KR20240017427A - Recombinant microorganism for producing 3'-fucosyllactose and methods using the same - Google Patents

Abstract

The present invention relates to an α-1,3-fucosyltransferase gene; GDP-D-mannose-dehydratase gene; GDP-L-fucose synthase gene; and a lactose permease gene; are introduced, and each of the genes is operably linked to an expression control sequence. A microorganism for producing 3-fucosyllactose and a microorganism derived from Parabacteroides johnsonii . Provided is a host cell into which an α-1,3-fucosyltransferase gene has been introduced.
The present invention discloses a novel α-1,3-fucosyltransferase derived from non-pathogenic bacteria that can be expressed and active in both prokaryotes and eukaryotes. In addition, the microorganism according to the present invention can efficiently produce 3-Foucault room lactose at a low cost, and especially when the microorganism is yeast ( Saccharomyces cerevisiae ) recognized as safe for food through GRAS certification, it stably produces 3-Foucault room lactose. Silactose can be safely produced with high yield.

Description

Genetically modified microorganism for producing 3-FUCOSYLLACTOSE and method using the same {RECOMBINANT MICROORGANISM FOR PRODUCING 3'-FUCOSYLLACTOSE AND METHODS USING THE SAME}

The present invention relates to a genetically modified microorganism for producing 3-fucosyllactose and a method of using the same, and more specifically, to an α-1,3-fucosyltransferase gene; GDP-D-mannose-dehydratase gene; GDP-L-fucose synthase gene; and a lactose permease gene; relates to a microorganism for producing 3-fucosyllactose.

The World Health Organization recommends exclusive breastfeeding, excluding other foods, for the first six months of life. This is because breast milk has been found to be superior to powdered milk in terms of effects on the baby's health, such as improving immunity, anti-inflammatory/immune regulation, prebiotic effect, strengthening the barrier function of the mucous membrane, and promoting the development of the intestines and brain (Ray et al ., Int J Pediatr. (2019), 2019:2390240). As an active ingredient involved in these various biological activities, human milk oligosaccharides (HMO), which are oligosaccharides with a unique structure that are contained in particularly large amounts in human breast milk among mammals, have recently been attracting attention.

More than 200 types of HMO have been reported so far, and they basically include D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (N-acetylglucosamine (GlcNAc), and L-fu. It consists of a combination of two or more of the five monosaccharides: cos (L-fucose, Fuc) and sialic acid (N-acetylneuraminic acid, Neu5Ac). These monosaccharides are linked by various glycosidic bonds and degrees of polymerization (DP 3-20) to form various oligosaccharides, but they have lactose (Galβ1-4Glc) at the reducing end in common, and lacto- N-biose or N-acetyllactosamine can be further extended to form a chain structure, and most of the ends or middle of the chain are fucosylated (50-80% of over 200 HMOs) or sialylation (out of over 200 HMOs). It is characteristic that it is 10-20%). Representative examples include 2-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), 3-sialyllactose, and 6-sialyl. Lactose (6'-sialyllactose), lactodifucotetraose, lacto-N-fucopentaose, lacto-N-neotetraose, lacto-N -Tetraose (lacto-N-tetraose), lacto-N-difucohexaose, etc. Among them, the HMO with the highest content of 5-15 g/L in breast milk is 1. It has been reported that 2-FL and 3-FL account for -2 g/L (Smilowitz et al., J Nutr . (2013), 143(11):1709-1718).

According to the various functions of HMO, the possibility of using HMO as baby food, health functional food for the elderly, and pharmaceutical material is being explored. In particular, it has begun to be known that among fucosylated HMOs, 3-FL regulates immune responses, inhibits tumor metastasis, and exhibits bacteriostatic and antibacterial effects against various pathogens. However, despite these efficacy, industrial mass production of 3-FL is difficult and research is not easy. For example, in the case of breast milk extraction, there are limitations in the supply of raw materials and low productivity, chemical synthesis has problems with expensive substrates, low isomer selectivity and production yield, and enzymatic synthesis using glycosyltransferase The synthesis method uses GDP-L-fucose, the active nucleic acid sugar form of sialic acid or fucose, as a substrate, but these substances are expensive and the cost of purifying the glycosyltransferase is also high, so it is not economically feasible. There are low problems. On the other hand, when using microorganisms, mass production at high concentrations is possible with inexpensive substrates, but no microorganisms that naturally produce HMO have yet been identified, so recombinant microorganisms are produced using metabolic engineering methods that introduce metabolic enzymes into microorganisms. (Jin Young-wook, Livestock and Food Science and Industry (2018), 7(1):34-41).

Most existing methods for producing 3-fucosyllactose using metabolic engineering use bacteria, especially Escherichia coli . For example, Republic of Korea Patent No. 10-1794971 includes α-1,3-fucosyltransferase derived from Helicobacter pylori ATCC 26695 and GDP-D-mannose-4. ,6-dehydratase (GDP-D-mannose-4,6-dehydratase), GDP-L-fucose synthase and mannose-1-phosphate guanyl transferase ( A recombinant Escherichia coli for the production of 3-fucosyllactose in which mannose-1-phoaphate guanyltransferase was introduced and UDP-glucose lipid carrier transferase was removed is described. However, the α-1,3-fucosyltransferase is derived from pathogenic bacteria, and products produced using this enzyme may be difficult to obtain approval or may not be preferred by consumers, and E. coli naturally produces endotoxin. ), so there is a risk that the final product may contain endotoxin.

