtatttcgcatccctaagtcgagcgtcccgcaagcacttcacgatccagatcatttatttacccagcagtttgtcgcaaatggccgttgtttagttgcctgcgacggcattcttgttaacaccttcgacgccttcgagcccgatgcagtaaccgcactgcgccaagggtcgatcacagtctctggcggttttccaccagttttcaccgtgggcccaatgcttcccgttcgcttccaggcagaggagacggctgactacatgcgttggttgtctgcacaaccaccccgcagtgtcgtctatgtctcgtttggaagtcgcaaggcgattcctcgcgaccagttacgtgaattggccgcagggttagaggctagtggcaagcgttttctgtgggtagtaaagtcgaccatcgtcgaccgcgatgataccgccgatctgggcggcttgttgggggacggctttcttgagcgcgtccaaggtcgtgcatttgtgactatgggatgggtggaacaggaagagattttgcaacatggctcggttggcttgtttatctcgcattgtgggtggaatagccttaccgaagccgccgcgttcggggtaccagttcttgcctggcctcgtttcggagatcagcgtgtgaacgccgccttagttgcgcgctctggattgggagcgtgggaagaagggtggacttgggatggtgaggagggacttactacacgcaaggaagtggcgaaaaagatcaagggcatgatggggtacgatgctgtagccgaaaaggcggccaaagttggtgacgcagctgcggcagcaattgcaaaatgtggcacgagttatcaatctttggaagagtttgtacaacgttgccgcgacgccgagcgtaagtaa

8. OsCGT from rice (SEQ ID NO: 44)

atgccttcctcaggagacgctgccggtcgtcgccctcacgtcgtgctgatcccttcagccggaatggggcacctggtcccgtttggtcgtctggctgtcgcactttcctctgggcacggatgtgatgtgtctttagtaacagttcttcctactgtgagtacagcggagtcaaagcatcttgatgcactttttgacgcattccccgcagttcgccgtcttgacttcgagttggcgccatttgacgcatcagagtttcccggtgctgaccctttcttccttcgttttgaggcgatgcgccgttcggctccattgcttggccctttgctgacgggcgcgggcgctagcgcactggcgacggacattgctttaacgtctgtcgtaattccagtagcaaaagagcaagggcttccgtgtcacattttattcactgcgtcggccgcaatgttatcattgtgtgcctacttcccaacttatttggatgccaacgctggcggagggggcggtgtgggcgacgtggatattcctggagtgtatcgcattccgaaggcatcaattccacaagccttacatgatcccaaccacttgtttactcgtcagtttgtggcgaatggtcgtagtcttacctcggcggccggtattctggtgaacactttcgatgcgttagagccggaggcagtagctgcattgcagcaaggaaaggtagcctccggctttccaccagtattcgcggtggggccgttgctgcctgcctctaaccaggccaaggatccgcaggcaaattacatggagtggctggacgcccagcccgcccgcagcgtagtttatgtaagtttcgggagtcgcaaggcgatttcacgtgaacaacttcgcgagctggctgctggcttagaggggagcggccaccgttttctgtgggtcgtgaaatccaccgtcgtggatcgtgacgacgcggccgagctgggagagctgttggacgagggttttttagagcgtgtcgagaagcgtggattggtgacaaaggcatgggtcgaccaggaggaggtactgaaacatgaaagcgtagccctgtttgtctcacattgcggctggaacagcgtgactgaggcggcggcgagcggtgtgcctgtcctggccttaccccgcttcggggaccaacgtgttaattcaggagtggtggcacgtgcaggattaggagtatgggcggatacttggtcgtgggagggcgaagcaggcgtgattggtgcggaggaaatctcagagaaggtcaaagcagctatggccgacgaagctttacgtatgaaagctgcatcccttgcagaggcagccgccaaggcagtggctggcggtgggagtagtcatcgctgtttagcggaatttgcccgtctgtgtcaaggtggaacttgccgtactaattaa

9. UGT708D1 (GmCGT) from soybean (SEQ ID NO: 45)

atgagttctagtgaaggagtggtacatgtagcttttcttccaagtgcaggaatgggccacttgaaccctttccttcgcttggcggcgaccttcattcgttatggttgtaaagtaacgttaatcaccccgaagcctactgtatccctggcagaatcgaatttaatttcacgcttttgttccagctttccacatcaggttacgcaactggacctgaatttagtcagcgttgatccaacgaccgttgacacaatcgacccattcttcttacaatttgaaaccatccgccgtagtctgcatcttttacctcccattttaagtcttcttagcactcctttgtctgccttcatttatgacattactcttatcacgcctttgctttctgtaatcgagaagctgtcgtgccccagctacttgtattttacatcttcagcacgtatgttctctttcttcgcacgtgtctccgtgttgtccgcatctaatcccgggcagactccctcgtcatttatcggtgacgatggagttaagatccctgggttcacaagccccatcccacgcagcagtgttccgcctgcgattcttcaagcgtcctcaaatctttttcagcgcattatgttagaagacagcgcgaacgttaccaagcttaataatggggtcttcatcaatagctttgaagaactggagggcgaagctttagccgctttaaacggggggaaagttcttgaaggtctgccgcccgtgtacggggtgggcccccttatggcgtgtgaatatgagaaaggcgacgaggagggtcaaaagggctgcatgtcttcgatcgtgaagtggctggatgaacagtcgaagggaagcgtggtatacgtgtccttgggcaatcgtacggaaacgcgccgtgagcagattaaggatatggcccttggtttgatcgagtgtggctatggattcttgtgggtcgtcaaactgaagcgcgtcgataaagaagatgaggaaggcttagaagaggtgttaggtagcgagctgagttccaaggttaaggagaagggtgttgtagttaaggaatttgttgaccaagtcgaaattttgggccacccaagtgttgggggatttttgtcgcacgggggttggaacagcgtaactgaaactgtatggaagggagtgccttgtctgtcatggccacagcatagtgatcagaagatgtctgcggaggtaatccgtatgtccggaatgggtatctggcccgaggagtggggctgggggacgcaagatgttgtgaagggagacgaaatcgccaaacgcattaaggaaatgatgtcgaacgaatcgttgcgcgtaaaggcgggagaattgaaggaagcggcgttaaaggcggcaggggtaggggggagttgtgaagtgactattaaacgtcagatcgaagagtggaaacgcaatgcccaggctaattaa

10. GtUF6CGT1 (GtCGT) from gentian trislot (SEQ ID NO: 46)

atggggagtttgactaacaacgataatcttcatatttttcttgtgtgcttcatcggccagggcgtggtcaatcccatgttacgtttggggaaggcgttcgcctccaaagggttacttgtcactttaagcgcaccggaaatcgttggaactgagatccgtaaggcgaataaccttaatgatgaccaaccaatcaaggtgggttccgggatgattcgtttcgaatttttcgacgatggatgggaatccgtaaacggtagcaaaccgtttgacgtatgggtctacatcaatcacttagaccagacaggccgtcaaaaacttccgattatgttaaagaaacatgaggagacagggactcctgtatcttgcttgatcctgaatcccttagtcccttgggtcgcggacgtagccgattcacttcagatcccctgcgctaccttgtgggtccaatcttgtgcaagtttttcagcatattaccactaccaccacgggttagtgcctttcccaaccgaatcagagcccgagatcgacgtacaacttcctgggatgccacttttgaaatatgatgaagtgcccgactacctgcatccgcgcacaccctaccccttttttggcacgaacattttaggtcaattcaagaatttatccaagaacttctgtatcctgatggataccttctacgagttggaacacgagatcatcgataatatgtgtaaattgtgtccgattaagccaattggcccgttgtttaagattccgaaagacccaagctccaacggaatcacgggtaatttcatgaaagtggatgactgcaaggagtggctggacagccgtccaacatcaactgtggtttacgttagtgtcgggtctgttgtatatttgaagcaggagcaggttacagaaatggcatacggcattttaaattcggaagtttcgtttttgtgggtgctgcgcccgccgagcaaacgcatcggtacggaaccgcatgtactgcccgaggagttctgggagaaggccggagatcgtggcaaggtggtgcaatggtcaccccaggagcaggtgcttgctcaccccgccactgtcggttttttaacacactgtggatggaatagcactcaagaggcgatttcgagcggagtgcccgtcatcactttcccacaatttggggaccaagtgaccaatgctaagttccttgtggaggaatttaaggtcggggtccgtttaggccgcggagagttagaaaatcgcatcatcacacgcgacgaagtagaacgcgctttacgcgagattacttcaggccccaaggctgaagaggtaaaagagaacgccttaaaatggaagaagaaggcagaagagacagtagctaaaggcggctactccgaacgtaatcttgtaggcttcattgaagaggtggctcgtaagactggtacaaagtaa

11. AvCGT (SEQ ID NO: 47) from aloe vera

atggaggaaatttccagtaaagtggagttcttatcccttaagcccagcatgtcaggaagtccccgttacagccccacatttcgtaaaatcggaagcggtcgcaattcccgccgcgactcccgtgctcatgcagggaatttcccctggattcgcaacaatcgtgtttttttttggctgcttttaatcaccatctgggcgtacatcggctttcacgtccaatctcaatgggcacatggcgaccataaagctgagttcgttggatacaagtcggaggtagggaagatgggtgaggacgtcaagtcggtaaatagtacgactacgttctccattgtacacaagggcaatttaactgttgaaggaaagaaagaccccgattccaattttggtatttcactgttgaaaaagggtaaacaggttctttcccgtttaaattcacgcaaaaagggccatcgttcgcgcaaggtgtcggaaaaactggaagaagaaacggacgacaatgggacgggagaaatggatgaggtccttatccagcgcaaaaacacatcttatggcttaattgtagggccttttgccaaactggaagagagtgtgcttgagtggagcccaggcaagcgccgtggtgtctgctatcgtaagggagaatttgcccgcgcggtgtcttctcagcgctttatgttgatcttccacgaattgtcaatgactggcgccccattgtccatgttggaattggccacggagatcctgtcttgcggtgggtctgtgagcgctattgtattatctaagaagggagggttaatgccggaactgaagaagcgtggtattaaggttttgcaagaccgtgacaaggtgagtttcaaggtcgccacgaaagtagacctgattattgcgggatctgctgtatgtagtagctggatcgagccatatctggagtatttccccgctgggtccggacatattgtctggtggatcatggaaaaccgtcgcgaatacttcgaccgtagcaagcatcttttaaaccgtgtgaaaattttggcatttcttagcgatagccagtcaaagcagtggctttcttggtgtgaggaagagaagattaaattcctgatccagccaatgttagtgccgttgtcagttaacgatgagctggccttcgttgccggtattccttgtagcttgaatactccagcattatcagtggagaaaatgatcgaaaagcgtgatttattacgtcacgcagtccgtaaggaaatggggttgggggacaatgacatgcttgtgatgagtttaagtagcatcaacccagccaagggtcagcgctttctgcttgaggcagccttactggtagctgaacacaatgtatcattgaaagatgctaacagttacagtcttatggaggaggagaagttatccgggaacgcacctcaaaatcaaaccatcatgatcggtcaactgaatcctggccacgtacttcagatcgccaatgacactaataagcccgtcaatgcgttacagaagattggcgccacacgtgtctcgtcgaagcgtcgcggcaagctgcatacgaatacagtcacgggcgtgcttcagaaaagccgcaaacttttgtccgaggcagcaggtatgaaggaggaaaccctgaaagtccttgtaggttccgtcggatcgaaatcgaataaggttctgtatgtaaaggcaatcatggaatacatcagccaacattctaatttgtctaaggtcgttctttggaccccagccaccacgtctatcgcagcactgtacgccgccgcggacgtgtacgtcattaacgctcagggacatggagagacattcggtcgcgtgacgatcgaggcgatggcctttggcctgccagtgctggggactgacgccggagggactaaagaaatcatcgaccaccgtgttacgggacttctgcatcctgtgggtcccgagggcactgtactgttagcgcaacacattcaatatcttttaaaaaatcccagcgtgcgcaagaaaatgggtatcaatggtcgccgcaaagtacaagataaatacttaaaacaccagacttacgagtcccttggcaaagtcatgttcaaatcgatgcgtccccgttaa

12. From Streptomyces wave ATCC 29050 dnrF(SEQ ID NO:48)

gtggccttgacgaagccggatgtcgatgtcctcgtggtgggcggcggtctcggggggctgtccaccgccctgttcctcgcccgccggggggcgcgggtcctgctggtggagcggcatgccagcacctcggtcctgcccaaggcggcaggccagaacccgcgcaccatggaactgttccgcttcggcggcgtggccgacgagatcctggccacggacgacatccgcggcgcccagggcgacttcaccatcaaggtcgtggagcgcgtgggcggtcgcgtcctgcacagcttcgcggagagcttcgaggaactggtcggtgcgacggaacagtgcacgcccatgccctgggcgctcgctccccaggaccgggtggagcccgtcctggtggcccacgccgccaagcacggcgcggagatccggttcgccaccgaactgacctccttccaggcgggcgacgacggtgtcacggcccgcctgcgcgacctgggcacgggagcggagagcaccgtgagcgcccgctacctggtcgccgccgacggaccccgcagcgcgatccgggagagcctgggcatcacccggcacggtcacggcaccctggcccacttcatgggcgtcatcttcgaggccgacctcaccgccgtcgtaccgcccgggtccaccggctggtactacctgcagcacccggacttcaccggcacgttcggccccaccgaccggcccaaccggcacaccttctacgtccgctacgaccccgaacgcggcgagaggccggaggactacacaccgcagcgctgcaccgagctgatccggctggctgtcgacgcgcccgggctcgtcccggacatcctcgacatccaggcctgggacatggcggcgtacatcgccgaccggtggcgcgaagggccggtgctgctggtcggcgatgccgccaaggtcaccccgcccaccgggggcatgggcggcaacaccgccatcggcgacgggttcgacgtggcctggaagctggccgccgtgctgcgcggcgaggcgggcgagcggctcctcgacagctacggggcggagcggtcgctcgtgtcccgcctcgtcgtcgacgagtcactcgccatctacgcccagcgcatggctccccacctgctcggcagcgttcccgaggaacgcggtacggcgcaggtcgtcctgggcttccgctaccgctccaccgccgtcgccgccgaggacgacgaccccgagccgaccgaggatccgcgacgcccgtccgggcgccccggcttccgcgcaccccacgtctggatcgaacaggacggcacacggcgttccaccgtcgagttgttcggcgactgctgggtgctcctggccgcaccggagggcggcgcctggggccaggcggccgcccgcgccgccgcggatctgggcgtccgcctcgacgtccatctcgtcggccgcgatgtcgccgccccctccggcgaactgacgcggacctacgggatcggccgggcgggggccagcttggtgcgcccggacggcgtggtcgcctggcgtacggcagtagcgccgggagcggaggcccaggaccagctgagcaccctgctcacccggctgctggcccgctga

13. Anti-def from light emitting bacilli(SEQ ID NO:49)

The underlined parts indicate the parts of the frames of antD and antE that overlap (i.e., the start codon and the stop codon overlap), and the bold lower case letters at the ends indicate the sequence between antE and antF.