Therefore, there is a need for a low-cost, high-yield, and safe 3-fucosyllactose production method. The inventors of the present invention selected α-1,3-fucosyltransferase from the non-pathogenic bacterium Parabacteroides johnsonii for the production of 3-fucosyllactose, and the enzyme was used in prokaryotes. It was confirmed that expression and activity can be shown in both eukaryotes and eukaryotes. In addition, the metabolic process of yeast ( Saccharomyces cerevisiae ), which has been recognized as safe for food through GRAS certification, has been analyzed using the newly discovered α-1,3-fucosyltransferase as described above. The present invention was completed by confirming that 3-fucosyllactose can be effectively produced by changing it through recombination, such as introduction.

The technical problem to be achieved by the present invention is to develop a recombinant microorganism that can effectively and safely produce 3-fucosyllactose at low cost and an α-1,3-fucosyltransferase gene derived from Parabacteroides johnsonii . The introduced host cells are provided.

The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above technical problem, an embodiment of the present invention provides an α-1,3-fucosyltransferase gene; GDP-D-mannose-dehydratase gene; GDP-L-fucose synthase gene; and a lactose permease gene; are introduced, and each of the genes is operably linked to an expression control sequence.

Here, the α-1,3-fucosyltransferase gene, the GDP-D-mannose-dehydratase gene, the GDP-L-fucose synthase gene, and the lactose permease gene are one or more It may be characterized as being introduced and included on a non-integrating vector.

The α-1,3-fucosyltransferase gene is on a vector different from the GDP-D-mannose-dehydratase gene, the GDP-L-fucose synthase gene, and the lactose permease gene. Included, the vector containing the α-1,3-fucosyltransferase gene is a high copy number vector, the GDP-D-mannose-dehydratase gene, the GDP-L -The vector containing the fucose synthase gene and the lactose permease gene may be characterized as one or more low copy number vectors.

The low copy number vector may be characterized as a centromere vector.

The α-1,3-fucosyltransferase gene may be derived from Parabacteroides johnsonii .

The α-1,3-fucosyltransferase gene may be characterized as comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 1.

The α-1,3-fucosyltransferase gene may be characterized as comprising the base sequence of SEQ ID NO: 2.

The GDP-D-mannose-dehydratase and GDP-L-fucose synthase genes may be derived from E. coli.

The lactose permease gene may be derived from Kluyveromyces lactis .

The microorganism may be characterized as yeast.

Another embodiment of the present invention provides a host cell into which an α-1,3-fucosyltransferase gene derived from Parabacteroides johnsonii has been introduced.

Another embodiment of the present invention provides 3-fucosyllactose produced using the above-mentioned microorganism.

The above-described means for solving the problem are merely illustrative and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be present in the drawings and detailed description of the invention.

The α-1,3-fucosyltransferase discovered in the present invention can be expressed and active in both prokaryotes and eukaryotes. This α-1,3-fucosyltransferase is derived from the non-pathogenic bacterium Parabacteroides johnsonii , and 3-fucosyllactose prepared using it is α-1 derived from existing pathogenic microorganisms. , Compared to the case of using 3-fucosyltransferase, it can be used as a food or pharmaceutical raw material without problems such as additional licensing or consumer resistance.

In addition, the α-1,3-fucosyltransferase gene of the present invention; GDP-D-mannose-dehydratase gene; GDP-L-fucose synthase gene; and lactose permease genes; can efficiently produce 3-fucosyllactose at low cost. In particular, these genes are used in yeast ( Saccharomyces cerevisiae ), which is recognized as safe for food through GRAS certification. When introduced, 3-fucosyllactose can be safely produced in a stable and high yield.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

Figure 1 shows the results of 3-FL production from E. coli cell lysate expressing futA or Pj3ft .
Figure 2 is a schematic diagram showing the 3-FL-related sugar metabolism pathway (black) existing in S. cerevisiae and the pathway (red) introduced in the present invention.
Figure 3 shows pGAL10 in S. cerevisiae Y2805 or BY4741. Alternatively, this is the Western blot result of expressing Pj3ft or futA using pGAPDH.
Figure 4 shows the results of comparing cell growth and 3-FL production of S. cerevisiae Y2805 or CEN.PK2-1C strains into which Pj3ft , gmd , gmer , and lac12 were introduced.
Figure 5 shows the results of comparing 3-FL production when Pj3ft , gmd , gmer , and lac12 were expressed with different promoters.
Figure 6 shows the results of PCR confirmation of the gmd and gmer gene portions on the episome-like plasmid according to culture time.
Figure 7 is a schematic diagram of the episome-like plasmid and centromere vector introduced for the two-vector expression system in yeast.
Figure 8 is a schematic diagram of an episome-like plasmid and two centromere vectors introduced for a 3-vector expression system in yeast.
Figure 9 shows the results of confirming the 3-FL production in yeast into which Pj3ft , gmd , gmer , and lac12 were introduced using only an episome-like plasmid or a 2-vector or 3-vector including a centromere vector.
Figure 10 shows the results of confirming the cell growth and intra/extracellular 3-FL production of yeast into which Pj3ft , gmd , gmer , and lac12 were introduced using a 2-vector or 3-vector containing both an episome-like plasmid and a centromere vector. am.
Figure 11 shows the results of confirming the 3-FL production volume obtained by scaling up in S. cerevisiae FCCL-3 .

Hereinafter, the present invention will be described with reference to the attached drawings. However, the present invention may be implemented in various different forms and, therefore, is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts that are not related to the description are omitted, and similar parts are given similar reference numerals throughout the specification.

Throughout the specification, when a part is said to be "connected (connected, contacted, combined)" with another part, this means not only "directly connected" but also "indirectly connected" with another member in between. "Includes cases where it is. Additionally, when a part is said to “include” a certain component, this does not mean that other components are excluded, but that other components can be added, unless specifically stated to the contrary.