14. anti-B from Photobacterium luminescense(SEQ ID NO:50)

ATGGACGATATTTCTTTATCATCTGATTTTTTTGATCTTTGGATTATCAAAATCGACGATATTGATTTAGCTTCTATTGAACAGTTAATTCACTGTTCTGATATAGTTCGCCATAACCAAATTTGTTTAGCGGATAGAAGAAAGAGATTTATATTTAGACGGGCTGCATTACGTTATGTTTTGAGTCAATATTTATCTGATTATGAAATCATAACGAATGATAACGGAAAACCTTATATATCCACGGAGCAAGACTTCAAATATTATTTTTCACTGAGTGCTTCAGGAAACTATTGTGCCATTGGTTTTAGCTCAAGGGAAATAGGTGTTGATATTGAAGTCACTCCTTCTAAGGTAAAATTTTCAGAAATTATTGAACGTTTTATTAAGGATAAAGATTTGGAATATATGAAAGGTATAATGTTAAAACAACTATCAGGAGTTAGTCTCGGATTTAATAACTATTATCATTTAATGTCATTATATTATTGGGTTAGACTTGAAGCATATATTAAATTATTTGCTTCGACTTTACATGAGAAATTATTGGTTAATAACTCTGATTCTGTTAAAGATATGAAAGAATTGGAGGCAAGCACATTATTGATTCATAGTCAGCAATTTGTTTGTGCCTTATCTCAAAAGAAAGTCATTTCTACACCAAATATCAAGGAAATAAATTATTCCGAAATTATAAGGAACAAAGATGAGTAA

15. anti-G from Photobacterium luminescense(SEQ ID NO:51)

ATGAAACTAATCTCTATGTTGTTACATTCAGAGCATGATAACTTACATCATGATTGTATTGTCACTAAGGATTATCATTATACAAGAAAAGAGGTGATATCTTCTGTTTCCCATTTAATTGATGATTTATTGAGTCGAGGAGTGCAAAAAGGTAATAAAGTCATTGTTATATTTGAACATGATGAATTAGGTGTTTTCTTTTTGGCTGCCGCCAGTGCTATGGGGTTGCATTTATTAATGCCCTATAATTTATCATCAGCGACAATCGATGAATGGATTAATTTTACCAATGAAGTGCAATACGATTTTGTTGTTTATCTCAAAAAAGATAAACATTTTGTTGGAAAATTAAAAGAAAACAACATTAATGTTATTGATATTTCAGATCATAAGATCAGAGTTAGTGATGATATTGCGGAAATCCCAATGATAACTTATTCTCCGCAACCTATTGCTAACTTTATTGTCCTGTTCACCAGTGGGAGTACAGGCAAACCAAAAGCCATTAGTATTTCAGAATCGTTAGTATGTCGTCGAATTTATTCGGTGACCGAGAAATTAAAATTTACGCAAGATGCCAAAATATTCATGTCAGGTTTGTTGAATAATACAACTGGAGTGATTTTTTCTTTCGGCTCATTATTGCATCAATCAACACTTTTTATACCCGAAGATAGAAATGTAGAGAGATGGCCTGATTATCTTTCTCGCAATAAAATCACTCATATTATGTTACGCCCAGAATCAATGAAATTATTCGTTAAATCGACAGCAGAACTTAATATTGATCTCTCTTGTTTACGGGTGGTTGCTTATGGCGCTGCGGCGATGCCTCCTAGCGTACTTGAGAAAGGGCGACAATTAATTGGCTGTGAATGGGTGCAGGGATATGGGTTAAGTGAAACTTATGGTCCTTTCTGTTGGGTGGATGAGCAAGATCATCGTGATAAAAGATATCTCAATTCAATTTATTGTGTTGGTAAGATTGATAATACATTGGAAGTGGCAGTTAAACCTATTATAGGTTCATCGGATAATATCGGAGAAATTATACTAAGGGGTAAAAGTATTATGGAAGGATATTATGATGTCCTTTCTGGAGAAATAACGCCTCCTGATGAATGGTTTGCCACTGGTGATCTTGGTTATATAGATGAAGAGGGTTATTTAGTTTTGAAAGGACGTAAGCAAAATACGTTTATGAGTGCTAACGGACACAGAATTTATCCTGAAGAAATTGAATCTATTTTATCCCGAATACCCAATGTGAATGTCGCTACGGTTGTTGGTTTTTCTTTCCATGAAAATGGTGTTGCTATTGATCAGCCGGTTGCTTGCATGAGTGGAGAGATATCTAAGAAGTCATTACCTGAAATTGAAGATATTATTTCATCATTTTTAATGAGTAAACTCAGTCGAGAAAAATGGCCGGATTGGTTCTATGTTACTGATGAATGCTTTCCGAAAAGCCATAATGATAAGATATTGAAATCAGAGTTAATTAAATCAATCGATCCTAAGAAATTATTTACATTGAGGAATCAATAA

16. From Streptomyces coelicolor (Streptomyces) coomcat of coelicolor)(SEQ ID NO:52)

Atgctcgtactcgtcgctcccggccagggcgcccagacgcccggcttcctgactgactggctcgccctccccggtgccgctgaccgcgtcgccgcgtggtcggacgccatcggactcgatctcgcccacttcggcaccaaggccgacgcggacgagatccgagacacgtccgtggcccagccgctgctggtcgccgccggaatcctgtccgccgcggcactcggtacgcagacatctgtcgctgacgcgacgggccccgggttcacccccggcgcggtcgccggacacagcgtcggcgagatcaccgccgccgtcttcgcgggcgtcctcgacgacaccgccgcgctgtccctcgtacgccgtcgcggcctggccatggccgaggccgcggcggtcaccgagaccggcatgtcggcgctgctcgggggcgaccccgaggtgagcgtcgcgcacctggagcggctcggcctgaccccggcgaacgtgaacggcgccggtcagatcgtggcggcgggcaccatggagcagctggccgcgctgaacgaggacaagcccgagggtgtgcgcaaggtcgtcccgctgaaggtggccggcgcgttccacacccgccacatggcccccgccgtggacaagctcgccgaggccgccaaggcgctgacgccggccgacccgaaggtgacgtacgtctccaacaaggacgggcgggccgtcgcctccggcaccgaggtgctggaccggctggtcggccaggtcgccaacccggtgcgctgggacctgtgcatggagacgttcaaggagctgggcgtcaccgcgatcatcgaggtgtgtccgggcggcacgctgaccgggctggccaagcgggcgctgcccggagtgaagacgctggccctgaagacccccgacgacctcgacgcggcccgtgagctcgtcgccgagcacacccaggcctaa

17actVA-orf5 from Streptomyces coelicolor(SEQ ID NO:53)

Atgagcgaggacacgatgacccaggagcggccgtccctgacggcacacgcccgccggatcgccgaactcgccgggaagcgggcggccgacgccgaacagcagcgccggctgagccccgacgtcgtcgacgcggtccttcgagccggtttcgccgcccacttcgtaccggtggcgcacggcggccgggccgcgacgttcggggagctggtggagcccgtcgcggtgctcggcgaggcctgtgcctcgaccgcctggtacgcctcgctcacggcgagcctcggccggatggccgcctacctgccggacgagggccaggccgagctgtggtccgacggccccgacgccctgatcgtcggtgccctgatgccgctgggccgggccgagaagaccccgggcggctggcacgtgtcgggcacctggccgttcgtcagcgtcgtggatcactccgactgggcgctgatctgcgccaaggtcggcgaggagccgtggttcttcgcggtgccgcgacaggagtacgggatcgtcgacagctggtacccgatgggtatgcgcggaacgggcagcaacacgctcgtcctcgacggggtgttcgtgccggatgcgcgggcctgcacccgtgcggccatcgcggcaggtctcggtccggatgccgaggcgatctgtcacaccgtgcccatgagggcggtcaacgggctggccttcgcactgccgatgctcggcgcggcccgcggggccgcggccgtgtggacctcgtggaccgccggaagactggccgggccgaccgggcagaacgccgtctcgtcccaggaccgcgtggtgtacgagcacacgctggcccgggccacgggtgagatcgacgcggcccagctgctgttggagcgggtcgcggcggtcgccgacgccggctcggcgaccggcgtactggtcggccgcggggcgcgggactgcgccctggcggcggagctgctgaccgccgcgaccgaccggctgttcgcctcggcgggcacccgggcacaggcccaggacagcccgatgcagcgcctgtggcgcgatgtgcacgcggcgggcagccatatcgggctgcagttcgggcccggggcggcgctgtacgccggagagctgttgaggaggagcaacgatggctga

18. actVB from Streptomyces coelicolor(SEQ ID NO:54)

Atggcagccgaccagggaatgctccgggacgccatggcccgggtgccggccggggtggcgctcgtcaccgcccatgaccgcgggggagtcccgcacggtttcaccgccagttcgttcgtgtccgtctcgatggagccgccactggcactggtctgcctggctcgtacggccaactccttcccggtgttcgacagttgcggcgagttcgcggtgagcgtgctgcgcgaggaccacacggacctggccatgcgcttcgcgcgcaagtccgcggacaagttcgcgggcggggagttcgtccgtaccgcgcggggagcgaccgtgctcgacggagcggtcgcggtcgtcgagtgcacggtccacgagcgctacccggcgggcgaccacatcatcctgctcggcgaggtccagtccgtgcacgtcgaggagaagggcgtaccggcggtctacgtggaccgccggttcgccgccctgtgctcggcggcgggtgcctgcccgtccgccaccgggcggggcgtgcccgcgcatgccggctaa

19. From Pseudomonas fluorescens (Pseudomonas fluoroscens) pobA(SEQ ID NO:55)

ATGAAAACGCTAAAAACCCAAGTCGCCATTATTGGCGCCGGTCCCTCCGGATTGCTGCTCGGCCAGTTACTGCACAACGCGGGTATCCAGACCCTGATTCTAGAGCGCCAGAGCGCCGACTACGTGCAAGGCCGCATCCGTGCCGGGGTGCTGGAGCAAGGCATGGTCGACCTGCTGCGCGAAGCGGGCGTCAGCCGACGCATGGACGCCGAGGGCCTTGTGCATGACGGTTTCGAATTGGCACTCAATGGCGAACTCACCCACATCGACCTCAAGGCGCTCACCGGCGGCCAGTCGGTGATGATCTACGGCCAGACCGAAGTCACCCGTGACTTGATGGCCGCCCGCGAAGCGGCGGGTGGCATCACTCTATACGAAACGCAGAACGTGCAGCCTCATGGTCACAAAACTGATCGACCCTGGCTGACCTTCGAGCACCAGGGTGAAGCTTTTCGCCTGGAGTGCGACTACATCGCGGGCTGTGATGGTTTTCACGGTGTGGCGCGGCAGTCGATTCCGGCGCAGTCGTTGAAGGTCTTCGAGCGCGTCTATCCCTTCGGTTGGCTGGGCGTCCTCGCCGACACACCGCCGGTGCATGACGAACTGGTGTACGCCAAACATGCGCGTGGCTTTGCCCTGTGCAGCATGCGCTCGCCGACCCGCAGCCGCTATTACCTGCAAGTGCCGGTTGAAGAAGCGCTGGATGAATGGTCGGATCAGCGCTTCTGGGATGAGCTGAAAACCCGTTTGCCCAGTGCACTGGCGGCCCAACTGGTCACCGGGCCATCCATCGAGAAGAGCATCGCGCCGCTGCGCAGCTTTGTGGTCGAGCCGATGCAATACGGGCGCCTGTTCCTGCTGGGGGACGCCGCGCATATCGTGCCGCCCACCGGGGCCAAGGGCTTGAACCTGGCGGCCAGCGACGTGAGTACGCTGTTTCGGATCTTGCTCAAGGTCTATCGCGAGGGGCGGGTGGACCTGCTGGAACAGTACTCAGCGATCTGCTTGCGCCGCGTATGGAAAGCCGAACGGTTTTCCTGGTGGATGACTTCGATGTTGCACCAGTTTCCGGAGGCCGACGGGTTCAGCCAGCGCATTGCCGAGAGCGAGCTTGCGTATTTCATCAGCTCCGAGGCGGGCCGCAAAACCATCGCAGAAAATTACGTCGGGCTTCCTTACGAAGCTATCGAATAA

^P217K 20. dnrF from Streptomyces wave (final dnrF mutant)(SEQ ID NO:56)

gtggccttgacgaagccggatgtcgatgtcctcgtggtgggcggcggtctcggggggctgtccaccgccctgttcctcgcccgccggggggcgcgggtcctgctggtggagcggcatgccagcacctcggtcctgcccaaggcggcaggccagaacccgcgcaccatggaactgttccgcttcggcggcgtggccgacgagatcctggccacggacgacatccgcggcgcccagggcgacttcaccatcaaggtcgtggagcgcgtgggcggtcgcgtcctgcacagcttcgcggagagcttcgaggaactggtcggtgcgacggaacagtgcacgcccatgccctgggcgctcgctccccaggaccgggtggagcccgtcctggtggcccacgccgccaagcacggcgcggagatccggttcgccaccgaactgacctccttccaggcgggcgacgacggtgtcacggcccgcctgcgcgacctgggcacgggagcggagagcaccgtgagcgcccgctacctggtcgccgccgacggaccccgcagcgcgatccgggagagcctgggcatcacccggcacggtcacggcaccctggcccacttcatgggcgtcatcttcgaggccgacctcaccgccgtcgtaccgAAGgggtccaccggctggtactacctgcagcacccggacttcaccggcacgttcggccccaccgaccggcccaaccggcacaccttctacgtccgctacgaccccgaacgcggcgagaggccggaggactacacaccgcagcgctgcaccgagctgatccggctggctgtcgacgcgcccgggctcgtcccggacatcctcgacatccaggcctgggacatggcggcgtacatcgccgaccggtggcgcgaagggccggtgctgctggtcggcgatgccgccaaggtcaccccgcccaccgggggcatgggcggcaacaccgccatcggcgacgggttcgacgtggcctggaagctggccgccgtgctgcgcggcgaggcgggcgagcggctcctcgacagctacggggcggagcggtcgctcgtgtcccgcctcgtcgtcgacgagtcactcgccatctacgcccagcgcatggctccccacctgctcggcagcgttcccgaggaacgcggtacggcgcaggtcgtcctgggcttccgctaccgctccaccgccgtcgccgccgaggacgacgaccccgagccgaccgaggatccgcgacgcccgtccgggcgccccggcttccgcgcaccccacgtctggatcgaacaggacggcacacggcgttccaccgtcgagttgttcggcgactgctgggtgctcctggccgcaccggagggcggcgcctggggccaggcggccgcccgcgccgccgcggatctgggcgtccgcctcgacgtccatctcgtcggccgcgatgtcgccgccccctccggcgaactgacgcggacctacgggatcggccgggcgggggccagcttggtgcgcccggacggcgtggtcgcctggcgtacggcagtagcgccgggagcggaggcccaggaccagctgagcaccctgctcacccggctgctggcccgctga

^V93Q/Y193F 21. GtCGT from gentian trislot (final GtCGT mutant)(SEQ ID NO:57)

atggggagtttgactaacaacgataatcttcatatttttcttgtgtgcttcatcggccagggcgtggtcaatcccatgttacgtttggggaaggcgttcgcctccaaagggttacttgtcactttaagcgcaccggaaatcgttggaactgagatccgtaaggcgaataaccttaatgatgaccaaccaatcaaggtgggttccgggatgattcgtttcgaatttttcgacgatggatgggaatccgtaaacggtagcaaaccgtttgacgtatggCAAtacatcaatcacttagaccagacaggccgtcaaaaacttccgattatgttaaagaaacatgaggagacagggactcctgtatcttgcttgatcctgaatcccttagtcccttgggtcgcggacgtagccgattcacttcagatcccctgcgctaccttgtgggtccaatcttgtgcaagtttttcagcatattaccactaccaccacgggttagtgcctttcccaaccgaatcagagcccgagatcgacgtacaacttcctgggatgccacttttgaaatatgatgaagtgcccgactTcctgcatccgcgcacaccctaccccttttttggcacgaacattttaggtcaattcaagaatttatccaagaacttctgtatcctgatggataccttctacgagttggaacacgagatcatcgataatatgtgtaaattgtgtccgattaagccaattggcccgttgtttaagattccgaaagacccaagctccaacggaatcacgggtaatttcatgaaagtggatgactgcaaggagtggctggacagccgtccaacatcaactgtggtttacgttagtgtcgggtctgttgtatatttgaagcaggagcaggttacagaaatggcatacggcattttaaattcggaagtttcgtttttgtgggtgctgcgcccgccgagcaaacgcatcggtacggaaccgcatgtactgcccgaggagttctgggagaaggccggagatcgtggcaaggtggtgcaatggtcaccccaggagcaggtgcttgctcaccccgccactgtcggttttttaacacactgtggatggaatagcactcaagaggcgatttcgagcggagtgcccgtcatcactttcccacaatttggggaccaagtgaccaatgctaagttccttgtggaggaatttaaggtcggggtccgtttaggccgcggagagttagaaaatcgcatcatcacacgcgacgaagtagaacgcgctttacgcgagattacttcaggccccaaggctgaagaggtaaaagagaacgccttaaaatggaagaagaaggcagaagagacagtagctaaaggcggctactccgaacgtaatcttgtaggcttcattgaagaggtggctcgtaagactggtacaaagtaa

22. ALS from Rheum palmatum(SEQ ID NO:58)

atggcagatgtcctgcaggagatccgcaactcgcagaaggcgagcgggcccgccacggtgctcgccatcggcactgcccatccaccgacgtgctaccctcaggccgactaccccgacttctacttccgagtttgcaagagcgagcacatgaccaaactcaagaagaaaatgcaattcatttgtgacagatcggggataaggcagcggtttatgttccacacggaagagaacctggggaagaacccggggatgtgcacattcgacgggccatcgctgaacgcgcggcaggacatgctgatcatggaagtgccgaagctgggggcggaggcggcggagaaggcgatcaaggagtgggggcaggacaagtcccggatcacccacctcatcttctgcaccaccacgagcaacgacatgcccggggcggactaccagttcgccaccctgttcgggctgaaccccggcgtgagccgcaccatggtctaccagcagggctgcttcgccgggggcaccgtgctgcgcctggtcaaggacatcgcggagaacaacaagggggcgcgcgtgctggtggtgtgctcggagatcgtggccttcgccttccgcgggccccacgaggaccacatcgactccctcatcgggcagctcctgttcggggacggggccgccgccctcgtggtcgggacagacatcgacgagagcgtcgagaggcccatcttccagatcatgtcggcgacccaggcgaccatccccaactcgctgcacaccatggctctccatctgacggaggcggggctgaccttccatctcagcaaggaggtgcccaaggtggtgagcgacaacatggaggagctcatgctcgaggccttcaagccgctcgggataaccgattggaactccatattctggcaagtgcatcccgggggtagagccatccttgacaagatcgaggagaagctggagctcaccaaggataagatgcgggattcccgctacatcttgagcgagtacgggaatctcaccagcgcctgtgtgctctttgtcatggacgagatgaggaagaggtccttccgggaagggaagcagaccaccggagacggctacgagtggggtgtcgccatcggattggggcccggtcttaccgtcgagaccgttgtcttgcgtagcgtccccattccctaa