The terms used herein are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to indicate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

When a change is made that causes a sequence difference while maintaining the characteristics of each domain according to the present invention, the amino acid sequence included in the present specification is at least 80%, preferably at least 90%, and more preferably at least 95% of the sequence. , most preferably can encompass sequences having more than 99% homology. The change may be one or more selected from addition, substitution, or deletion of some amino acid residues.

Microorganisms for producing 3-fucosyllactose according to one aspect of the present invention include an α-1,3-fucosyltransferase gene; GDP-D-mannose-dehydratase gene; GDP-L-fucose synthase gene; and a lactose permease gene; are introduced, and each of the genes is operably linked to an expression control sequence.

3-Fucosyllactose (3-FL) is a substance with the same structure as chemical structure 1 below, and is one of the two fucosyllactoses that account for the largest proportion of human breast milk oligosaccharides. It has been reported that 3-FL exhibits various physiological activities such as antibacterial, bacteriostatic, promoting the growth of beneficial intestinal bacteria, and anti-inflammatory effects.

[Chemical structure 1]

The α-1,3-fucosyltransferase (α-1,3-fucosyltransferase) is an enzyme classified with enzyme number EC 2.4.1.152, and guanosine diphosphofucose-glucoside alpha1->3-fucose. It is also called guanosine diphosphofucose-glucoside alpha1->3-fucosyltransferase, galactoside 3-fucosyltransferase, etc., and GDP-β-L-fucose (GDP-β) It is an enzyme that can catalyze the α 1->3 bond of -L-fucose) and galactose.

The α-1,3-fucosyltransferase gene may be derived from Parabacteroides johnsonii . The α-1,3-fucosyltransferase gene derived from P. johnsonii can be expressed and active not only in prokaryotic microorganisms but also in eukaryotic microorganisms.

The α-1,3-fucosyltransferase gene may be characterized as comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 1.

The α-1,3-fucosyltransferase gene may be characterized as comprising the base sequence of SEQ ID NO: 2.

The GDP-D-mannose-dehydratase (GDP-D-mannose-dehydratase) is an enzyme classified as enzyme number EC 4.2.1.47, GDP-D-mannose 4,6-dehydratase (GDP -D-mannose 4,6-dehydratase), guanosine 5'-diphosphate-D-mannose oxidoreductase (Guanosine 5'-diphosphate-D-mannose oxidoreductase), guanosine diphosphomannose 4,6-di Also called hydratase (Guanosine diphosphomannose 4,6-dehydratase) or guanosine diphosphomannose oxidoreductase, GDP-α-mannose and GDP-4-keto It is an enzyme that has the activity of catalyzing the conversion between -6-deoxymannose (GDP-4-keto-6-deoxymannose).

The GDP-L-fucose synthase is an enzyme classified with enzyme number EC 1.1.1.271, GDP-4-keto-6-deoxy-D-mannose-3,5-epi. Also called merase-4-reductase (GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase), GDP-4-dehydro-α-D-rhamnose (GDP-4-dehydro-α-D-rhamnose) It is an enzyme that catalyzes the conversion between -4-dehydro-α-D-rhamnose) and GDP-L-fucose through NADPH/NADP(+).

The GDP-D-mannose-dehydratase and GDP-L-fucose synthase genes may be derived from E. coli. The GDP-D-mannose-dehydratase ( gmd ) and GDP-L-fucose synthase ( gmer ) genes derived from E. coli can be expressed and active in not only prokaryotic microorganisms but also eukaryotic microorganisms.

Lactose permease is a membrane protein that can transport lactose across cell membranes. The lactose permease gene may be derived from Kluyveromyces lactis . Lactose is essential as a substrate for fucosyllactose biosynthesis in the microorganism of the present invention, and lactose permease is required for its absorption. K. lactis is a yeast with an excellent ability to utilize lactose, and lactose permease derived therefrom , preferably lac12 , can induce lactose absorption in the medium with high efficiency.

Vectors can be broadly divided into integrating vectors and non-integrating vectors. Integrative vectors are inserted into the genome of a host cell and have the advantage of amplifying the host cell and stably passing it to daughter cells. However, depending on the insertion location, they may have unintended effects such as inhibiting the normal function of the host cell. Non-integrative vectors are vectors that are not inserted into the genome of a host cell and exist separately, that is, in the form of an episome. They do not exhibit the above-mentioned side effects of integrative vectors, but may generally be less stable than integrative vectors.

The α-1,3-fucosyltransferase gene, the GDP-D-mannose-dehydratase gene, the GDP-L-fucose synthase gene and the lactose permease gene are one or more non-integral It may be characterized as being introduced and included on a (non-integrating) vector.

The α-1,3-fucosyltransferase gene is on a vector different from the GDP-D-mannose-dehydratase gene, the GDP-L-fucose synthase gene, and the lactose permease gene. Included, the vector containing the α-1,3-fucosyltransferase gene is a high copy number vector, the GDP-D-mannose-dehydratase gene, the GDP-L -The vector containing the fucose synthase gene and the lactose permease gene may be characterized as one or more low copy number vectors.

The low copy number vector may be characterized as a centromere vector. Centromere vectors can be amplified synchronized with the host cell's genome replication like additional chromosomes, including centromere, and are generally known to have higher mitotic stability than non-integrative vectors. .

An expression control sequence refers to a nucleic acid sequence of a functional unit that can regulate gene expression at the level of transcription, mRNA kinetics, and/or translation. These expression control sequences include promoter, enhancer, insulator, silencer, 5' untranslated region, 3' untranslated region, polyA The polyadenylation signal, internal ribosome entry site (IRES), and splicing regulatory element are known.

The four genes described above, namely α-1,3-fucosyltransferase, GDP-D-mannose-dehydratase, GDP-L-fucose synthase, and lactose permease, are involved in the transformation or movement of substrates. As an enzyme that catalyzes, its presence at high concentrations may be advantageous for the production of 3-FL. The expression control sequence may preferably induce highly efficient expression. The expression control sequence may be a promoter, and the expression control sequence may be selected so that the gene can be appropriately expressed in the introduced microorganism. In yeast, various promoters that can induce high expression are known, and when the microorganism is yeast, housekeeping genes such as GPD (glyceraldehyde-3-phosphate dehydrogenase), TEF (Translational elongation factor), PGK (phosphoglycerate kinase), etc. Housekeeping gene) promoters, promoters for inducible expression derived from pGAL1-10, pGAL10, etc. can be used, and preferably those selected from pGAPDH, pGAL1-10, or pGAL10 can be used.

Each gene may be operably linked to one or more expression control sequences, and one expression control sequence may be operably linked to one or more genes.

The microorganism may be yeast, preferably Saccharomyces cerevisiae . S. cerevisiae can express the four genes described above, namely α-1,3-fucosyltransferase, GDP-D-mannose-dehydratase, GDP-L-fucose synthase, and lactose permease. If introduced, it can not only effectively produce 3-FL, but also have advantages in terms of safety, such as avoiding risks such as endotoxin contamination of E. coli used for 3-FL biosynthesis, and is GRAS (generally It is recognized as safe for food through certification (food ingredient classification by the U.S. FDA), so if 3-FL produced from it is used as a food or pharmaceutical raw material, it may be advantageous in terms of licensing or consumer awareness. The yeast may preferably be derived from S. cerevisiae Y2805, and more preferably may be S. cerevisiae FCCL-3.

Another aspect of the present invention provides a host cell into which an α-1,3-fucosyltransferase gene derived from Parabacteroides johnsonii has been introduced.

The α-1,3-fucosyltransferase gene may preferably include a base sequence encoding the amino acid sequence of SEQ ID NO: 1.

The host cell may be, for example, for expressing the α-1,3-fucosyltransferase gene, may be for producing 3-fucosyllactose, and may be for producing the α-1,3-fucosyltransferase gene. It may be for amplifying a vector containing.

The α-1,3-fucosyltransferase gene may be integrated into the host cell genome. The α-1,3-fucosyltransferase gene may preferably be introduced through a non-integrating vector, and the non-integrating vector is synchronized with or independent of cell proliferation within the host cell. can be amplified.

The host cell may be a prokaryotic cell or a eukaryotic cell, and preferably a microbial cell. The host cell may preferably be selected from Eubacteria or Fungi, and more preferably E. coli or S. cerevisiae .

When it is desired to express the α-1,3-fucosyltransferase gene in the host cell, the α-1,3-fucosyltransferase gene is preferably codon-optimized (codon) to facilitate expression in the host cell. optimization), and an appropriate expression control sequence can be selected as described above. For example, the host cell may be yeast, such as S. cerevisiae , and in this case, the α-1,3-fucosyltransferase gene may include the base sequence of SEQ ID NO: 2.

Another aspect of the present invention provides 3-fucosyllactose produced using the above-mentioned microorganism.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example 1. Discovery of a new α-1,3-fucosyltransferase (3-FT) gene for the production of 3-fucosyllactose (3-FL)>

1) In silico discovery and expression construct for α-1,3-fucosyltransferase (α-1,3-fucosyltransferase, hereinafter referred to as 3-FT) candidate

Representatively known 3-FT is from Helicobacter pylori and Bacteroides nordii. These bacteria are all pathogenic bacteria of BSL2 or higher. Recently, 3-FT enzyme from Azospirillum brasilense of BSL1 was discovered, and the enzyme's Using the sequence, 382 candidate sequences were initially obtained from the database of the U.S. National Center for Biotechnology Information (NCBI).

From this, excluding sequences with potential risk of conflict and sequences derived from pathogenic organisms above BSL2, the amino acid sequence was compared with the H. pylori- derived enzyme for which the active site and substrate binding site were known, and sequences in which important amino acid residues were conserved were identified. Several factors, including enzymes predicted to be located in the cytoplasm in yeast cells, were identified using the DeepLoc software of the Technical University of Denmark, with high homology in the active site and substrate binding site with 3-FT enzymes that were experimentally confirmed to be active. Ten genes selected after a comprehensive review were synthesized according to yeast preferred codons, and one gene from one strain available from a domestic resource bank was amplified by PCR.

2) Confirmation of expression and activity of 3-FT in E. coli and yeast

3-FT candidate enzymes were expressed in E. coli ( E. coli BL21Δ ), then reacted with lactose and GDP-fucose in vitro with the E. coli cell lysate expressing each enzyme, and the substrate was identified using TLC (Thin Layer Chromatography). -The result of checking whether it could be converted to Foucault room lactose was as shown in Figure 1. At this time, green and white in Figure 1 indicate TLC observed under UV and general white light, and GDP-fucose appears clearly at the UV wavelength. Through this, it was confirmed that in the case of E. coli expression, only Pj3ft, derived from Parabacteroides johnsonii, showed the same 3-FT activity as the positive control, futA .