23. accBC from Corynebacterium glutamicum(SEQ ID NO:59)

gtgtcagtcgagactaggaagatcaccaaggttcttgtcgctaaccgtggtgagattgcaatccgcgtgttccgtgcagctcgagatgaaggcatcggatctgtcgccgtctacgcagagccagatgcagatgcaccattcgtgtcatatgcagacgaggcttttgccctcggtggccaaacatccgctgagtcctaccttgtcattgacaagatcatcgatgcggcccgcaagtccggcgccgacgccatccaccccggctacggcttcctcgcagaaaacgctgacttcgcagaagcagtcatcaacgaaggcctgatctggattggaccttcacctgagtccatccgctccctcggcgacaaggtcaccgctcgccacatcgcagataccgccaaggctccaatggctcctggcaccaaggaaccagtaaaagacgcagcagaagttgtggctttcgctgaagaattcggtctcccaatcgccatcaaggcagctttcggtggcggcggacgtggcatgaaggttgcctacaagatggaagaagtcgctgacctcttcgagtccgcaacccgtgaagcaaccgcagcgttcggccgcggcgagtgcttcgtggagcgctacctggacaaggcacgccacgttgaggctcaggtcatcgccgataagcacggcaacgttgttgtcgccggaacccgtgactgctccctgcagcgccgtttccagaagctcgtcgaagaagcaccagcaccattcctcaccgatgaccagcgcgagcgtctccactcctccgcgaaggctatctgtaaggaagctggctactacggtgcaggcaccgttgagtacctcgttggctccgacggcctgatctccttcctcgaggtcaacacccgcctccaggtggaacacccagtcaccgaagagaccaccggcatcgacctggtccgcgaaatgttccgcatcgcagaaggccacgagctctccatcaaggaagatccagctccacgcggccacgcattcgagttccgcatcaacggcgaagacgctggctccaacttcatgcctgcaccaggcaagatcaccagctaccgcgagccacagggcccaggcgtccgcatggactccggtgtcgttgaaggttccgaaatctccggacagttcgactccatgctggcaaagctgatcgtttggggcgacacccgcgagcaggctctccagcgctcccgccgtgcacttgcagagtacgttgtcgagggcatgccaaccgttatcccattccaccagcacatcgtggaaaacccagcattcgtgggcaacgacgaaggcttcgagatctacaccaagtggatcgaagaggtttgggataacccaatcgcaccttacgttgacgcttccgagctcgacgaagatgaggacaagaccccagcacagaaggttgttgtggagatcaacggccgtcgcgttgaggttgcactcccaggcgatctggcactcggtggcaccgctggtcctaagaagaaggccaagaagcgtcgcgcaggtggtgcaaaggctggcgtatccggcgatgcagtggcagctccaatgcagggcactgtcatcaaggtcaacgtcgaagaaggcgctgaagtcaacgaaggcgacaccgttgttgtcctcgaggctatgaagatggaaaaccctgtgaaggctcataagtccggaaccgtaaccggccttactgtcgctgcaggcgagggtgtcaacaagggcgttgttctcctcgagatcaagtaa

24. accD1 from Corynebacterium glutamicum(SEQ ID NO:60)

atgaccatttcctcacctttgattgacgtcgccaaccttccagacatcaacaccactgccggcaagatcgccgaccttaaggctcgccgcgcggaagcccatttccccatgggtgaaaaggcagtagagaaggtccacgctgctggacgcctcactgcccgtgagcgcttggattacttactcgatgagggctccttcatcgagaccgatcagctggctcgccaccgcaccaccgctttcggcctgggcgctaagcgtcctgcaaccgacggcatcgtgaccggctggggcaccattgatggacgcgaagtctgcatcttctcgcaggacggcaccgtattcggtggcgcgcttggtgaggtgtacggcgaaaagatgatcaagatcatggagctggcaatcgacaccggccgcccattgatcggtctttacgaaggcgctggcgctcgcattcaggacggcgctgtctccctggacttcatttcccagaccttctaccaaaacattcaggcttctggcgttatcccacagatctccgtcatcatgggcgcatgtgcaggtggcaacgcttacggcccagccctgaccgacttcgtggtcatggtggacaagacctccaagatgttcgttaccggcccagacgtgatcaagaccgtcaccggcgaggaaatcacccaggaagagcttggcggagcaaccacccacatggtgaccgctggcaactcccactacaccgctgcgaccgatgaggaagcactggattgggtacaggacctggtgtccttcctcccatccaacaatcgctcttacacaccactggaagacttcgacgaggaagaaggcggcgttgaagaaaacatcaccgctgacgatctgaagctcgacgagatcatcccagattccgcgaccgttccttacgacgtccgcgatgtcatcgaatgcctcaccgacgatggcgaatacctggaaatccaggcagaccgcgcagaaaacgttgttattgcattcggccgcatcgaaggccagtccgttggatttgttgccaaccagccaacccagttcgctggctgcctggacatcgactcctctgagaaggcagctcgcttcgtccgcacctgcgacgcgtttaacatcccaatcgtcatgcttgtcgacgtccccggcttccttccaggcgcaggccaggagtatggtggcatcctgcgtcgtggcgcaaagctgctctacgcatacggcgaagcaaccgttccaaagattaccgtcaccatgcgtaaggcttacggcggagcgtactgcgtgatgggttccaagggcttgggctctgacatcaaccttgcatggccaaccgcacagatcgccgtcatgggcgctgctggcgcagtcggattcatctaccgcaaggagctcatggcagctgatgccaagggcctcgataccgtagctctggctaagtccttcgagcgcgagtacgaagaccacatgctcaacccgtaccacgctgcagaacgtggcctgatcgacgccgtgatcctgccaagcgaaacccgcggacagatttcccgcaaccttcgcctgctcaagcacaagaacgtcactcgccctgctcgcaagcacggcaacatgccactgtaa

^V93Q/Y193F ^V93Q/Y193F GtCGT (GtUF 6CGT 1) variants(SEQ ID NO:61)

26. Codon optimized zhuIJ of escherichia coli(SEQ ID NO:62)

Description of the abbreviations

FK: yellow carminic acid

KA: nopalinic acid

CA: carminic acid

Industrial applicability

According to the invention, the variants of C-glucosyltransferase have an increased glycosidic bond formation capacity compared to wild-type C-glucosyltransferase, thereby enhancing the glycoside producing effect of polyketides and similar natural products, in particular type I, type II and type III polyketides, non-ribosomal peptides, phenylpropanoids and other aromatic natural products. Thus, the variant of C-glucosyltransferase according to the present invention can be used for producing pharmaceuticals, food additives, nutritional supplements, etc. containing an increased amount of C-glycoside compounds as constituent components by polyketide production of natural products.

The free text of the sequence list is attached with an electronic file.

<110> Korean science and technology institute

<120> C-glycosyltransferase variants and uses thereof

<130> PP-B2717

<150> KR 2021-0011326

<151> 2021-01-27

<160> 122

<170> KoPatentIn 3.0

<210> 1

<211> 477

<212> PRT

<213> gentiana straminea (Gentiana triflora)

<400> 1

Met Gly Ser Leu Thr Asn Asn Asp Asn Leu His Ile Phe Leu Val Cys

1 5 10 15

Phe Ile Gly Gln Gly Val Val Asn Pro Met Leu Arg Leu Gly Lys Ala

20 25 30

Phe Ala Ser Lys Gly Leu Leu Val Thr Leu Ser Ala Pro Glu Ile Val

35 40 45

Gly Thr Glu Ile Arg Lys Ala Asn Asn Leu Asn Asp Asp Gln Pro Ile

50 55 60

Lys Val Gly Ser Gly Met Ile Arg Phe Glu Phe Phe Asp Asp Gly Trp

65 70 75 80

Glu Ser Val Asn Gly Ser Lys Pro Phe Asp Val Trp Val Tyr Ile Asn

85 90 95

His Leu Asp Gln Thr Gly Arg Gln Lys Leu Pro Ile Met Leu Lys Lys

100 105 110

His Glu Glu Thr Gly Thr Pro Val Ser Cys Leu Ile Leu Asn Pro Leu

115 120 125

Val Pro Trp Val Ala Asp Val Ala Asp Ser Leu Gln Ile Pro Cys Ala

130 135 140

Thr Leu Trp Val Gln Ser Cys Ala Ser Phe Ser Ala Tyr Tyr His Tyr

145 150 155 160

His His Gly Leu Val Pro Phe Pro Thr Glu Ser Glu Pro Glu Ile Asp

165 170 175

Val Gln Leu Pro Gly Met Pro Leu Leu Lys Tyr Asp Glu Val Pro Asp

180 185 190

Tyr Leu His Pro Arg Thr Pro Tyr Pro Phe Phe Gly Thr Asn Ile Leu

195 200 205

Gly Gln Phe Lys Asn Leu Ser Lys Asn Phe Cys Ile Leu Met Asp Thr

210 215 220

Phe Tyr Glu Leu Glu His Glu Ile Ile Asp Asn Met Cys Lys Leu Cys

225 230 235 240

Pro Ile Lys Pro Ile Gly Pro Leu Phe Lys Ile Pro Lys Asp Pro Ser

245 250 255

Ser Asn Gly Ile Thr Gly Asn Phe Met Lys Val Asp Asp Cys Lys Glu

260 265 270

Trp Leu Asp Ser Arg Pro Thr Ser Thr Val Val Tyr Val Ser Val Gly

275 280 285

Ser Val Val Tyr Leu Lys Gln Glu Gln Val Thr Glu Met Ala Tyr Gly

290 295 300

Ile Leu Asn Ser Glu Val Ser Phe Leu Trp Val Leu Arg Pro Pro Ser

305 310 315 320

Lys Arg Ile Gly Thr Glu Pro His Val Leu Pro Glu Glu Phe Trp Glu

325 330 335

Lys Ala Gly Asp Arg Gly Lys Val Val Gln Trp Ser Pro Gln Glu Gln

340 345 350

Val Leu Ala His Pro Ala Thr Val Gly Phe Leu Thr His Cys Gly Trp

355 360 365

Asn Ser Thr Gln Glu Ala Ile Ser Ser Gly Val Pro Val Ile Thr Phe

370 375 380

Pro Gln Phe Gly Asp Gln Val Thr Asn Ala Lys Phe Leu Val Glu Glu

385 390 395 400

Phe Lys Val Gly Val Arg Leu Gly Arg Gly Glu Leu Glu Asn Arg Ile

405 410 415

Ile Thr Arg Asp Glu Val Glu Arg Ala Leu Arg Glu Ile Thr Ser Gly

420 425 430

Pro Lys Ala Glu Glu Val Lys Glu Asn Ala Leu Lys Trp Lys Lys Lys

435 440 445

Ala Glu Glu Thr Val Ala Lys Gly Gly Tyr Ser Glu Arg Asn Leu Val

450 455 460

Gly Phe Ile Glu Glu Val Ala Arg Lys Thr Gly Thr Lys

465 470 475

<210> 2

<211> 536

<212> PRT

<213> Streptomyces wave (Streptomyces peucetius)