Next, an in vivo experiment was performed by culturing 50 ml of yeast transformed with a 2μ vector into which the centromeric vector (pRS416_EcGMD_EcGMER_Lac12) and the FT gene was inserted for 72 hours. In this case, only Pj3ft (3FT-2 in Table 1) was 3- It was confirmed that it exhibited FT activity (Table 1). At this time, the activity was confirmed using the L-fucose assay kit.

gene derived organism active result E. coli
( in vivo ) leaven
( in vivo ) futA H. pylori + - 3FT-1 Fusicatenibacter saccharivorans MGYG-HGUT-00251 - - 3FT-2 Parabacteroides johnsonii 82F11 + + 3FT-3 Helicobacter himalayensis DSM28742 - - 3FT-4 Helicobacter himalayensis DSM28742 - - 3FT-5 Flavobacterium saccharophilum DSM1811 - - 3FT-6 Bacteroides reticulotermitis KCTC15367 - - 3FT-7 Cyclobacterium xiamenense KCTC32253 - - 3FT-8 Algoriphagus locisalis DSM23445 - - 3FT-9 Akkermansia glycaniphila DSM100705 - - 3FT-10 Gramella aestuarii JCM17790 - - 3FT-11 Anaeromyxobacter dehalogenans DSM21875 - -

At this time, the amino acid sequence and nucleic acid sequence of the introduced Pj3ft gene were in the same order as SEQ ID NO: 1 and SEQ ID NO: 2 (Table 2).

amino acid sequence MKTIKIYFVDFWSGFDPIDNFFTKLLSVKYNVIIDPVSPDYVFYSCFSFNIYKYPNAVKIYFTGENDVPNFNLADYALGFYYIDFEDRYLRFPLYLLDHYSWNDLDTLSSKSVSSNLVNRKFCNFVYSNKKNADPIRDKFFFELSKYKKVDSGGRLYNNIGGPVKDKRAFLRDYKFTIAFENSLVNGYTTEKVIEPMLVNSIPIYWG NKLIGKDFNKSSMLVVEDEKDFGKIIDKIRFLDENDDAYLSKLRENWFVRVNEKEYWKCLFIDFFDNIFSQDVKKAKRCPQYGFVKNLLKEEERVSYLKKNYLWDKMWGLFDRLRNKK* nucleic acid sequence ATGAAAACAATCAAGATATATTTTGTTGACTTTTGGAGTGGATTCGACCCGATTGACAATTTCTTTACCAAAATTATTATCCGTTAAATATAACGTAATAATAGACCCCGTAAGTCCTGATTATGTGTTTTATAGTTGCTTTTCTTTTAACATCTACAAATATCCTAACGCAGTTAAAATATATTTTACCGGGGAAAACGATGTCCCCAATTTCAATCTTGCCGATTACGCTTTTGGGCTTCTACTATATCGACTTTGAGGATAGGT ATTTGAGATTCCCTCTGTACCTGTTGGATCATTACTCCTGGAACGATCTTGACACGCTTAGCTCCAAATCAGTCAGTAGTAACTTAGTGAACAGAAAATTCTGTAATTTCGTCTACAGCAACAAGAAGAACGCCGACCCAATCCGTGACAAATTCTTCTTCGAACTTAGTAAGTATAAGAAAGTGGACTCAGGCGGTCGTCTTTACAATAATATAGGGGGTCCGGTCAAGGACAAGAGGGCCTTCTTGAGGGACTATAAATTTACAATA GCGTTTGAGAATAGTCTTGTTAATGGGTATACGACCGAAAAGGTGATAGAGCCCATGTTGGTGAACTCTATACCGATTTACTGGGGAAACAAGTTGATAGGTAAAGACTTTAATAAATCTTCAATGCTTGTTGTGGAAGACGAAAAGGATTTTGGCAAAATTATCGACAAGATAAGGTTTCTGGACGAGAATGACGATGCTTATCTAAGCAAACTAAGGGAGAACTGGTTGTGCGTGTGAACGAGAAGGAATATTGGTGAAA TTTATTTATTGATTTCTTCGATAATATATTTTCCCAGGACGTAAAGAAGGCTAAGAGGGTGTCCTCAATACGGGTTTCGTAAAGAATTTACTTAAAGAGGAGGAGCGTGTGAGCTACCTAAAAAAAAAAATTATCTGTGGGATAAAATGTGGGGGTTATTTGACCGTCTGAGAAACAAAAAGTAA

<Example 2. Construction of an expression system for production of 3-foucault room lactose>

1) Yeast capable of synthesizing GDP-fucose and absorbing lactose ( S. cerevisiae )Manufacture of

To prepare yeast ( S. cerevisiae ) for the production of 3-FL, first make GDP-fucose, a biosynthetic precursor of 3-FL, and then establish a metabolic pathway to absorb lactose, another precursor of 3-FL. Engineered recombinant yeast was prepared (except Pj3ft in Figure 2). In more detail, yeast ( S. cerevisiae ) can synthesize GDP-mannose, so it has a GDP-D-mannose-dehydratase gene that can convert it into GDP-4-keto-6-deoxymannose and this again into GDP. -Yeast capable of synthesizing GDP-fucose and absorbing lactose by introducing a GDP-L-fucose synthase gene that can be converted to fucose, and then introducing a lactose permease gene that can introduce lactose into yeast cells. ( S. cerevisiae ). In this process, gmd , a GDP-D-mannose-dehydratase from E. coli, gmer, a GDP-L-fucose synthase from E. coli, and lac12 genes from Kluyveromyces lactis , a yeast with excellent lactose utilization, were obtained from the corresponding genome. It was introduced into yeast ( S. cerevisiae ), and the nucleic acid sequences of the three introduced genes are shown in Table 3.

gmd ATGTCCAAAGTTGCGTTAATCACTGGCGTGACAGGGCAAGATGGTTCATACCTAGCCGAATTTTTACTGGAAAAAGGGTACGAAGTACACGGCATTAAGAGGCGTGCAAGCTCCTTTAACACTGAACGTGTGGATCACATATATCAGGACCCACACACGTGCAACCCGAAGTTTCATCTGCATTACGGCGACTTGTCAGACACCAGTAACTTAACTAGGATTTTGAGAGAGGTTCAGCCTGATGAGGTCTATAATCTGGGGTG CAATGAGCCACGTGGCAGTTTCTTTTGAGAGCCCCGAATACACGGCCGACGTTGATGCAATGGGAACGTTAAGGTTACTTGAGGCGATTAGGTTCTTAGGTTTGGAAAAGAAGACCAGGTTTTTACCAGGCAAGTACAAGTGAACTATATGGACTGGTCCAAGAAATCCCCCAGAAAGAAACGACCCCTTTCTATCCTAGGAGCCCTTACGCGGTTGCCAAATTATACGCCTACTGGATTACCGTCAATTACCGTGAGTCTTAC GGGATGTACGCTTGCAATGGGATACTATTCAATCATGAAAGTCCGCGTAGAGGAGAAACGTTTGTCACAAGAAAAATCACCAGGGCCATAGCTAATATTGCCCAAGGGTTGGAGTCATGCCTATACTTAGGGAATATGGATTCTTTAAGAGACTGGGGGCCATGCCAAAGATTACGTTAAGATGCAATGGATGATGCTGCAACAGGAGCAACCCGAGGATTTCGTCATAGCTACAGGGGTGCAGTACTCTGTCAGACAGTTCGTGG AGATGCCGCCGCCCAATTGGGTATAAAATTGAGATTCGAGGGCACTGGAGTGGAAGAGAAGGGTATTGTGGTATCTGTCACGGGACACGACGCTCCAGGCGTAAAGCCCGGTGATGTAATAATTGCGGTAGATCCCAGATACTTTCGTCCGGCTGAAGTGGAAACCCTTCTTGGGGACCCAACTAAGGCGCACGAGAAATTAGGGTGGAAGCCTGAGATAACACTAAGAGAAATGGTTAGCGAAATGGTCGCGAACGACTTA GAAGCTGCGAAGAAACACAGTCTACTAAAATCTCATGGATATGACGTTGCAATAGCACTTGAGTCCTAA gmer ATGAGCAAACAAAGGGTCTTCATTGCTGGTCACCGTGGGATGGTGGGTAGCGCAATTCGTAGGCAGTTGGAACAGAGGGGAGATGTGGAGCTAGTTCTTAGGACTAGGGACGAGCTTAACTTGCTTGACTCCAGGGCAGTACACGATTTCTTCGCAAGTGAAAGGATTGACCAGGTGTATTTGGCTGCTGCAAAGGTAGGGGGTATAGTCGCAAATAACACTTACCCGGCCGACTTCATTTACCAGAATATGATGATTGAATCAAATA TCATACACGCCGCCCACCAAAACGACGTGAACAAACTATTGTTCTTAGGTAGTTCCTGCATCTACCCTAAGTTGGCAAAACAGCCAATGGCCGAAAGTGAACTGCTGCAGGGCACTTTGGAGCCTACTAACGAACCGTATGCCATTGCCAAGATAGCGGGCATTAAATTATGTGAATCATACAATAGGCAATATGGAAGGGATTATAGAAGCGTAATGCCAACGAATTTATACGGACCCCATGACAACTTTCATCCATCAAATAGCCACGTAATACC CGCGCTGCTTCGTCGTTTTCACGAAGCGACAGCTCAGAATGCCCCGGACGTGGTGGTATGGGGATCAGGGACCCCAATGAGAGAATTTCTACACGTAGATGATATGGCTGCTGCAAGTATTCACGTCATGGAACTAGCACACGAAGTCTGGCTGGAGAACACGCAACCCATGTTGAGCCACATTAACGTAGGCACCGGCGTAGATTGCACGATCCGTGAATTAGCGCAGACTATCGCTAAGGTCGTGGGGGTATAAGGG GAGGGTCGTGTTTGATGCTTCAAAACCAGACGGCACCCCTCGTAAGTTGCTAGATGTCACTCGTCTTCATCAATTAGGTTGGTATCATGAAATTTCACTGGAGGGCGGGATTAGCCAGTACCTATCAATGGTTTTTGGAGAATCAAGACCGTTTTAGAGGTTAA lac12 ATGGCAGATCATTCGAGCAGCTCATCTTCGCTGCAGAAGAAGCCAATTAATACTATCGAGCATAAAGACACTTTGGGCAATGATCGGGATCACAAGGAAGGCCTTGAACAGTGATAATGATAATACTTCTGGATTGAAAATCAATGGTGTCCCCATCGAGGACGCTAGAGAGGAAGTGCTCTTACCAGGTTACTTGTCGAAGCAATATTACAAATTGTACGGTTTATGTTTTATAACATATCTGTGTGCTACTATGCAAGGTT ATGATGGGGCTTTAATGGGTTCTATCTATACCGAAGATGCATATTTGAAATACTACCATTTGGATTTAACTCATCCTCTGGTACTGGTCTAGTGTTCTCTATTTTCAACGTTGGTCAAATTTGGCGGTGCATTCTTTGTTCCTCTTATGGATTGGAAAGGTAGAAAACCTGCTATTTTAATTGGGTGTCTGGGTGTTGTTATTGGTGCTATTATTTCGTCTTTAACAACAACAAAGAGTGCATTAATTGGTGGTAGATGGTTCGTGGCC TTTTTCGCTACAATCGCTAATGCAGCAGCTCCAACATACTGTGCAGAAGTGGCTCCAGCTCACTTAAGAGGTAAGGTTGCAGGTCTTTATAACACCCTTTGGGCTGTCGGTTCCATTGTTGCTGCCTTTTCCACTTACGGTACCAACAAAAACTTCCCTAACTCCTCCAAGGCTTTTAAGATTCCATTATACTTACAAATGATGTTCCCAGGTCTTGTGTGTATATTTGGTTGGTTAATCCCAGAATCTCCAAGATGGTTGGTTGGTGTT GGCCTGAGGAAGAAGCTCGTGAATTCATTATCAAATACCACTTAAATGGCGATAGAACTCATCCATTATTGGATATGGAGATGGCAGAAATAATAGAATCTTTCCATGGTACAGATTTATCAAACCCTCTAGAAATGTTAGATGTAAGGAGCTTATTCAGAACGAGATCGGATAGGTACAGAGCAATGTTGGTTATACTTATGGCTTGGTTCGGTCAATTTTCCGGTAACAATGTGTGTTCGTACTATTTGCCTACCATGTTGAGAAA TGTTGGTATGAAGAGTGTCTCATTGAATGTGTTAATGAATGGTGTTTTATTCCATCGTCACTTGGATTTCTTCAATTTGCGGTGCATTCTTTATTGATAAGATTGGTAGAAGGGAAGGTTTCCTTGGTTCTATCTCAGGTGCTGCATTAGCATTGACAGGTCTATCTATCTGTACTGCTCGTTATGAGAAGACTAAGAAGAAGAGTGCTTCCAATGGTGCATTGGTGTTCATTTATCTCTTTGGTGGTATCTTTTTTTTTT GCTTTCACTCCAATGCAATCCATGTACTCAACAGAAGTGTCTACAAACTTGACGAGATCTAAGGCCCAACTCCTCAACTTTGTGGTTTCTGTGTTGCCCAATTTGTTAATCAATTTGCTACTCCAAAGGCAATGAAGAATATCAAATATTGGTTCTATGTGTTCTACGTTTTCTTCGATATTTTCGAATTTATTGTTATCTACTTCTTCTTCGTTGAAACTAAGGGTAGAAGCTTAGAAGAATTAGAAGTTGTCTTTGAAGCTTCAA ACCCAAGAAAGGCATCCGTTGATCAAGCATTCTTGGCTCAAGTCAGGGCAACTTTGGTCCAACGAAATGACGTTAGAGTTGCAAATGCTCAAAATTTGAAAGAGCAAGAGCCTCTAAAGAGCGATGCTGATCATGTCGAAAAGCTTTCAGAGGCAGAATCTGTTTAA