<400> 2

Met Ala Leu Thr Lys Pro Asp Val Asp Val Leu Val Val Gly Gly Gly

1 5 10 15

Leu Gly Gly Leu Ser Thr Ala Leu Phe Leu Ala Arg Arg Gly Ala Arg

20 25 30

Val Leu Leu Val Glu Arg His Ala Ser Thr Ser Val Leu Pro Lys Ala

35 40 45

Ala Gly Gln Asn Pro Arg Thr Met Glu Leu Phe Arg Phe Gly Gly Val

50 55 60

Ala Asp Glu Ile Leu Ala Thr Asp Asp Ile Arg Gly Ala Gln Gly Asp

65 70 75 80

Phe Thr Ile Lys Val Val Glu Arg Val Gly Gly Arg Val Leu His Ser

85 90 95

Phe Ala Glu Ser Phe Glu Glu Leu Val Gly Ala Thr Glu Gln Cys Thr

100 105 110

Pro Met Pro Trp Ala Leu Ala Pro Gln Asp Arg Val Glu Pro Val Leu

115 120 125

Val Ala His Ala Ala Lys His Gly Ala Glu Ile Arg Phe Ala Thr Glu

130 135 140

Leu Thr Ser Phe Gln Ala Gly Asp Asp Gly Val Thr Ala Arg Leu Arg

145 150 155 160

Asp Leu Gly Thr Gly Ala Glu Ser Thr Val Ser Ala Arg Tyr Leu Val

165 170 175

Ala Ala Asp Gly Pro Arg Ser Ala Ile Arg Glu Ser Leu Gly Ile Thr

180 185 190

Arg His Gly His Gly Thr Leu Ala His Phe Met Gly Val Ile Phe Glu

195 200 205

Ala Asp Leu Thr Ala Val Val Pro Pro Gly Ser Thr Gly Trp Tyr Tyr

210 215 220

Leu Gln His Pro Asp Phe Thr Gly Thr Phe Gly Pro Thr Asp Arg Pro

225 230 235 240

Asn Arg His Thr Phe Tyr Val Arg Tyr Asp Pro Glu Arg Gly Glu Arg

245 250 255

Pro Glu Asp Tyr Thr Pro Gln Arg Cys Thr Glu Leu Ile Arg Leu Ala

260 265 270

Val Asp Ala Pro Gly Leu Val Pro Asp Ile Leu Asp Ile Gln Ala Trp

275 280 285

Asp Met Ala Ala Tyr Ile Ala Asp Arg Trp Arg Glu Gly Pro Val Leu

290 295 300

Leu Val Gly Asp Ala Ala Lys Val Thr Pro Pro Thr Gly Gly Met Gly

305 310 315 320

Gly Asn Thr Ala Ile Gly Asp Gly Phe Asp Val Ala Trp Lys Leu Ala

325 330 335

Ala Val Leu Arg Gly Glu Ala Gly Glu Arg Leu Leu Asp Ser Tyr Gly

340 345 350

Ala Glu Arg Ser Leu Val Ser Arg Leu Val Val Asp Glu Ser Leu Ala

355 360 365

Ile Tyr Ala Gln Arg Met Ala Pro His Leu Leu Gly Ser Val Pro Glu

370 375 380

Glu Arg Gly Thr Ala Gln Val Val Leu Gly Phe Arg Tyr Arg Ser Thr

385 390 395 400

Ala Val Ala Ala Glu Asp Asp Asp Pro Glu Pro Thr Glu Asp Pro Arg

405 410 415

Arg Pro Ser Gly Arg Pro Gly Phe Arg Ala Pro His Val Trp Ile Glu

420 425 430

Gln Asp Gly Thr Arg Arg Ser Thr Val Glu Leu Phe Gly Asp Cys Trp

435 440 445

Val Leu Leu Ala Ala Pro Glu Gly Gly Ala Trp Gly Gln Ala Ala Ala

450 455 460

Arg Ala Ala Ala Asp Leu Gly Val Arg Leu Asp Val His Leu Val Gly

465 470 475 480

Arg Asp Val Ala Ala Pro Ser Gly Glu Leu Thr Arg Thr Tyr Gly Ile

485 490 495

Gly Arg Ala Gly Ala Ser Leu Val Arg Pro Asp Gly Val Val Ala Trp

500 505 510

Arg Thr Ala Val Ala Pro Gly Ala Glu Ala Gln Asp Gln Leu Ser Thr

515 520 525

Leu Leu Thr Arg Leu Leu Ala Arg

530 535

<210> 3

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 3

aaacactacg tggctagcca aaaaacccct caagacc 37

<210> 4

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 4

aaagcatgca ctagttaata cgactcacta taggg 35

<210> 5

<211> 47

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 5

ctttaagaag gagatataca tatgataata aataacagaa atgaatc 47

<210> 6

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 6

cttgtcgacg gagctcgaat tcattaattt ttatcgttta aacttgatg 49

<210> 7

<211> 61

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 7

gaattcgagc tccgtcgaca aataaggaga tataccatgg acgatatttc tttatcatct 60

g 61

<210> 8

<211> 53

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 8

gtggtgctcg agtgcggccg caagcttatt actcatcttt gttccttata atc 53

<210> 9

<211> 46

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 9

ctttaagaag gagatataca tatgaaacta atctctatgt tgttac 46

<210> 10

<211> 47

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 10

cttgtcgacg gagctcgaat tcattattga ttcctcaatg taaatag 47

<210> 11

<211> 45

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 11

ctttaagaag gagatataca tatgcgtcat gtagagcata cagtc 45

<210> 12

<211> 47

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 12

cttgtcgacg gagctcgaat tcttattaat cctcttcctc ttgctcg 47

<210> 13

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 13

ctttaagaag gagatataca tatggccttg acgaagccgg atg 43

<210> 14

<211> 41

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 14

caagcttgtc gacggagctc gagttgtcgg agcggctggc c 41

<210> 15

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 15

ctttaagaag gagatataca tatg 24

<210> 16

<211> 41

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 16

caaaacagcc aagcttgcat gcaagcttgt cgacggagct c 41

<210> 17

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 17

catgcaagct tggctgtttt g 21

<210> 18

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 18

catatgtata tctccttctt aaag 24

<210> 19

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 19

gttaagtata agaaggagat atacatatgc gtcgactgcc tgatttag 48

<210> 20

<211> 52

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 20

gatatccaat tgagatctgc cttatgatta ccctttctgt accattgtga tc 52

<210> 21

<211> 47

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 21

caatttcaca caggaaacag aattcatggc tgccattaat acgaaag 47

<210> 22

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 22

ccgggtaccg agctcgaatt cattacttct taatgcccat ctc 43

<210> 23

<211> 64

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 23

gtaatgaatt cgagctcggt acccaatttc acacaggaaa cagaatggca atccacaatc 60

gtgc 64

<210> 24

<211> 44

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 24

ctctagagga tccccgggta ccattacgcg tttttcagaa cttc 44

<210> 25

<211> 68

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 25

ctagagtcga cctgcaggca tgccaatttc acacaggaaa cagaatggct attgaacgta 60

ctttttcc 68

<210> 26

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 26

caaaacagcc aagcttgcat gcattaacgg gtgcgcgggc ac 42

<210> 27

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 27

gtcgtaccga aggggtccac cggctggtac 30

<210> 28

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 28

ggtggacccc ttcggtacga cggcggtgag 30

<210> 29

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 29

gacgtatggc aatacatcaa tcacttagac 30

<210> 30

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 30

gattgatgta ttgccatacg tcaaacggtt 30

<210> 31

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 31

gtgcccgact tcctgcatcc gcgcacaccc 30

<210> 32

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 32

cggatgcagg aagtcgggca cttcatcata 30

<210> 33

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 33

ctttaataag gagatatacc atggccttga cgaagccgga tg 42

<210> 34

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 34

ccgagctcga attcggatcc caagcttgtc gacggagctc 40

<210> 35

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 35

gtataagaag gagatataca tatggcagat gtcctgcagg ag 42

<210> 36

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 36

gatatccaat tgagatctgc caattaggga atggggacgc tac 43

<210> 37

<211> 261

<212> DNA

<213> unknown

<220>

<223> Streptomyces griseo

<400> 37

atgtccagtt tcagtattga tgatctgaag cgtatcttgc gcgaaggggc aggggcaacg 60

gctgagttag acggtgacat tttagacgcc tcctttgatg atttggggta tgattctttg 120

gctcttcttg aaacgggttc gcgcatcgga cgtgaatacg gtttggaatt tgaggataca 180

gctttcgccg acgtggaaac acctcgtgac ttggtcggcg tagttaatgc tcagttatcg 240

gccccggctc cgcgtgggta a 261

<210> 38

<211> 1321

<212> DNA

<213> unknown

<220>

<223> Streptomyces (Streptomyces sp.) R1128

<400> 38

atgcgtcatg tagagcatac agtcaccgtt gcggccccag cagacttggt ttgggaggta 60

cttgccgatg tcttaggcta tgctgacatc ttcccaccga cggaaaaagt tgaaattctt 120

gaggaggggc aaggatacca ggtagtgcgc cttcacgtcg atgttgcggg tgagattaat 180

acatggacca gtcgtcgcga tttagaccct gcgcgccgcg taattgctta ccgccaactt 240

gagacggctc cgatcgtggg ccacatgagc ggggaatggc gtgctttcac actggatgcc 300

gaacgtaccc aattagtcct gactcacgat ttcgtaaccc gtgcagccgg ggatgacggt 360

ttagtcgccg gaaaattgac cccagatgag gcgcgcgaaa tgttagaagc ggtggtagaa 420

cgtaactctg tcgccgactt aaacgcggtc cttggagaag ctgagcgtcg cgtccgcgca 480

gccggtggag ttggtaccgt aactgcgtaa taataatttt gtttaacttt aagaaggaga 540

tatatccatg tcagggcgca aaaccttttt agacttaagt tttgctaccc gcgacacacc 600

gtcggaggcg actccggtgg tggtagattt gctggaccac gtaactggag ccaccgtatt 660

aggattatca cctgaggatt tccccgatgg tatggctatt tccaatgaga ccgttacgtt 720

gacgacccac actggcacgc acatggatgc gccactgcac tatggtccct taagtggggg 780

agttccggca aagtcgattg accaagtgcc cttggaatgg tgctatggac ctggagttcg 840

tttggatgtt cgccacgtgc cggcaggaga tggtattact gtcgatcatt tgaacgccgc 900

gttggatgca gcagagcacg atttggcccc cggtgacatt gtgatgctgt ggaccggcgc 960

ggacgctctg tggggaaccc gcgaatactt gagcacgttt ccggggttaa ctgggaaggg 1020

gacacaattt ttggtcgagg cgggtgttaa agtcattggc attgatgcat ggggactgga 1080

tcgcccgatg gcagctatga tcgaagaata ccgtcgtacg ggcgataaag gagcattatg 1140

gccggctcac gtctatggac gcacacgcga atacctgcaa ttagagaagc ttaataattt 1200

gggcgcttta ccaggagcta cagggtatga catttcatgc tttccggttg cggttgcagg 1260

cactggagct gggtggactc gtgtggtcgc cgttttcgag caagaggaag aggattaata 1320

a 1321

<210> 39

<211> 1482

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 39

atgtcccatc gccgcaaccg tagccacaac aatcgtttac ccccgggacc taatccctgg 60

cccattatcg gaaacctgcc gcacatgggt acgaagcccc accgtacgtt gtcggctatg 120

gttacgacct atggcccaat tctgcacctg cgtctgggtt ttgtagacgt ggtcgtagcg 180

gcgtccaagt cggtcgccga gcagttcttg aagattcacg acgctaactt cgcttcacgt 240

ccccccaact caggagccaa acacatggcg tataattatc aagacttggt attcgccccc 300

tatggacatc gctggcgctt attgcgcaag atcagctcgg tgcatctgtt ctcggcaaaa 360

gcgttggagg actttaaaca cgtgcgtcag gaagaggtcg gaacactgac tcgcgaatta 420

gtacgtgtcg gcaccaaacc agtgaacctt gggcaactgg tgaatatgtg cgttgtcaac 480

gccttaggac gcgaaatgat cggacgccgc ttattcgggg ccgatgccga tcataaggcg 540

gatgagttcc gctcgatggt cacggagatg atggcgttag cgggggtctt taatatcggc 600

gactttgtac cgtcattaga ctggcttgat ctgcaagggg tcgctggaaa gatgaagcgt 660

ttacataagc gttttgatgc gttcttaagt tcgattttaa aagaacatga aatgaatggg 720

caagaccaaa agcataccga tatgttatcg accttaatca gccttaaggg tacagatctg 780

gatggggatg gcggctcctt aacggatact gaaattaagg cgcttttatt aaacatgttc 840

acagccggaa ccgacacatc agccagtaca gtagattggg caatcgctga attgatccgc 900

caccccgata tcatggtgaa ggctcaggaa gaattagata ttgttgtagg tcgcgaccgc 960

cctgtgaatg agtctgatat cgcccaactg ccgtacttac aggcggtaat taaggaaaat 1020

tttcgtctgc atccacctac tcccctgtct ttgccgcaca ttgcgagtga atcctgtgag 1080

attaacggtt accatattcc caaaggttca acattactta ccaacatctg ggctatcgcc 1140

cgtgatccgg accagtggag cgatccgtta gcttttaaac cagaacgttt tctgccagga 1200

ggagaaaaat ctggggttga tgtaaaaggt agtgatttcg agctgattcc gttcggtgca 1260

ggccgtcgca tttgtgcagg cctgtctctg ggtcttcgca cgatccagtt cttaacagcg 1320

actttagtac aagggtttga ttgggagtta gctgggggag tgacgcccga aaaactgaac 1380

atggaagaat cgtacgggtt aactttgcaa cgcgctgtcc ctctggtagt acacccgaaa 1440

cctcgtttgg ccccgaatgt gtacgggctg ggcagtggct aa 1482

<210> 40

<211> 2136

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 40

atgtcttctt cttcttcttc ttctacctct atgatcgacc tgatggctgc tatcatcaaa 60

ggtgaaccgg ttatcgtttc tgacccggct aacgcttctg cttacgaatc tgttgctgct 120

gaactgtctt ctatgttaat tgagaatcgt cagtttgcta tgatcgttac aacatccatc 180

gcggtcctta ttggttgtat cgttatgttg gtctggcgcc gctctggttc cggtaactct 240

aaacgtgtgg aaccgcttaa accgctggtg atcaaacctc gtgaggagga aatcgacgat 300

ggacgtaaaa aagtaacaat ctttttcgga acgcagactg gcactgcgga aggttttgcc 360

aaggcattag gtgaggaagc taaagctcgt tatgaaaaga cgcgcttcaa gattgttgat 420

ctggacgatt acgctgcaga cgatgatgaa tacgaggaaa aattaaaaaa agaggatgta 480

gctttcttct tcttagctac gtatggcgat ggtgaaccga cagataatgc cgctcgtttt 540

tataagtggt ttaccgaagg caatgatcgt ggtgagtggt tgaaaaactt aaaatatggg 600

gttttcgggc tgggcaatcg tcaatacgag cactttaaca aggtcgcgaa agtggtcgat 660

gacattctgg ttgagcaagg cgcacagcgt ctggtacaag tagggttagg ggatgatgac 720

cagtgtatcg aagatgattt cacagcttgg cgcgaagcat tgtggcccga gttggatacg 780

attctgcgcg aagagggcga tacggctgtt gccacaccct acacagccgc agtattagag 840

tatcgcgtaa gcatccatga tagcgaggat gccaaattca atgatattaa ccttgctaac 900

ggaaacgggt atacagtttt tgacgctcaa catccgtata aggccaacgt tgcggtcaaa 960

cgtgaattgc acaccccgga gtccgaccgt tcctgtatcc atctggaatt tgatattgcg 1020

ggatcaggtt taacatacga aactggagat cacgttggtg ttctgtgcga taacttatcc 1080

gagacggtgg atgaggcact gcgcctttta gacatgtccc ctgacacgta ttttagcttg 1140

catgctgaaa aagaggacgg tactccgatc agtagctcgc tgccaccgcc gtttccaccg 1200

tgcaatttac gcacggcttt aacacgttac gcgtgcctgt tgtcatctcc taagaaatcc 1260

gccttagtgg ctttggctgc acacgctagt gatcccactg aggccgagcg cttgaaacac 1320

ttagcaagcc ctgcaggtaa agacgagtac tccaagtggg tagtagagtc acagcgtagt 1380

ttattggagg tgatggccga gtttcctagt gcgaagccac cgttgggagt tttctttgcc 1440

ggggtggctc cgcgtttgca accacgtttt tatagcatca gtagttctcc aaaaatcgcc 1500

gagactcgca ttcacgttac atgtgccctg gtctacgaaa aaatgccgac tgggcgcatc 1560

cacaagggtg tatgctcgac ttggatgaag aacgccgtac cctacgaaaa gtctgaaaac 1620

tgcagctcgg cgccaatctt cgtacgccag tccaatttca agttgccgtc agattcaaag 1680

gtaccgatca ttatgatcgg tccaggaacg gggttagctc cgttccgtgg gttcttacag 1740

gaacgcttag cactggtcga gtcgggggta gaattgggcc cctccgtctt gtttttcggg 1800

tgtcgtaacc gtcgcatgga cttcatctat gaagaagagc tgcaacgttt cgtggaaagt 1860

ggggcgcttg ctgaactgtc ggtggcgttt tcccgcgaag gacccacgaa agaatatgtt 1920

caacacaaaa tgatggacaa agcgtcggat atctggaaca tgatttcaca gggcgcttat 1980

ttatatgtat gtggcgatgc gaaaggcatg gcgcgtgacg tccaccgttc tctgcacacc 2040

attgcgcaag agcaaggtag catggattca acgaaagcag aaggcttcgt gaagaattta 2100

caaacctctg ggcgctatct tcgtgatgtg tggtaa 2136

<210> 41

<211> 1098

<212> DNA

<213> unknown

<220>

<223> Streptomyces fradiae (Streptomyces fradiae) Tu2717

<400> 41

atgtttgccc tggctccgct ggccacagca gctcgtaatg caggtcatca ggtagtaatg 60

gcagcaaacc aggacatggg acctgtcgta accggggttg gccttccagc cgtagcaacc 120

actgatcttc cgatccgtca tttcatcact accgatcgtg aaggacgtcc cgaggccatt 180

ccttctgacc cggtcgcgca ggcccgtttc actggtcgct ggttcgcccg tatggctgcc 240

agttccttgc cccgtatgct tgatttttca cgtgcatggc gcccagactt aatcgtcggt 300

ggtactatga gctatgtcgc tccgctgtta gctcttcacc tgggagtccc gcacgcccgt 360

cagacttggg atgcggtaga cgctgatgga atccacccag gtgctgatgc tgagcttcgc 420

ccagagttaa gcgaattggg attggagcgc cttcccgcac ccgatttgtt catcgacatt 480

tgcccgccct cgttacgtcc tgccaacgca gcaccagctc gcatgatgcg ccacgtagcc 540

acgagccgcc aatgcccgtt agagccgtgg atgtatacac gtgacactcg ccagcgtgtt 600

ttagtgacgt cgggatcgcg tgttgcaaaa gaatcttacg atcgtaattt cgatttttta 660

cgtggattag cgaaggattt ggtgcgctgg gatgttgaat taattgtggc tgctcctgac 720

accgtggctg aggctcttcg tgccgaggtg ccacaagctc gcgtagggtg gaccccttta 780

gacgtcgtgg cccctacatg cgatttattg gtgcatcacg ccggcggagt ctctacgctg 840

actggtttat cggctggcga gccccaatta ttaatcccaa agggcagtgt attggaagct 900

cctgcgcgcc gcgtagcaga ttacggcgcg gcgattgcac tgttgcctgg tgaggactcg 960

acggaagcta tcgccgatag ttgtcaggag ttgcacgcca aggacactta tgcccgccgc 1020

gctcaagact taagccgcga aatttcaggg atgcctctgc cggccacagt ggtgactgca 1080

ctggaacagt tagcctaa 1098

<210> 42

<211> 1548

<212> DNA

<213> unknown

<220>

<223> cochineal (Dactylopius coccus)

<400> 42

atggagttcc gcttattgat tctggcactg tttagtgtct taatgagtac gtcaaatggt 60

gccgagattc ttgccctgtt tccaattcac ggaatttcca actacaacgt agcagaggca 120

cttcttaaaa cgcttgccaa ccgtggacat aacgtcactg tggtcacgtc attcccccag 180

aagaaacccg tgccaaactt gtatgaaatc gacgtatctg gggccaaagg tcttgctacc 240

aactcaattc actttgagcg tcttcagact atcattcaag acgtgaagtc caacttcaag 300

aatatggtgc gtctttcacg cacttactgc gagattatgt tctccgatcc tcgcgtactt 360

aacatccgcg acaaaaagtt tgatttggta atcaacgcgg tgttcggatc ggattgtgac 420

gctggctttg cgtggaaaag ccaagcaccc ctgatctcaa ttttaaatgc acgccacacc 480

ccgtgggcgc ttcaccgtat gggaaaccct tctaaccctg cgtacatgcc agtaatccac 540

tcacgcttcc cagtaaaaat gaatttcttc cagcgtatga tcaatacagg ctggcattta 600

tactttttgt atatgtactt ttattatggc aacggagaag acgcaaataa aatggcgcgt 660

aagtttttcg gtaacgacat gcccgacatt aacgaaatgg tctttaacac atcgttgctt 720

tttgttaata ctcactttag tgttgatatg ccctaccctc ttgttcctaa ctgcattgaa 780

attggtggga ttcacgtcaa agagccccaa ccgcttcccc tggagattca aaaattcatg 840

gatgaagcag agcatggtgt aatctttttc actttgggct cgatggtccg cactagcacc 900

tttcccaatc agactatcca agcgtttaaa gaggcgttcg cagaacttcc acagcgtgtt 960

ctttggaagt ttgaaaacga gaatgaagac atgccttcta acgttttaat ccgcaaatgg 1020

tttccacaga acgatatctt tggacataag aacattaagg cgtttatctc gcatggcggt 1080

aactcagggg cccttgaagc cgtgcatttc ggcgtgccca tcatcggcat tcctctgttc 1140

tatgatcagt atcgtaatat cttgagcttc gtgaaagaag gggttgcggt actgctggac 1200

gttaatgatt tgacaaagga taacattctg tcgtctgttc gtacagtagt gaacgataaa 1260

tcttacagcg aacgcatgaa agctctgtcc cagctgtttc gcgatcgtcc aatgagtccc 1320

ctggacacgg ctgtctattg gacggaatat gtaatccgtc accgcggagc acatcatctg 1380

aagactgctg gcgctttttt gcactggtat cagtatttgt tgctggatgt gattactttc 1440

cttttggtaa cattctgtgc cttttgcttc attgtcaagt acatctgtaa ggcgctgatt 1500

caccattatt ggtcgagttc caagagtgaa aaattaaaga aaaactaa 1548

<210> 43

<211> 1428

<212> DNA

<213> corn (Zea mays)