2) Pj3ft Confirmation of 3-FL production in yeast through gene introduction

The Pj3ft gene (Table 2) discovered in Example 1 was introduced (Figure 2) into yeast ( S. cerevisiae ) capable of synthesizing GDP-fucose and absorbing lactose (Figure 2) to confirm whether 3-FL could be produced. At this time, the yeast ( S. cerevisiae ) strains used were BY4741 and Y2805, which were prepared by introducing a centromeric vector (pRS416_EcGMD_EcGMER_Lac12) and a 2μ vector into which 3FT was inserted. The promoter used for the expression of Pj3ft was pGAL10 pGAL10 or pGAPDH, which are known to induce high expression. As a control, yeast into which futA was introduced instead of Pj3ft was used and cultured in 50 ml YPD medium at 30°C for 72 hours. At this time, lactose was supplied four times at 48 hours of culture, with a final dosage of 2 g/L. It was done as much as possible.

futA showed excellent activity in E. coli, but when expressed in yeast ( S. cerevisiae ), which has the ability to synthesize GDP-fucose and absorb lactose, there were cases where it was not expressed depending on the yeast strain, and expression was confirmed by Western blot. The target substance, 3-FL, was not detected in all of the strains used in the experiment, including (Figure 3 and Table 4). On the other hand, the Pj3ft gene from Parabacteroides johnsonii (3FT-2 in Figure 3 and Table 4) was confirmed to be expressed in all strains used for expression in the recombinant yeast capable of producing GDP-fucose, and was expressed in 3-FL, the target substance. It was confirmed that .

strain promoter enzyme(source) manifestation 3-FL generation
(mg/gDW) S. cerevisiae BY4741 pGAL10 futA (H. pylori) - n.d. 3FT-2 (P. johnsonii) + n.d. pGAPDH futA (H. pylori) - n.d. 3FT-2 (P. johnsonii) +++ n.d. S. cerevisiae Y2805 pGAL10 futA (H. pylori) +++ n.d. 3FT-2 (P. johnsonii) + 0.024 pGAPDH futA (H. pylori) - n.d. 3FT-2 (P. johnsonii) +++ 0.12 *n.d. Not detected

Additionally, the growth rate and 3-FL production were checked at different lactose injection times for Y2805 and CEN.PK2-1C strains introduced with centromeric vector (pRS416_EcGMD_EcGMER_Lac12) and 2μ vector inserting 3FT (Table 5). At this time, the time of lactose introduction was divided into 8 hours and 24 hours after culture, and the culture was finally continued for 80 hours. At this time, 3-FL present outside and inside the cells were analyzed separately. The culture medium was centrifuged and divided into a supernatant and a pellet, and the outside of the cells was analyzed using the L-fucose assay kit for the supernatant. Inside the cells, after a washing step of resuspending the pellet in distilled water and repeating the centrifugation process twice, the obtained pellet was resuspended in distilled water, heated in boiling water for 10 minutes to disrupt the cells, and cooled in ice water for 2 minutes. , the lysate was centrifuged and the supernatant was analyzed using an L-fucose assay kit.

code name strain Timing of lactose addition Y-8h-80 S.cerevisiae Y2805 8 hours after incubation Y-24h-80 S.cerevisiae Y2805 24 hours after incubation C-8h-80 S.cerevisiae CEN.PK2-1C 8 hours after incubation C-24h-80 S.cerevisiae CEN.PK2-1C 24 hours after incubation

As a result, it was confirmed that both strains could produce 3-FL. However, more 3-FL is released into the culture medium in Y2805 than in CEN.PK2-1C, with approximately 38 to 62% of 3-FL present in the culture medium, and the total 3-FL production is also higher in Y-2805, so the two strains It was found that there was a difference in productivity (Figure 4).