<400> 43

atggctgcta atggggggga tcatacctcc gcgcgcccac atgtggtgtt gcttccatcc 60

gctggcatgg gacatcttgt ccctttcgcc cgcttagctg tggctttatc tgagggacac 120

ggctgcaacg taagtgtagc tgcagttcaa ccaacggttt cctctgcgga gtcgcgtctg 180

ttagacgcac ttttcgtcgc cgccgcccca gccgtccgcc gtcttgattt ccgcctggcc 240

cccttcgatg aatccgagtt ccccggtgca gacccttttt tcttacgctt cgaggcgaca 300

cgtcgctcgg caccgcttct ggggccgtta ttagatgcgg cggaagcctc cgcacttgtg 360

actgatattg tccttgcttc ggtagcgttg ccagtggcgc gcgagcgtgg agttccctgc 420

tatgtgcttt ttacgtcatc ggccgcaatg ctgtcgttgt gtgcgtattt tccagcttat 480

ttagatgcac atgcagcggc cggctcggtc ggggtcggag tagggaacgt cgacattcca 540

ggggtatttc gcatccctaa gtcgagcgtc ccgcaagcac ttcacgatcc agatcattta 600

tttacccagc agtttgtcgc aaatggccgt tgtttagttg cctgcgacgg cattcttgtt 660

aacaccttcg acgccttcga gcccgatgca gtaaccgcac tgcgccaagg gtcgatcaca 720

gtctctggcg gttttccacc agttttcacc gtgggcccaa tgcttcccgt tcgcttccag 780

gcagaggaga cggctgacta catgcgttgg ttgtctgcac aaccaccccg cagtgtcgtc 840

tatgtctcgt ttggaagtcg caaggcgatt cctcgcgacc agttacgtga attggccgca 900

gggttagagg ctagtggcaa gcgttttctg tgggtagtaa agtcgaccat cgtcgaccgc 960

gatgataccg ccgatctggg cggcttgttg ggggacggct ttcttgagcg cgtccaaggt 1020

cgtgcatttg tgactatggg atgggtggaa caggaagaga ttttgcaaca tggctcggtt 1080

ggcttgttta tctcgcattg tgggtggaat agccttaccg aagccgccgc gttcggggta 1140

ccagttcttg cctggcctcg tttcggagat cagcgtgtga acgccgcctt agttgcgcgc 1200

tctggattgg gagcgtggga agaagggtgg acttgggatg gtgaggaggg acttactaca 1260

cgcaaggaag tggcgaaaaa gatcaagggc atgatggggt acgatgctgt agccgaaaag 1320

gcggccaaag ttggtgacgc agctgcggca gcaattgcaa aatgtggcac gagttatcaa 1380

tctttggaag agtttgtaca acgttgccgc gacgccgagc gtaagtaa 1428

<210> 44

<211> 1416

<212> DNA

<213> Rice (Oryza sativa)

<400> 44

atgccttcct caggagacgc tgccggtcgt cgccctcacg tcgtgctgat cccttcagcc 60

ggaatggggc acctggtccc gtttggtcgt ctggctgtcg cactttcctc tgggcacgga 120

tgtgatgtgt ctttagtaac agttcttcct actgtgagta cagcggagtc aaagcatctt 180

gatgcacttt ttgacgcatt ccccgcagtt cgccgtcttg acttcgagtt ggcgccattt 240

gacgcatcag agtttcccgg tgctgaccct ttcttccttc gttttgaggc gatgcgccgt 300

tcggctccat tgcttggccc tttgctgacg ggcgcgggcg ctagcgcact ggcgacggac 360

attgctttaa cgtctgtcgt aattccagta gcaaaagagc aagggcttcc gtgtcacatt 420

ttattcactg cgtcggccgc aatgttatca ttgtgtgcct acttcccaac ttatttggat 480

gccaacgctg gcggaggggg cggtgtgggc gacgtggata ttcctggagt gtatcgcatt 540

ccgaaggcat caattccaca agccttacat gatcccaacc acttgtttac tcgtcagttt 600

gtggcgaatg gtcgtagtct tacctcggcg gccggtattc tggtgaacac tttcgatgcg 660

ttagagccgg aggcagtagc tgcattgcag caaggaaagg tagcctccgg ctttccacca 720

gtattcgcgg tggggccgtt gctgcctgcc tctaaccagg ccaaggatcc gcaggcaaat 780

tacatggagt ggctggacgc ccagcccgcc cgcagcgtag tttatgtaag tttcgggagt 840

cgcaaggcga tttcacgtga acaacttcgc gagctggctg ctggcttaga ggggagcggc 900

caccgttttc tgtgggtcgt gaaatccacc gtcgtggatc gtgacgacgc ggccgagctg 960

ggagagctgt tggacgaggg ttttttagag cgtgtcgaga agcgtggatt ggtgacaaag 1020

gcatgggtcg accaggagga ggtactgaaa catgaaagcg tagccctgtt tgtctcacat 1080

tgcggctgga acagcgtgac tgaggcggcg gcgagcggtg tgcctgtcct ggccttaccc 1140

cgcttcgggg accaacgtgt taattcagga gtggtggcac gtgcaggatt aggagtatgg 1200

gcggatactt ggtcgtggga gggcgaagca ggcgtgattg gtgcggagga aatctcagag 1260

aaggtcaaag cagctatggc cgacgaagct ttacgtatga aagctgcatc ccttgcagag 1320

gcagccgcca aggcagtggc tggcggtggg agtagtcatc gctgtttagc ggaatttgcc 1380

cgtctgtgtc aaggtggaac ttgccgtact aattaa 1416

<210> 45

<211> 1443

<212> DNA

<213> Soybean (Glycine max)

<400> 45

atgagttcta gtgaaggagt ggtacatgta gcttttcttc caagtgcagg aatgggccac 60

ttgaaccctt tccttcgctt ggcggcgacc ttcattcgtt atggttgtaa agtaacgtta 120

atcaccccga agcctactgt atccctggca gaatcgaatt taatttcacg cttttgttcc 180

agctttccac atcaggttac gcaactggac ctgaatttag tcagcgttga tccaacgacc 240

gttgacacaa tcgacccatt cttcttacaa tttgaaacca tccgccgtag tctgcatctt 300

ttacctccca ttttaagtct tcttagcact cctttgtctg ccttcattta tgacattact 360

cttatcacgc ctttgctttc tgtaatcgag aagctgtcgt gccccagcta cttgtatttt 420

acatcttcag cacgtatgtt ctctttcttc gcacgtgtct ccgtgttgtc cgcatctaat 480

cccgggcaga ctccctcgtc atttatcggt gacgatggag ttaagatccc tgggttcaca 540

agccccatcc cacgcagcag tgttccgcct gcgattcttc aagcgtcctc aaatcttttt 600

cagcgcatta tgttagaaga cagcgcgaac gttaccaagc ttaataatgg ggtcttcatc 660

aatagctttg aagaactgga gggcgaagct ttagccgctt taaacggggg gaaagttctt 720

gaaggtctgc cgcccgtgta cggggtgggc ccccttatgg cgtgtgaata tgagaaaggc 780

gacgaggagg gtcaaaaggg ctgcatgtct tcgatcgtga agtggctgga tgaacagtcg 840

aagggaagcg tggtatacgt gtccttgggc aatcgtacgg aaacgcgccg tgagcagatt 900

aaggatatgg cccttggttt gatcgagtgt ggctatggat tcttgtgggt cgtcaaactg 960

aagcgcgtcg ataaagaaga tgaggaaggc ttagaagagg tgttaggtag cgagctgagt 1020

tccaaggtta aggagaaggg tgttgtagtt aaggaatttg ttgaccaagt cgaaattttg 1080

ggccacccaa gtgttggggg atttttgtcg cacgggggtt ggaacagcgt aactgaaact 1140

gtatggaagg gagtgccttg tctgtcatgg ccacagcata gtgatcagaa gatgtctgcg 1200

gaggtaatcc gtatgtccgg aatgggtatc tggcccgagg agtggggctg ggggacgcaa 1260

gatgttgtga agggagacga aatcgccaaa cgcattaagg aaatgatgtc gaacgaatcg 1320

ttgcgcgtaa aggcgggaga attgaaggaa gcggcgttaa aggcggcagg ggtagggggg 1380

agttgtgaag tgactattaa acgtcagatc gaagagtgga aacgcaatgc ccaggctaat 1440

taa 1443

<210> 46

<211> 1434

<212> DNA

<213> gentiana straminea (Gentiana triflora)

<400> 46

atggggagtt tgactaacaa cgataatctt catatttttc ttgtgtgctt catcggccag 60

ggcgtggtca atcccatgtt acgtttgggg aaggcgttcg cctccaaagg gttacttgtc 120

actttaagcg caccggaaat cgttggaact gagatccgta aggcgaataa ccttaatgat 180

gaccaaccaa tcaaggtggg ttccgggatg attcgtttcg aatttttcga cgatggatgg 240

gaatccgtaa acggtagcaa accgtttgac gtatgggtct acatcaatca cttagaccag 300

acaggccgtc aaaaacttcc gattatgtta aagaaacatg aggagacagg gactcctgta 360

tcttgcttga tcctgaatcc cttagtccct tgggtcgcgg acgtagccga ttcacttcag 420

atcccctgcg ctaccttgtg ggtccaatct tgtgcaagtt tttcagcata ttaccactac 480

caccacgggt tagtgccttt cccaaccgaa tcagagcccg agatcgacgt acaacttcct 540

gggatgccac ttttgaaata tgatgaagtg cccgactacc tgcatccgcg cacaccctac 600

cccttttttg gcacgaacat tttaggtcaa ttcaagaatt tatccaagaa cttctgtatc 660

ctgatggata ccttctacga gttggaacac gagatcatcg ataatatgtg taaattgtgt 720

ccgattaagc caattggccc gttgtttaag attccgaaag acccaagctc caacggaatc 780

acgggtaatt tcatgaaagt ggatgactgc aaggagtggc tggacagccg tccaacatca 840

actgtggttt acgttagtgt cgggtctgtt gtatatttga agcaggagca ggttacagaa 900

atggcatacg gcattttaaa ttcggaagtt tcgtttttgt gggtgctgcg cccgccgagc 960

aaacgcatcg gtacggaacc gcatgtactg cccgaggagt tctgggagaa ggccggagat 1020

cgtggcaagg tggtgcaatg gtcaccccag gagcaggtgc ttgctcaccc cgccactgtc 1080

ggttttttaa cacactgtgg atggaatagc actcaagagg cgatttcgag cggagtgccc 1140

gtcatcactt tcccacaatt tggggaccaa gtgaccaatg ctaagttcct tgtggaggaa 1200

tttaaggtcg gggtccgttt aggccgcgga gagttagaaa atcgcatcat cacacgcgac 1260

gaagtagaac gcgctttacg cgagattact tcaggcccca aggctgaaga ggtaaaagag 1320

aacgccttaa aatggaagaa gaaggcagaa gagacagtag ctaaaggcgg ctactccgaa 1380

cgtaatcttg taggcttcat tgaagaggtg gctcgtaaga ctggtacaaa gtaa 1434

<210> 47

<211> 2112

<212> DNA

<213> Aloe (Aloe vera)

<400> 47

atggaggaaa tttccagtaa agtggagttc ttatccctta agcccagcat gtcaggaagt 60

ccccgttaca gccccacatt tcgtaaaatc ggaagcggtc gcaattcccg ccgcgactcc 120

cgtgctcatg cagggaattt cccctggatt cgcaacaatc gtgttttttt ttggctgctt 180

ttaatcacca tctgggcgta catcggcttt cacgtccaat ctcaatgggc acatggcgac 240

cataaagctg agttcgttgg atacaagtcg gaggtaggga agatgggtga ggacgtcaag 300

tcggtaaata gtacgactac gttctccatt gtacacaagg gcaatttaac tgttgaagga 360

aagaaagacc ccgattccaa ttttggtatt tcactgttga aaaagggtaa acaggttctt 420

tcccgtttaa attcacgcaa aaagggccat cgttcgcgca aggtgtcgga aaaactggaa 480

gaagaaacgg acgacaatgg gacgggagaa atggatgagg tccttatcca gcgcaaaaac 540

acatcttatg gcttaattgt agggcctttt gccaaactgg aagagagtgt gcttgagtgg 600

agcccaggca agcgccgtgg tgtctgctat cgtaagggag aatttgcccg cgcggtgtct 660

tctcagcgct ttatgttgat cttccacgaa ttgtcaatga ctggcgcccc attgtccatg 720

ttggaattgg ccacggagat cctgtcttgc ggtgggtctg tgagcgctat tgtattatct 780

aagaagggag ggttaatgcc ggaactgaag aagcgtggta ttaaggtttt gcaagaccgt 840

gacaaggtga gtttcaaggt cgccacgaaa gtagacctga ttattgcggg atctgctgta 900

tgtagtagct ggatcgagcc atatctggag tatttccccg ctgggtccgg acatattgtc 960

tggtggatca tggaaaaccg tcgcgaatac ttcgaccgta gcaagcatct tttaaaccgt 1020

gtgaaaattt tggcatttct tagcgatagc cagtcaaagc agtggctttc ttggtgtgag 1080

gaagagaaga ttaaattcct gatccagcca atgttagtgc cgttgtcagt taacgatgag 1140

ctggccttcg ttgccggtat tccttgtagc ttgaatactc cagcattatc agtggagaaa 1200

atgatcgaaa agcgtgattt attacgtcac gcagtccgta aggaaatggg gttgggggac 1260

aatgacatgc ttgtgatgag tttaagtagc atcaacccag ccaagggtca gcgctttctg 1320

cttgaggcag ccttactggt agctgaacac aatgtatcat tgaaagatgc taacagttac 1380

agtcttatgg aggaggagaa gttatccggg aacgcacctc aaaatcaaac catcatgatc 1440

ggtcaactga atcctggcca cgtacttcag atcgccaatg acactaataa gcccgtcaat 1500

gcgttacaga agattggcgc cacacgtgtc tcgtcgaagc gtcgcggcaa gctgcatacg 1560

aatacagtca cgggcgtgct tcagaaaagc cgcaaacttt tgtccgaggc agcaggtatg 1620

aaggaggaaa ccctgaaagt ccttgtaggt tccgtcggat cgaaatcgaa taaggttctg 1680

tatgtaaagg caatcatgga atacatcagc caacattcta atttgtctaa ggtcgttctt 1740

tggaccccag ccaccacgtc tatcgcagca ctgtacgccg ccgcggacgt gtacgtcatt 1800

aacgctcagg gacatggaga gacattcggt cgcgtgacga tcgaggcgat ggcctttggc 1860

ctgccagtgc tggggactga cgccggaggg actaaagaaa tcatcgacca ccgtgttacg 1920

ggacttctgc atcctgtggg tcccgagggc actgtactgt tagcgcaaca cattcaatat 1980

cttttaaaaa atcccagcgt gcgcaagaaa atgggtatca atggtcgccg caaagtacaa 2040

gataaatact taaaacacca gacttacgag tcccttggca aagtcatgtt caaatcgatg 2100

cgtccccgtt aa 2112

<210> 48

<211> 1611

<212> DNA

<213> unknown

<220>

<223> Streptomyces wave (Streptomyces peucetius) ATCC 29050

<400> 48

gtggccttga cgaagccgga tgtcgatgtc ctcgtggtgg gcggcggtct cggggggctg 60

tccaccgccc tgttcctcgc ccgccggggg gcgcgggtcc tgctggtgga gcggcatgcc 120

agcacctcgg tcctgcccaa ggcggcaggc cagaacccgc gcaccatgga actgttccgc 180

ttcggcggcg tggccgacga gatcctggcc acggacgaca tccgcggcgc ccagggcgac 240

ttcaccatca aggtcgtgga gcgcgtgggc ggtcgcgtcc tgcacagctt cgcggagagc 300

ttcgaggaac tggtcggtgc gacggaacag tgcacgccca tgccctgggc gctcgctccc 360

caggaccggg tggagcccgt cctggtggcc cacgccgcca agcacggcgc ggagatccgg 420

ttcgccaccg aactgacctc cttccaggcg ggcgacgacg gtgtcacggc ccgcctgcgc 480

gacctgggca cgggagcgga gagcaccgtg agcgcccgct acctggtcgc cgccgacgga 540

ccccgcagcg cgatccggga gagcctgggc atcacccggc acggtcacgg caccctggcc 600

cacttcatgg gcgtcatctt cgaggccgac ctcaccgccg tcgtaccgcc cgggtccacc 660

ggctggtact acctgcagca cccggacttc accggcacgt tcggccccac cgaccggccc 720

aaccggcaca ccttctacgt ccgctacgac cccgaacgcg gcgagaggcc ggaggactac 780

acaccgcagc gctgcaccga gctgatccgg ctggctgtcg acgcgcccgg gctcgtcccg 840

gacatcctcg acatccaggc ctgggacatg gcggcgtaca tcgccgaccg gtggcgcgaa 900

gggccggtgc tgctggtcgg cgatgccgcc aaggtcaccc cgcccaccgg gggcatgggc 960

ggcaacaccg ccatcggcga cgggttcgac gtggcctgga agctggccgc cgtgctgcgc 1020

ggcgaggcgg gcgagcggct cctcgacagc tacggggcgg agcggtcgct cgtgtcccgc 1080

ctcgtcgtcg acgagtcact cgccatctac gcccagcgca tggctcccca cctgctcggc 1140

agcgttcccg aggaacgcgg tacggcgcag gtcgtcctgg gcttccgcta ccgctccacc 1200

gccgtcgccg ccgaggacga cgaccccgag ccgaccgagg atccgcgacg cccgtccggg 1260

cgccccggct tccgcgcacc ccacgtctgg atcgaacagg acggcacacg gcgttccacc 1320

gtcgagttgt tcggcgactg ctgggtgctc ctggccgcac cggagggcgg cgcctggggc 1380

caggcggccg cccgcgccgc cgcggatctg ggcgtccgcc tcgacgtcca tctcgtcggc 1440

cgcgatgtcg ccgccccctc cggcgaactg acgcggacct acgggatcgg ccgggcgggg 1500

gccagcttgg tgcgcccgga cggcgtggtc gcctggcgta cggcagtagc gccgggagcg 1560

gaggcccagg accagctgag caccctgctc acccggctgc tggcccgctg a 1611

<210> 49

<211> 2678

<212> DNA

<213> Photobacterium luminescence (Photorhabdus luminescens)

<400> 49

atgataataa ataacagaaa tgaatctcaa ccacgtagag ttgtggtgac agggctaggt 60

gttgtcgcac cgacaggtgt tggcgttaat gaattttgga acaatattca taacggcaaa 120

tcgggggtaa gtgaatatga gtggggaaga aaaaaatttg gttttaaaag cggagcaata 180

ggaaaagttc acggtaacga tagcgatagc aaagagtttg tgctgaaaag tgagcgtaaa 240

tatcttgagt ttgcgctaga agcctctgag atggcaatgc aagatgcaaa tttaaaacct 300

tcagacattg atggccggcg ttttggcgtt gcgatagcaa cagcgattgc cgatgctgcg 360

ggaatggaag agtgtttgct caggatcacc aaagggggca aagagaatat tcatcctgat 420

ttaattaaat cagaggatta tgacagcttt gatttcagct ctgccgccac ctctgttgcg 480

aaaaaatatg gcgcatcgat gtccgtcagt aacatatcaa ctgggtgtgc ggcaggactt 540

gatgcattag gcattgcgat ggagcatatc cgttatggca gagcggatgt gatgctggct 600

ggcgccagtg aagcgccgct ttgtccactt tctatcggct cttttgaagc tttaggggcg 660

ctatcatcaa gagaattgga aaatcagcaa gcagcgactt gtcctttttc ccttgagcgg 720

gatggatttg tgattgctga agggtgtgga atattaattt tagagtctta tgaacatgct 780

aagcagcgtg gagcacatat ctatgctgaa ttagcagggt atgcgtccgt gaataacgct 840

tatcatatga ccgacttgcc tgcggatgga atggcaatgg cgcggtgcat tgatatggcg 900

ttgaaggatg cccagatatc gccatcagcg gtcaattata ttagtgctca tggcagttct 960

acggctcaaa atgatattaa cgaatcaaat gcgattaaat ttgttttggg agaaaatgca 1020

tttgatattc caattaactc attaaagtca atgacaggtc atgctttagc tgccgctaat 1080

gcgatcgagt ctgtagcgtt atgtctggaa atagaaaagc aatatattca tccaacaatt 1140

aattatcaaa cgccggaccc tgattgcgat ttagattata ttcctaatca aggttgcgca 1200

tatccaatta agaccgcatt aaaattatcg agtggttttt ctggtattca cagtgttatt 1260

gttatgaggg cagtagacaa tgcgtaaaag agttgttgtt accggcgttg gcgcagtaca 1320

tcctgatggc aatgatgtca ccgctataaa aacaaaagtg attcagaaat tattgggtca 1380

ggaatcgata aataatacca acaaaagttc tgtaataagg acattgaatg atttcgatgg 1440

ggcaaaatat atcaataacc gcttaagacg taaaattgat gaattttcag tttatggtat 1500

cgtcgccgtt gaaatggcat taaaagcgag cagattggat gtagataagc ttgatcctaa 1560

tcgtgttggc atatatgttg gaaactgttt tggcggatgg cagcatattg aggatgaagt 1620

taaagcgctc catgttgaag gcatatcggg gatgggacct tatgttgcta cggcatggtt 1680

ccctgctgcg cttcaagggc aattgtcact gctttatggt tttagtgcgc aatctaagac 1740

attttccacc tccgatgtag cagggatgca agcaataggc tatgcggctg aagcgatttc 1800

taatggtgtt gccgaagtga tgttatgtgg cgcgtcagaa catctttcca gcccgttagt 1860

taaaagttta ctggagaaag agtcaagcca gaaacactct gaggtttttg gcgaaagaca 1920

gccaggggac ttttccgaag gcgctgcatt tctagtgctg gaagagaggc aacatgcttt 1980

agaacgcggc gcttcgatat tgtgtgaatt aacgggtttt gttgattatt tttcaccgga 2040

taaaaataca agaaataaca ccttagaata tactgctgaa ctattcaacc ataatgagaa 2100

tgctgtattt attatggatg gaatatatga tgatgaaaaa gaaataacga gtaaggcttt 2160

ctccaataaa gagataaaaa catcatttat aaatctgagg ccttacttga ataatcaatt 2220

ttcagtcagc ggcgtaattg attcagtcct ggcatcatca tttttatcag aaaataacgg 2280

ggatggagaa caacaatcta ataaaataaa tgaactttca aatactaacc aaataataat 2340

tcagcgcttt agtaaccagg gtcatgtatg tgcgttgagt ttttcagcaa tttaatctct 2400

aaaatattta attacgcgag gaaaaatata tgaataataa cccagaagta aaaataaaaa 2460

cgattttgtc tctttttctt aacgttaata ttgatgattt caatatggat gcaaaccttg 2520

ctgatgccta tgatatggat tctacggaat tggctgactt ggcaaaagag attacgaaag 2580

agttcggtat ttccgtgacg aaaagtcagt tcagtcattg ggaaacagga agagccgttc 2640

ttgatttcgt ctcatcaagt ttaaacgata aaaattaa 2678

<210> 50

<211> 714

<212> DNA

<213> Photobacterium luminescence (Photorhabdus luminescens)

<400> 50

atggacgata tttctttatc atctgatttt tttgatcttt ggattatcaa aatcgacgat 60

attgatttag cttctattga acagttaatt cactgttctg atatagttcg ccataaccaa 120

atttgtttag cggatagaag aaagagattt atatttagac gggctgcatt acgttatgtt 180

ttgagtcaat atttatctga ttatgaaatc ataacgaatg ataacggaaa accttatata 240

tccacggagc aagacttcaa atattatttt tcactgagtg cttcaggaaa ctattgtgcc 300

attggtttta gctcaaggga aataggtgtt gatattgaag tcactccttc taaggtaaaa 360

ttttcagaaa ttattgaacg ttttattaag gataaagatt tggaatatat gaaaggtata 420

atgttaaaac aactatcagg agttagtctc ggatttaata actattatca tttaatgtca 480

ttatattatt gggttagact tgaagcatat attaaattat ttgcttcgac tttacatgag 540

aaattattgg ttaataactc tgattctgtt aaagatatga aagaattgga ggcaagcaca 600

ttattgattc atagtcagca atttgtttgt gccttatctc aaaagaaagt catttctaca 660

ccaaatatca aggaaataaa ttattccgaa attataagga acaaagatga gtaa 714

<210> 51

<211> 1548

<212> DNA

<213> Photobacterium luminescence (Photorhabdus luminescens)

<400> 51

atgaaactaa tctctatgtt gttacattca gagcatgata acttacatca tgattgtatt 60

gtcactaagg attatcatta tacaagaaaa gaggtgatat cttctgtttc ccatttaatt 120

gatgatttat tgagtcgagg agtgcaaaaa ggtaataaag tcattgttat atttgaacat 180

gatgaattag gtgttttctt tttggctgcc gccagtgcta tggggttgca tttattaatg 240

ccctataatt tatcatcagc gacaatcgat gaatggatta attttaccaa tgaagtgcaa 300

tacgattttg ttgtttatct caaaaaagat aaacattttg ttggaaaatt aaaagaaaac 360

aacattaatg ttattgatat ttcagatcat aagatcagag ttagtgatga tattgcggaa 420

atcccaatga taacttattc tccgcaacct attgctaact ttattgtcct gttcaccagt 480

gggagtacag gcaaaccaaa agccattagt atttcagaat cgttagtatg tcgtcgaatt 540

tattcggtga ccgagaaatt aaaatttacg caagatgcca aaatattcat gtcaggtttg 600

ttgaataata caactggagt gattttttct ttcggctcat tattgcatca atcaacactt 660

tttatacccg aagatagaaa tgtagagaga tggcctgatt atctttctcg caataaaatc 720

actcatatta tgttacgccc agaatcaatg aaattattcg ttaaatcgac agcagaactt 780

aatattgatc tctcttgttt acgggtggtt gcttatggcg ctgcggcgat gcctcctagc 840

gtacttgaga aagggcgaca attaattggc tgtgaatggg tgcagggata tgggttaagt 900

gaaacttatg gtcctttctg ttgggtggat gagcaagatc atcgtgataa aagatatctc 960

aattcaattt attgtgttgg taagattgat aatacattgg aagtggcagt taaacctatt 1020

ataggttcat cggataatat cggagaaatt atactaaggg gtaaaagtat tatggaagga 1080

tattatgatg tcctttctgg agaaataacg cctcctgatg aatggtttgc cactggtgat 1140

cttggttata tagatgaaga gggttattta gttttgaaag gacgtaagca aaatacgttt 1200

atgagtgcta acggacacag aatttatcct gaagaaattg aatctatttt atcccgaata 1260

cccaatgtga atgtcgctac ggttgttggt ttttctttcc atgaaaatgg tgttgctatt 1320

gatcagccgg ttgcttgcat gagtggagag atatctaaga agtcattacc tgaaattgaa 1380

gatattattt catcattttt aatgagtaaa ctcagtcgag aaaaatggcc ggattggttc 1440

tatgttactg atgaatgctt tccgaaaagc cataatgata agatattgaa atcagagtta 1500

attaaatcaa tcgatcctaa gaaattattt acattgagga atcaataa 1548

<210> 52

<211> 951

<212> DNA

<213> Streptomyces coelicolor (Streptomyces coelicolor)

<400> 52

atgctcgtac tcgtcgctcc cggccagggc gcccagacgc ccggcttcct gactgactgg 60

ctcgccctcc ccggtgccgc tgaccgcgtc gccgcgtggt cggacgccat cggactcgat 120

ctcgcccact tcggcaccaa ggccgacgcg gacgagatcc gagacacgtc cgtggcccag 180

ccgctgctgg tcgccgccgg aatcctgtcc gccgcggcac tcggtacgca gacatctgtc 240

gctgacgcga cgggccccgg gttcaccccc ggcgcggtcg ccggacacag cgtcggcgag 300

atcaccgccg ccgtcttcgc gggcgtcctc gacgacaccg ccgcgctgtc cctcgtacgc 360

cgtcgcggcc tggccatggc cgaggccgcg gcggtcaccg agaccggcat gtcggcgctg 420

ctcgggggcg accccgaggt gagcgtcgcg cacctggagc ggctcggcct gaccccggcg 480

aacgtgaacg gcgccggtca gatcgtggcg gcgggcacca tggagcagct ggccgcgctg 540

aacgaggaca agcccgaggg tgtgcgcaag gtcgtcccgc tgaaggtggc cggcgcgttc 600

cacacccgcc acatggcccc cgccgtggac aagctcgccg aggccgccaa ggcgctgacg 660

ccggccgacc cgaaggtgac gtacgtctcc aacaaggacg ggcgggccgt cgcctccggc 720

accgaggtgc tggaccggct ggtcggccag gtcgccaacc cggtgcgctg ggacctgtgc 780

atggagacgt tcaaggagct gggcgtcacc gcgatcatcg aggtgtgtcc gggcggcacg 840

ctgaccgggc tggccaagcg ggcgctgccc ggagtgaaga cgctggccct gaagaccccc 900

gacgacctcg acgcggcccg tgagctcgtc gccgagcaca cccaggccta a 951

<210> 53

<211> 1146

<212> DNA

<213> Streptomyces coelicolor (Streptomyces coelicolor)

<400> 53

atgagcgagg acacgatgac ccaggagcgg ccgtccctga cggcacacgc ccgccggatc 60

gccgaactcg ccgggaagcg ggcggccgac gccgaacagc agcgccggct gagccccgac 120

gtcgtcgacg cggtccttcg agccggtttc gccgcccact tcgtaccggt ggcgcacggc 180

ggccgggccg cgacgttcgg ggagctggtg gagcccgtcg cggtgctcgg cgaggcctgt 240

gcctcgaccg cctggtacgc ctcgctcacg gcgagcctcg gccggatggc cgcctacctg 300

ccggacgagg gccaggccga gctgtggtcc gacggccccg acgccctgat cgtcggtgcc 360

ctgatgccgc tgggccgggc cgagaagacc ccgggcggct ggcacgtgtc gggcacctgg 420

ccgttcgtca gcgtcgtgga tcactccgac tgggcgctga tctgcgccaa ggtcggcgag 480

gagccgtggt tcttcgcggt gccgcgacag gagtacggga tcgtcgacag ctggtacccg 540

atgggtatgc gcggaacggg cagcaacacg ctcgtcctcg acggggtgtt cgtgccggat 600

gcgcgggcct gcacccgtgc ggccatcgcg gcaggtctcg gtccggatgc cgaggcgatc 660

tgtcacaccg tgcccatgag ggcggtcaac gggctggcct tcgcactgcc gatgctcggc 720

gcggcccgcg gggccgcggc cgtgtggacc tcgtggaccg ccggaagact ggccgggccg 780

accgggcaga acgccgtctc gtcccaggac cgcgtggtgt acgagcacac gctggcccgg 840

gccacgggtg agatcgacgc ggcccagctg ctgttggagc gggtcgcggc ggtcgccgac 900

gccggctcgg cgaccggcgt actggtcggc cgcggggcgc gggactgcgc cctggcggcg 960

gagctgctga ccgccgcgac cgaccggctg ttcgcctcgg cgggcacccg ggcacaggcc 1020

caggacagcc cgatgcagcg cctgtggcgc gatgtgcacg cggcgggcag ccatatcggg 1080

ctgcagttcg ggcccggggc ggcgctgtac gccggagagc tgttgaggag gagcaacgat 1140

ggctga 1146

<210> 54

<211> 534

<212> DNA

<213> Streptomyces coelicolor (Streptomyces coelicolor)

<400> 54

atggcagccg accagggaat gctccgggac gccatggccc gggtgccggc cggggtggcg 60

ctcgtcaccg cccatgaccg cgggggagtc ccgcacggtt tcaccgccag ttcgttcgtg 120

tccgtctcga tggagccgcc actggcactg gtctgcctgg ctcgtacggc caactccttc 180

ccggtgttcg acagttgcgg cgagttcgcg gtgagcgtgc tgcgcgagga ccacacggac 240

ctggccatgc gcttcgcgcg caagtccgcg gacaagttcg cgggcgggga gttcgtccgt 300

accgcgcggg gagcgaccgt gctcgacgga gcggtcgcgg tcgtcgagtg cacggtccac 360

gagcgctacc cggcgggcga ccacatcatc ctgctcggcg aggtccagtc cgtgcacgtc 420

gaggagaagg gcgtaccggc ggtctacgtg gaccgccggt tcgccgccct gtgctcggcg 480

gcgggtgcct gcccgtccgc caccgggcgg ggcgtgcccg cgcatgccgg ctaa 534

<210> 55

<211> 1194

<212> DNA

<213> Pseudomonas fluorescens (Pseudomonas fluorescens)

<400> 55

atgaaaacgc taaaaaccca agtcgccatt attggcgccg gtccctccgg attgctgctc 60

ggccagttac tgcacaacgc gggtatccag accctgattc tagagcgcca gagcgccgac 120

tacgtgcaag gccgcatccg tgccggggtg ctggagcaag gcatggtcga cctgctgcgc 180

gaagcgggcg tcagccgacg catggacgcc gagggccttg tgcatgacgg tttcgaattg 240

gcactcaatg gcgaactcac ccacatcgac ctcaaggcgc tcaccggcgg ccagtcggtg 300

atgatctacg gccagaccga agtcacccgt gacttgatgg ccgcccgcga agcggcgggt 360

ggcatcactc tatacgaaac gcagaacgtg cagcctcatg gtcacaaaac tgatcgaccc 420

tggctgacct tcgagcacca gggtgaagct tttcgcctgg agtgcgacta catcgcgggc 480

tgtgatggtt ttcacggtgt ggcgcggcag tcgattccgg cgcagtcgtt gaaggtcttc 540

gagcgcgtct atcccttcgg ttggctgggc gtcctcgccg acacaccgcc ggtgcatgac 600

gaactggtgt acgccaaaca tgcgcgtggc tttgccctgt gcagcatgcg ctcgccgacc 660

cgcagccgct attacctgca agtgccggtt gaagaagcgc tggatgaatg gtcggatcag 720

cgcttctggg atgagctgaa aacccgtttg cccagtgcac tggcggccca actggtcacc 780

gggccatcca tcgagaagag catcgcgccg ctgcgcagct ttgtggtcga gccgatgcaa 840

tacgggcgcc tgttcctgct gggggacgcc gcgcatatcg tgccgcccac cggggccaag 900

ggcttgaacc tggcggccag cgacgtgagt acgctgtttc ggatcttgct caaggtctat 960

cgcgaggggc gggtggacct gctggaacag tactcagcga tctgcttgcg ccgcgtatgg 1020

aaagccgaac ggttttcctg gtggatgact tcgatgttgc accagtttcc ggaggccgac 1080

gggttcagcc agcgcattgc cgagagcgag cttgcgtatt tcatcagctc cgaggcgggc 1140

cgcaaaacca tcgcagaaaa ttacgtcggg cttccttacg aagctatcga ataa 1194

<210> 56

<211> 1611

<212> DNA

<213> artificial sequence

<220>

<223> dnrF P217K from Streptomyces wave (Streptomyces peucetius)

<400> 56

gtggccttga cgaagccgga tgtcgatgtc ctcgtggtgg gcggcggtct cggggggctg 60

tccaccgccc tgttcctcgc ccgccggggg gcgcgggtcc tgctggtgga gcggcatgcc 120

agcacctcgg tcctgcccaa ggcggcaggc cagaacccgc gcaccatgga actgttccgc 180

ttcggcggcg tggccgacga gatcctggcc acggacgaca tccgcggcgc ccagggcgac 240

ttcaccatca aggtcgtgga gcgcgtgggc ggtcgcgtcc tgcacagctt cgcggagagc 300

ttcgaggaac tggtcggtgc gacggaacag tgcacgccca tgccctgggc gctcgctccc 360

caggaccggg tggagcccgt cctggtggcc cacgccgcca agcacggcgc ggagatccgg 420

ttcgccaccg aactgacctc cttccaggcg ggcgacgacg gtgtcacggc ccgcctgcgc 480

gacctgggca cgggagcgga gagcaccgtg agcgcccgct acctggtcgc cgccgacgga 540

ccccgcagcg cgatccggga gagcctgggc atcacccggc acggtcacgg caccctggcc 600

cacttcatgg gcgtcatctt cgaggccgac ctcaccgccg tcgtaccgaa ggggtccacc 660

ggctggtact acctgcagca cccggacttc accggcacgt tcggccccac cgaccggccc 720

aaccggcaca ccttctacgt ccgctacgac cccgaacgcg gcgagaggcc ggaggactac 780

acaccgcagc gctgcaccga gctgatccgg ctggctgtcg acgcgcccgg gctcgtcccg 840

gacatcctcg acatccaggc ctgggacatg gcggcgtaca tcgccgaccg gtggcgcgaa 900

gggccggtgc tgctggtcgg cgatgccgcc aaggtcaccc cgcccaccgg gggcatgggc 960

ggcaacaccg ccatcggcga cgggttcgac gtggcctgga agctggccgc cgtgctgcgc 1020

ggcgaggcgg gcgagcggct cctcgacagc tacggggcgg agcggtcgct cgtgtcccgc 1080

ctcgtcgtcg acgagtcact cgccatctac gcccagcgca tggctcccca cctgctcggc 1140

agcgttcccg aggaacgcgg tacggcgcag gtcgtcctgg gcttccgcta ccgctccacc 1200

gccgtcgccg ccgaggacga cgaccccgag ccgaccgagg atccgcgacg cccgtccggg 1260

cgccccggct tccgcgcacc ccacgtctgg atcgaacagg acggcacacg gcgttccacc 1320

gtcgagttgt tcggcgactg ctgggtgctc ctggccgcac cggagggcgg cgcctggggc 1380

caggcggccg cccgcgccgc cgcggatctg ggcgtccgcc tcgacgtcca tctcgtcggc 1440

cgcgatgtcg ccgccccctc cggcgaactg acgcggacct acgggatcgg ccgggcgggg 1500

gccagcttgg tgcgcccgga cggcgtggtc gcctggcgta cggcagtagc gccgggagcg 1560

gaggcccagg accagctgag caccctgctc acccggctgc tggcccgctg a 1611

<210> 57

<211> 1434

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F from gentiana rigescens (Gentiana triflora)

<400> 57

atggggagtt tgactaacaa cgataatctt catatttttc ttgtgtgctt catcggccag 60

ggcgtggtca atcccatgtt acgtttgggg aaggcgttcg cctccaaagg gttacttgtc 120

actttaagcg caccggaaat cgttggaact gagatccgta aggcgaataa ccttaatgat 180

gaccaaccaa tcaaggtggg ttccgggatg attcgtttcg aatttttcga cgatggatgg 240

gaatccgtaa acggtagcaa accgtttgac gtatggcaat acatcaatca cttagaccag 300

acaggccgtc aaaaacttcc gattatgtta aagaaacatg aggagacagg gactcctgta 360

tcttgcttga tcctgaatcc cttagtccct tgggtcgcgg acgtagccga ttcacttcag 420

atcccctgcg ctaccttgtg ggtccaatct tgtgcaagtt tttcagcata ttaccactac 480

caccacgggt tagtgccttt cccaaccgaa tcagagcccg agatcgacgt acaacttcct 540

gggatgccac ttttgaaata tgatgaagtg cccgacttcc tgcatccgcg cacaccctac 600

cccttttttg gcacgaacat tttaggtcaa ttcaagaatt tatccaagaa cttctgtatc 660

ctgatggata ccttctacga gttggaacac gagatcatcg ataatatgtg taaattgtgt 720

ccgattaagc caattggccc gttgtttaag attccgaaag acccaagctc caacggaatc 780

acgggtaatt tcatgaaagt ggatgactgc aaggagtggc tggacagccg tccaacatca 840

actgtggttt acgttagtgt cgggtctgtt gtatatttga agcaggagca ggttacagaa 900

atggcatacg gcattttaaa ttcggaagtt tcgtttttgt gggtgctgcg cccgccgagc 960

aaacgcatcg gtacggaacc gcatgtactg cccgaggagt tctgggagaa ggccggagat 1020

cgtggcaagg tggtgcaatg gtcaccccag gagcaggtgc ttgctcaccc cgccactgtc 1080

ggttttttaa cacactgtgg atggaatagc actcaagagg cgatttcgag cggagtgccc 1140

gtcatcactt tcccacaatt tggggaccaa gtgaccaatg ctaagttcct tgtggaggaa 1200

tttaaggtcg gggtccgttt aggccgcgga gagttagaaa atcgcatcat cacacgcgac 1260

gaagtagaac gcgctttacg cgagattact tcaggcccca aggctgaaga ggtaaaagag 1320

aacgccttaa aatggaagaa gaaggcagaa gagacagtag ctaaaggcgg ctactccgaa 1380

cgtaatcttg taggcttcat tgaagaggtg gctcgtaaga ctggtacaaa gtaa 1434

<210> 58

<211> 1176

<212> DNA

<213> unknown

<220>

<223> Rheum palmatum (Rheum palmatum)

<400> 58

atggcagatg tcctgcagga gatccgcaac tcgcagaagg cgagcgggcc cgccacggtg 60

ctcgccatcg gcactgccca tccaccgacg tgctaccctc aggccgacta ccccgacttc 120

tacttccgag tttgcaagag cgagcacatg accaaactca agaagaaaat gcaattcatt 180

tgtgacagat cggggataag gcagcggttt atgttccaca cggaagagaa cctggggaag 240

aacccgggga tgtgcacatt cgacgggcca tcgctgaacg cgcggcagga catgctgatc 300

atggaagtgc cgaagctggg ggcggaggcg gcggagaagg cgatcaagga gtgggggcag 360

gacaagtccc ggatcaccca cctcatcttc tgcaccacca cgagcaacga catgcccggg 420

gcggactacc agttcgccac cctgttcggg ctgaaccccg gcgtgagccg caccatggtc 480

taccagcagg gctgcttcgc cgggggcacc gtgctgcgcc tggtcaagga catcgcggag 540

aacaacaagg gggcgcgcgt gctggtggtg tgctcggaga tcgtggcctt cgccttccgc 600

gggccccacg aggaccacat cgactccctc atcgggcagc tcctgttcgg ggacggggcc 660

gccgccctcg tggtcgggac agacatcgac gagagcgtcg agaggcccat cttccagatc 720

atgtcggcga cccaggcgac catccccaac tcgctgcaca ccatggctct ccatctgacg 780

gaggcggggc tgaccttcca tctcagcaag gaggtgccca aggtggtgag cgacaacatg 840

gaggagctca tgctcgaggc cttcaagccg ctcgggataa ccgattggaa ctccatattc 900

tggcaagtgc atcccggggg tagagccatc cttgacaaga tcgaggagaa gctggagctc 960

accaaggata agatgcggga ttcccgctac atcttgagcg agtacgggaa tctcaccagc 1020

gcctgtgtgc tctttgtcat ggacgagatg aggaagaggt ccttccggga agggaagcag 1080

accaccggag acggctacga gtggggtgtc gccatcggat tggggcccgg tcttaccgtc 1140

gagaccgttg tcttgcgtag cgtccccatt ccctaa 1176

<210> 59

<211> 1776

<212> DNA

<213> unknown

<220>

<223> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 59

gtgtcagtcg agactaggaa gatcaccaag gttcttgtcg ctaaccgtgg tgagattgca 60

atccgcgtgt tccgtgcagc tcgagatgaa ggcatcggat ctgtcgccgt ctacgcagag 120

ccagatgcag atgcaccatt cgtgtcatat gcagacgagg cttttgccct cggtggccaa 180

acatccgctg agtcctacct tgtcattgac aagatcatcg atgcggcccg caagtccggc 240

gccgacgcca tccaccccgg ctacggcttc ctcgcagaaa acgctgactt cgcagaagca 300

gtcatcaacg aaggcctgat ctggattgga ccttcacctg agtccatccg ctccctcggc 360

gacaaggtca ccgctcgcca catcgcagat accgccaagg ctccaatggc tcctggcacc 420

aaggaaccag taaaagacgc agcagaagtt gtggctttcg ctgaagaatt cggtctccca 480

atcgccatca aggcagcttt cggtggcggc ggacgtggca tgaaggttgc ctacaagatg 540

gaagaagtcg ctgacctctt cgagtccgca acccgtgaag caaccgcagc gttcggccgc 600

ggcgagtgct tcgtggagcg ctacctggac aaggcacgcc acgttgaggc tcaggtcatc 660

gccgataagc acggcaacgt tgttgtcgcc ggaacccgtg actgctccct gcagcgccgt 720

ttccagaagc tcgtcgaaga agcaccagca ccattcctca ccgatgacca gcgcgagcgt 780

ctccactcct ccgcgaaggc tatctgtaag gaagctggct actacggtgc aggcaccgtt 840

gagtacctcg ttggctccga cggcctgatc tccttcctcg aggtcaacac ccgcctccag 900

gtggaacacc cagtcaccga agagaccacc ggcatcgacc tggtccgcga aatgttccgc 960

atcgcagaag gccacgagct ctccatcaag gaagatccag ctccacgcgg ccacgcattc 1020

gagttccgca tcaacggcga agacgctggc tccaacttca tgcctgcacc aggcaagatc 1080

accagctacc gcgagccaca gggcccaggc gtccgcatgg actccggtgt cgttgaaggt 1140

tccgaaatct ccggacagtt cgactccatg ctggcaaagc tgatcgtttg gggcgacacc 1200

cgcgagcagg ctctccagcg ctcccgccgt gcacttgcag agtacgttgt cgagggcatg 1260

ccaaccgtta tcccattcca ccagcacatc gtggaaaacc cagcattcgt gggcaacgac 1320

gaaggcttcg agatctacac caagtggatc gaagaggttt gggataaccc aatcgcacct 1380

tacgttgacg cttccgagct cgacgaagat gaggacaaga ccccagcaca gaaggttgtt 1440

gtggagatca acggccgtcg cgttgaggtt gcactcccag gcgatctggc actcggtggc 1500

accgctggtc ctaagaagaa ggccaagaag cgtcgcgcag gtggtgcaaa ggctggcgta 1560

tccggcgatg cagtggcagc tccaatgcag ggcactgtca tcaaggtcaa cgtcgaagaa 1620

ggcgctgaag tcaacgaagg cgacaccgtt gttgtcctcg aggctatgaa gatggaaaac 1680

cctgtgaagg ctcataagtc cggaaccgta accggcctta ctgtcgctgc aggcgagggt 1740

gtcaacaagg gcgttgttct cctcgagatc aagtaa 1776

<210> 60

<211> 1321

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 60

atgcgtcatg tagagcatac agtcaccgtt gcggccccag cagacttggt ttgggaggta 60

cttgccgatg tcttaggcta tgctgacatc ttcccaccga cggaaaaagt tgaaattctt 120

gaggaggggc aaggatacca ggtagtgcgc cttcacgtcg atgttgcggg tgagattaat 180

acatggacca gtcgtcgcga tttagaccct gcgcgccgcg taattgctta ccgccaactt 240

gagacggctc cgatcgtggg ccacatgagc ggggaatggc gtgctttcac actggatgcc 300

gaacgtaccc aattagtcct gactcacgat ttcgtaaccc gtgcagccgg ggatgacggt 360

ttagtcgccg gaaaattgac cccagatgag gcgcgcgaaa tgttagaagc ggtggtagaa 420

cgtaactctg tcgccgactt aaacgcggtc cttggagaag ctgagcgtcg cgtccgcgca 480

gccggtggag ttggtaccgt aactgcgtaa taataatttt gtttaacttt aagaaggaga 540

tatatccatg tcagggcgca aaaccttttt agacttaagt tttgctaccc gcgacacacc 600

gtcggaggcg actccggtgg tggtagattt gctggaccac gtaactggag ccaccgtatt 660

aggattatca cctgaggatt tccccgatgg tatggctatt tccaatgaga ccgttacgtt 720

gacgacccac actggcacgc acatggatgc gccactgcac tatggtccct taagtggggg 780

agttccggca aagtcgattg accaagtgcc cttggaatgg tgctatggac ctggagttcg 840

tttggatgtt cgccacgtgc cggcaggaga tggtattact gtcgatcatt tgaacgccgc 900

gttggatgca gcagagcacg atttggcccc cggtgacatt gtgatgctgt ggaccggcgc 960

ggacgctctg tggggaaccc gcgaatactt gagcacgttt ccggggttaa ctgggaaggg 1020

gacacaattt ttggtcgagg cgggtgttaa agtcattggc attgatgcat ggggactgga 1080

tcgcccgatg gcagctatga tcgaagaata ccgtcgtacg ggcgataaag gagcattatg 1140

gccggctcac gtctatggac gcacacgcga atacctgcaa ttagagaagc ttaataattt 1200

gggcgcttta ccaggagcta cagggtatga catttcatgc tttccggttg cggttgcagg 1260

cactggagct gggtggactc gtgtggtcgc cgttttcgag caagaggaag aggattaata 1320

a 1321

<210> 61

<211> 477

<212> PRT

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F (GtUF 6CGT1V 93Q/Y193F) variants

<400> 61

Met Gly Ser Leu Thr Asn Asn Asp Asn Leu His Ile Phe Leu Val Cys

1 5 10 15

Phe Ile Gly Gln Gly Val Val Asn Pro Met Leu Arg Leu Gly Lys Ala

20 25 30

Phe Ala Ser Lys Gly Leu Leu Val Thr Leu Ser Ala Pro Glu Ile Val

35 40 45

Gly Thr Glu Ile Arg Lys Ala Asn Asn Leu Asn Asp Asp Gln Pro Ile

50 55 60

Lys Val Gly Ser Gly Met Ile Arg Phe Glu Phe Phe Asp Asp Gly Trp

65 70 75 80

Glu Ser Val Asn Gly Ser Lys Pro Phe Asp Val Trp Gln Tyr Ile Asn

85 90 95

His Leu Asp Gln Thr Gly Arg Gln Lys Leu Pro Ile Met Leu Lys Lys

100 105 110

His Glu Glu Thr Gly Thr Pro Val Ser Cys Leu Ile Leu Asn Pro Leu

115 120 125

Val Pro Trp Val Ala Asp Val Ala Asp Ser Leu Gln Ile Pro Cys Ala

130 135 140

Thr Leu Trp Val Gln Ser Cys Ala Ser Phe Ser Ala Tyr Tyr His Tyr

145 150 155 160

His His Gly Leu Val Pro Phe Pro Thr Glu Ser Glu Pro Glu Ile Asp

165 170 175

Val Gln Leu Pro Gly Met Pro Leu Leu Lys Tyr Asp Glu Val Pro Asp

180 185 190

Phe Leu His Pro Arg Thr Pro Tyr Pro Phe Phe Gly Thr Asn Ile Leu

195 200 205

Gly Gln Phe Lys Asn Leu Ser Lys Asn Phe Cys Ile Leu Met Asp Thr

210 215 220

Phe Tyr Glu Leu Glu His Glu Ile Ile Asp Asn Met Cys Lys Leu Cys

225 230 235 240

Pro Ile Lys Pro Ile Gly Pro Leu Phe Lys Ile Pro Lys Asp Pro Ser

245 250 255

Ser Asn Gly Ile Thr Gly Asn Phe Met Lys Val Asp Asp Cys Lys Glu

260 265 270

Trp Leu Asp Ser Arg Pro Thr Ser Thr Val Val Tyr Val Ser Val Gly

275 280 285

Ser Val Val Tyr Leu Lys Gln Glu Gln Val Thr Glu Met Ala Tyr Gly

290 295 300

Ile Leu Asn Ser Glu Val Ser Phe Leu Trp Val Leu Arg Pro Pro Ser

305 310 315 320

Lys Arg Ile Gly Thr Glu Pro His Val Leu Pro Glu Glu Phe Trp Glu

325 330 335

Lys Ala Gly Asp Arg Gly Lys Val Val Gln Trp Ser Pro Gln Glu Gln

340 345 350

Val Leu Ala His Pro Ala Thr Val Gly Phe Leu Thr His Cys Gly Trp

355 360 365

Asn Ser Thr Gln Glu Ala Ile Ser Ser Gly Val Pro Val Ile Thr Phe

370 375 380

Pro Gln Phe Gly Asp Gln Val Thr Asn Ala Lys Phe Leu Val Glu Glu

385 390 395 400

Phe Lys Val Gly Val Arg Leu Gly Arg Gly Glu Leu Glu Asn Arg Ile

405 410 415

Ile Thr Arg Asp Glu Val Glu Arg Ala Leu Arg Glu Ile Thr Ser Gly

420 425 430

Pro Lys Ala Glu Glu Val Lys Glu Asn Ala Leu Lys Trp Lys Lys Lys

435 440 445

Ala Glu Glu Thr Val Ala Lys Gly Gly Tyr Ser Glu Arg Asn Leu Val

450 455 460

Gly Phe Ile Glu Glu Val Ala Arg Lys Thr Gly Thr Lys

465 470 475

<210> 62

<211> 1321

<212> DNA

<213> artificial sequence

<220>

<223> codon optimized zhuIJ of E.coli (E.coli)

<400> 62

atgcgtcatg tagagcatac agtcaccgtt gcggccccag cagacttggt ttgggaggta 60

cttgccgatg tcttaggcta tgctgacatc ttcccaccga cggaaaaagt tgaaattctt 120

gaggaggggc aaggatacca ggtagtgcgc cttcacgtcg atgttgcggg tgagattaat 180

acatggacca gtcgtcgcga tttagaccct gcgcgccgcg taattgctta ccgccaactt 240

gagacggctc cgatcgtggg ccacatgagc ggggaatggc gtgctttcac actggatgcc 300

gaacgtaccc aattagtcct gactcacgat ttcgtaaccc gtgcagccgg ggatgacggt 360

ttagtcgccg gaaaattgac cccagatgag gcgcgcgaaa tgttagaagc ggtggtagaa 420

cgtaactctg tcgccgactt aaacgcggtc cttggagaag ctgagcgtcg cgtccgcgca 480

gccggtggag ttggtaccgt aactgcgtaa taataatttt gtttaacttt aagaaggaga 540

tatatccatg tcagggcgca aaaccttttt agacttaagt tttgctaccc gcgacacacc 600

gtcggaggcg actccggtgg tggtagattt gctggaccac gtaactggag ccaccgtatt 660

aggattatca cctgaggatt tccccgatgg tatggctatt tccaatgaga ccgttacgtt 720

gacgacccac actggcacgc acatggatgc gccactgcac tatggtccct taagtggggg 780

agttccggca aagtcgattg accaagtgcc cttggaatgg tgctatggac ctggagttcg 840

tttggatgtt cgccacgtgc cggcaggaga tggtattact gtcgatcatt tgaacgccgc 900

gttggatgca gcagagcacg atttggcccc cggtgacatt gtgatgctgt ggaccggcgc 960

ggacgctctg tggggaaccc gcgaatactt gagcacgttt ccggggttaa ctgggaaggg 1020

gacacaattt ttggtcgagg cgggtgttaa agtcattggc attgatgcat ggggactgga 1080

tcgcccgatg gcagctatga tcgaagaata ccgtcgtacg ggcgataaag gagcattatg 1140

gccggctcac gtctatggac gcacacgcga atacctgcaa ttagagaagc ttaataattt 1200

gggcgcttta ccaggagcta cagggtatga catttcatgc tttccggttg cggttgcagg 1260

cactggagct gggtggactc gtgtggtcgc cgttttcgag caagaggaag aggattaata 1320

a 1321

<210> 63

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I18P_F

<400> 63

tgtgcttccc aggccagggc gtggtcaat 29

<210> 64

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I18P_R

<400> 64

cgccctggcc tgggaagcac acaagaaaaa t 31

<210> 65

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_Q20M_F

<400> 65

ttcatcggca tgggcgtggt caatcccat 29

<210> 66

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_Q20M_R

<400> 66

tgaccacgcc catgccgatg aagcacacaa g 31

<210> 67

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50K_F

<400> 67

aatcgttgga aaggagatcc gtaaggcgaa 30

<210> 68

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50K_R

<400> 68

tacggatctc ctttccaacg atttccggtg 30

<210> 69

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50N_F

<400> 69

aatcgttgga aatgagatcc gtaaggcgaa 30

<210> 70

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50Q_F

<400> 70

tacggatctc atttccaacg atttccggtg 30

<210> 71

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50Q_R

<400> 71

aatcgttgga caggagatcc gtaaggcgaa 30

<210> 72

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50R_F

<400> 72

tacggatctc ctgtccaacg atttccggtg 30

<210> 73

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50R_R

<400> 73

aatcgttgga cgtgagatcc gtaaggcgaa 30

<210> 74

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50V_F

<400> 74

tacggatctc acgtccaacg atttccggtg 30

<210> 75

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50V_R

<400> 75

aatcgttgga gttgagatcc gtaaggcgaa 30

<210> 76

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_T50V_R

<400> 76

tacggatctc aactccaacg atttccggtg 30

<210> 77

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I95L_F

<400> 77

gtatggcaat acctcaatca cttagaccag acag 34

<210> 78

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I95L_R

<400> 78

ggtctaagtg attgaggtat tgccatacgt caaacg 36

<210> 79

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I95M_F

<400> 79

gtatggcaat acatgaatca cttagaccag acag 34

<210> 80

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I95M_R

<400> 80

ggtctaagtg attcatgtat tgccatacgt caaacg 36

<210> 81

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I95T_F

<400> 81

gtatggcaat acactaatca cttagaccag acag 34

<210> 82

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I95T_R

<400> 82

ggtctaagtg attagtgtat tgccatacgt caaacg 36

<210> 83

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_V290A_F

<400> 83

gtgtcgggtc tgctgtatat ttgaagcagg ag 32

<210> 84

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_V290A_R

<400> 84

gcttcaaata tacagcagac ccgacactaa cg 32

<210> 85

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_V290G_F

<400> 85

gtgtcgggtc tggtgtatat ttgaagcagg ag 32

<210> 86

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_V290G_R

<400> 86

gcttcaaata tacaccagac ccgacactaa cg 32

<210> 87

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I323A_F

<400> 87

agcaaacgcg ccggtacgga accgcatgt 29

<210> 88

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I323A_R

<400> 88

gttccgtacc ggcgcgtttg ctcgg 25

<210> 89

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I323S_F

<400> 89

agcaaacgca gcggtacgga accgcatgt 29

<210> 90

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT V93Q/Y193F_I323S_R

<400> 90

gttccgtacc gctgcgtttg ctcgg 25

<210> 91

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> V22A-f

<400> 91

cggccagggc gcggtcaatc ccatgttacg 30

<210> 92

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> V22A-R

<400> 92

tgggattgac cgcgccctgg ccgatgaagc 30

<210> 93

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> L29A-F

<400> 93

catgttacgt gcggggaagg cgttcgcctc 30

<210> 94

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> L29A-R

<400> 94

cgccttcccc gcacgtaaca tgggattgac 30

<210> 95

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> E46G-f

<400> 95

agcgcaccgg gcatcgttgg aactgagatc 30

<210> 96

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> E46G-R

<400> 96

tccaacgatg cccggtgcgc ttaaagtgac 30

<210> 97

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> V48G-f

<400> 97

ccggaaatcg gtggaactga gatccgtaag 30

<210> 98

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> V48G-R

<400> 98

ctcagttcca ccgatttccg gtgcgcttaa 30

<210> 99

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> E51C-f

<400> 99

cgttggaact tgcatccgta aggcgaataa c 31

<210> 100

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> E51C-R

<400> 100

ccttacggat gcaagttcca acgatttccg g 31

<210> 101

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> A55S-f

<400> 101

gatccgtaag tcgaataacc ttaatgatga ccaac 35

<210> 102

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> A55S-r

<400> 102

aaggttattc gacttacgga tctcagttcc 30

<210> 103

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> S86V-F

<400> 103

cgtaaacggt gtcaaaccgt ttgacgtatg g 31

<210> 104

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> S86V-r

<400> 104

caaacggttt gacaccgttt acggattccc a 31

<210> 105

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> D99G-F

<400> 105

caatcactta ggccagacag gccgtcaaaa ac 32

<210> 106

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> D99G-r

<400> 106

ggcctgtctg gcctaagtga ttgatgtatt gc 32

<210> 107

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> R103V-F

<400> 107

ccagacaggc gttcaaaaac ttccgattat g 31

<210> 108

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> R103V-R

<400> 108

ggaagttttt gaacgcctgt ctggtctaag tg 32

<210> 109

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> C151G-F

<400> 109

ggtccaatct ggtgcaagtt tttcagcata t 31

<210> 110

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> C151G-r

<400> 110

gaaaaacttg caccagattg gacccacaag g 31

<210> 111

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> L184G-F

<400> 111

ctgggatgcc aggtttgaaa tatgatgaag tg 32

<210> 112

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> L184G-r

<400> 112

catatttcaa acctggcatc ccaggaagtt g 31

<210> 113

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> L194A-F

<400> 113

cccgacttcg cgcatccgcg cacaccctac 30

<210> 114

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> L194A-r

<400> 114

gtgcgcggat gcgcgaagtc gggcacttca tc 32

<210> 115

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> E332P-F

<400> 115

tgtactgccc ccggagttct gggagaaggc c 31

<210> 116

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> E332P-r

<400> 116

cccagaactc cgggggcagt acatgcggtt c 31

<210> 117

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> I18A-F

<400> 117

tgtgtgcttc gccggccagg gcgtggtcaa t 31

<210> 118

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> I18A-r

<400> 118

cgccctggcc ggcgaagcac acaagaaaaa t 31

<210> 119

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> P385A-F

<400> 119

catcactttc gcacaatttg gggaccaagt g 31

<210> 120

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> P385A-r

<400> 120

ccccaaattg tgcgaaagtg atgacgggca c 31

<210> 121

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT_N_His_IV_F

<400> 121

caccatcacc atcaccatgg gagtttgact aacaacgata atc 43

<210> 122

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> GtCGT_N_His_IV_R

<400> 122

catatgtata tctccttctt atac 24

Claims

1. A C-glucosyltransferase variant comprising a mutation of at least one amino acid selected from the group consisting of F17, V93, V132, Y193, L164 and R322 in the C-glucosyltransferase set forth in SEQ ID No. 1.

2. The C-glucosyltransferase variant of claim 1, wherein the variant further comprises a mutation of at least one amino acid selected from the group consisting of the C-glucosyltransferases set forth in SEQ ID NO: 1: f17, V405, P107, L208, L164, P45, I305, L316, F401, Y94, N57, Y187, C16, P319, F167, V132, N206, R406, Q386, V129, L125, L194, I95, S215, L184, Y158, L29, L27, F202, H159, S370, H365, V329, M301, V315, V190, C366, W80, L58, Q210, F312, D61, I207, L363, P196, L106, V93, A394, W314, S155, P88, D99, Y284, E189, G49, H328, E399, T392, F387, A44, P199E 46, R28, V285, I124, R419, L306, Y157, Y200, E373, P191, L214, S376, V15, E332, E51, I417, L98, I323, H161, T383, P127, E309, N84, L313, Q104, T371, N213, G79, L330, N307, K105, L128, A152, I18, N59, W147, S86, L293, E296, S377, L185, K216, F89, S286, F396, F211, Y303, D223, R415, N96, V22, S153, F154, D192, Y193, H195, P201, Y292 and R322.

3. The C-glucosyltransferase variant of claim 1, wherein the mutation of amino acid comprises a mutation of amino acids V93 and Y193.

4. The C-glucosyltransferase variant of claim 1, comprising a substitution of at least one amino acid selected from the group consisting of F17G, V93Q, V132A, Y193F, L G and R322D.

5. The C-glucosyltransferase variant of claim 3, comprising substitutions of amino acids V93Q and Y193F.

6. The C-glucosyltransferase variant of claim 2, further comprising a substitution of at least one amino acid selected from the group consisting of: f17 405 107 208 45,107,316 45,401 94,57,187,57,187,187,319,132,132,206,386 129,194,194,29,202,159,370,329 301 315 190 366 80, 58, 312, 207, 363, 61, 196, 106, 93, 394, 155, 99, 284, 189, TH328, 399, 392, 44, 199, 46, 285, 124 419 306,157,373,373,201,191,214,376 15,332,417,98,323,383 127 309, 313, 104, 371, 213, 79, 330, 307, 105, 128, 152, 153, 59 147 86 293 296 377 185 216 89 286 396 211 223 96 93 93 93 93 154 193 195 195 292 292D and R322A.

7. The variant C-glycosyltransferase of claim 4, further comprising a step selected from the group consisting of I18P, Q20M, T50N, T Q, T50K, T50R, T95 5446 95M, I T, V290G, V290 79290 793 79323S, I323A, I L, V22A, L A, E46G, V48G, E51C, A86 5297 5299C, A103C, A151 184C, A194C, A52332C, A a and P385A.

8. The C-glucosyltransferase variant of claim 7, further comprising a substitution of at least one amino acid selected from the group consisting of I323S, T50R, T50V, I P, I T, Q20M, I323A, P385A, L194A and V48G.

9. A nucleic acid encoding the variant of any one of claims 1 to 8.

10. A recombinant microorganism into which the nucleic acid according to claim 9 is introduced.

11. The recombinant microorganism of claim 10, wherein expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and/or nucleoside-diphosphate kinase is enhanced in the recombinant microorganism.

12. The recombinant microorganism of claim 10, wherein the recombinant microorganism is used to produce polyketide and/or phenylpropanoid glycosides.

13. The recombinant microorganism of claim 12, wherein polyketide synthase or phenylpropanoid synthase is further introduced into the recombinant microorganism.

14. The recombinant microorganism of claim 12, wherein expression of the pabA gene is attenuated in the recombinant microorganism.

15. The recombinant microorganism of claim 12, wherein the polyketone is selected from the group consisting of:

a type III polyketone selected from the group consisting of aloin, 5, 7-dihydroxy-2-methyl chromone and aloeresin, and

phenylpropanoids selected from the group consisting of: non-ribosomes including the peptides neofumycin, bacitracin, daptomycin, vancomycin, thaumatin, casein, gramicidin, bifenthrin A, bleomycin, cyclosporin, pyovadine, enterobacterin, colistin A, indigoside and phycocyanin, pinocembrin, dihydrokaempferol, eriodictyol, dihydroquercetin, coniferol, silybin, isosilybin, silychristin, silrinide, 2, 3-dehydrosilybin, silybin, daidzein, genistein, apigenin, luteolin, kaempferol, quercetin, catechin, pelargonidin, anthocyanin, alfucatechin, myricetin, feissones, galangin, hesperetin, delrin, chrysin, resveratrol and naringenin.

16. The recombinant microorganism of claim 12, wherein at least one gene selected from the group consisting of: (i) a gene encoding a type II polyketide synthase, (II) a gene encoding a 4' -phosphopantetheinyl transferase, (iii) a gene encoding a cyclase, (iv) a gene encoding an acetyl-CoA carboxylase, and (v) a gene encoding an aclacinone 12-hydroxylase, and the polyketide is carminic acid.

17. The recombinant microorganism of claim 16, wherein the gene encoding type II polyketide synthase is at least one selected from the group consisting of antD (ketosynthase), antE (chain length factor), antF (ACP), antB (phosphopantetheinyl transferase), and antG (malonyl-CoA: ACP malonyl transferase); or a combination thereof.

18. The recombinant microorganism of claim 16, wherein the aclacinone 12-hydroxylase comprises a proline to lysine mutation (P217K) at amino acid position 217 in the amino acid sequence shown in SEQ ID No. 2.

19. The recombinant microorganism of claim 16, wherein:

the type II polyketide synthase is derived from photorhabdus luminescens (p.luminescens);

the 4' -phosphopantetheinyl transferase is derived from bacillus subtilis or photorhabdus luminescens (p.luminescens);

The cyclase is derived from Streptomyces sp;

the acetyl-CoA carboxylase is derived from Corynebacterium glutamicum (Corynebacterium glutamicum); and/or

The aclacinone 12-hydroxylase is derived from Streptomyces peucetius.

20. The recombinant microorganism of claim 12, wherein (i) a gene encoding aloe vera pine synthase is introduced and the polyketide is aloin.

21. The recombinant microorganism of claim 20, wherein the aloe vera pine synthase is derived from rheum palmatum.

22. A method of producing polyketide and/or phenylpropanoid glycosides, comprising:

(a) Producing polyketide and/or phenylpropanoid glycosides by culturing the recombinant microorganism of claim 10; and

(b) Recovering the polyketide and/or phenylpropanoid glycoside produced.

23. The method of claim 22, wherein the recombinant microorganism has the ability to produce precursors of the polyketide and/or the phenylpropanoid glycoside.

24. The method of claim 22, wherein step (a) comprises culturing the recombinant microorganism of claim 10 in a medium supplemented with polyketides and/or phenylpropanoids.

25. The method of claim 22, wherein the polyketide is carminic acid and in step (a) the microorganism is cultivated by adding ascorbic acid to the medium during the cultivation.

26. A method of producing polyketide and/or phenylpropanoid glycosides, comprising:

(a) Producing polyketide and/or phenylpropanoid glycosides by reacting the C-glucosyltransferase variant of any one of claims 1 to 8 or a microorganism expressing the C-glucosyltransferase variant with polyketides and/or phenylpropanoids; and

(b) Recovering the polyketide and/or phenylpropanoid glycoside produced.