3) Optimization of yeast strains for 3-fucosyllactose production

S. cerevisiae is traditionally the most commonly used yeast strain, and genetic manipulation methods have been developed, allowing a variety of gene expression vector/host cell systems to be used. The S. cerevisiae strain for producing 3-fucosyllactose according to the present invention can be obtained by introducing at least four foreign genes, including the above-described gmd , gmer , lac12 , and Pj3ft , and through this genetic manipulation, a stable and high 3 -We attempted to manufacture it to show the yield of Foucault room lactose. To achieve this, first select an appropriate high-efficiency expression promoter, and establish a gene expression system using an appropriate auxotrophic mutant strain to enable transformation with the auxotrophic selection marker of the plasmid, as at least two or more plasmids are required to accommodate at least four genes. I wanted to do it.

As highly efficient expression promoters in yeast, constitutive promoters for constitutive expression (GPD, TEF, PGK, etc.) and promoters for inducible expression (GAL1-10, GAL10, etc.) are known depending on the expression mode. First, using these, the three genes 3-FT, gmd , and gmer are 1) 3-FT, gmd , and gmer are all connected to the GAL1-10 promoter, 2) 3-FT is connected to the GAL1-10 promoter, and gmd and gmer are connected to the GPD promoter. 3) All three enzymes were linked to the GPD promoter and introduced into yeast, and the amount of 3-fucosyllactose biosynthesis of these recombinant yeasts was measured and compared using the L-fucose assay kit (Megazyme). The results showed that there was no difference between these two promoters (Figure 5).

Next, to select an expression vector, all genes were expressed using only the 2μ (pYEG-HIR525) vector, an episomal plasmid vector (pGPD-3FT-2 and pGPD_Gmd_pGPD_Gmer_pGPD_Lac12). As a result, it was discovered that there was a large difference in the production of 3-FL between transformant clones, ranging from 2 to 65 mg/L. As a result of amplifying the part containing both gmd and gmer on one plasmid by PCR, the production amount of 3-FL increased as culture time passed. It was confirmed that the sensitivity of PCR amplification gradually weakened (Figure 6), and it was found that the above difference was due to the low stability of the vector.

Accordingly, in order to increase vector stability, we tried using a centromere vector, which exists at the level of 1 to 2 copies and has a much lower copy number than the 2μ (pYEG-HIR525) vector, but has high stability. Specifically, 3-FT, which is considered a bottleneck reaction in 3-fucosyllactose biosynthesis, was expressed in a 2μ vector, and gmd , gmer , and lac12 for precursor and substrate supply were expressed in pRS, a centromere vector. At this time, since the number of genes that can be stably expressed in one vector is limited, gmd , gmer , and lac12 can all be expressed in one vector (pRS415) (Figure 7), or gmd and gmer are expressed in pRS416 and lac12 is expressed in pRS416. By allowing pRS415 to be expressed in two different vectors (FIG. 8), yeast were prepared in which a total of four genes, including 3-FT, were introduced through two or three vectors, respectively.

Compared to the case of using only the 2μ vector (pYEG x pYEG), the 2-vector (pYEG In the case of the introduced yeast, as shown in Figure 9 and Table 6, about 180 mg/L of 3-FL, which is about three times higher, was produced, and the relative standard deviation (RSD) value was also found to be significantly reduced from 50% to 1.3%. From this, it was indirectly confirmed that the expression stability of the corresponding enzymes was improved by introducing gmd , gmer , and lac12 into the centromere vector.

average Standard Deviation pYEG x pYEG 64 18.34 pYEG x CEN x gLac12 185.15 15.58 pYEG x CEN 180.41 2.40

Next, we checked whether there was a difference in 3-FL productivity between the 2-vector (pYEG x CEN) and 3-vector (pYEG x CEN x gLac12) systems in which the above three genes except 3-FT were introduced into the centromere vector. As a result, as shown in Figure 10, there was no statistically significant difference in both the growth and 3-FL production of the strain.

Additionally, the yeast strain into which the 3-vector system was introduced was deposited at the Korea Center for Biological Resources (KCTC) under the strain name Saccharomyces cerevisiae FCCL-3 (KCTC14730BP). In detail, this is a strain for 3-FL production prepared by constructing the S. cerevisiae Y2805 Ura-/Leu-/His- strain and introducing 2μ (pYEG-HIR525) and centromere (pRS415, pRS416) vectors.

4) Mass production of 3-fucosyllactose

3-Foucault room lactose production using Saccharomyces cerevisiae FCCL-3, a yeast strain introduced with the 3-vector system, was scaled up from 50 ml to 2.5 L using the same medium and temperature conditions (YPD medium, 30°C). It was carried out in At this time, lactose supply is maintained at a low concentration by fed-batch culture at the point of cell growth (OD ₆₀₀ ) 60-70 (administered from 48 hours so that the total amount of lactose added is 10 g/L). A method was used to increase the productivity of fermentation by using a device to add 0.5 g/L per hour for up to 72 hours, and the same method was used three times.

The yeast strain was cultured for a total of 72 hours, and the final cell growth reached 72 - 87, and the total production of 3-fucosyllactose was 1,200 - 1,650 mg/L in the analysis using the L-fucose assay kit, which was produced in 50 ml of test. It was found that production increased by about 5.7 to 7.8 times compared to the city (Figure 11).

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (12)

α-1,3-fucosyltransferase gene;
GDP-D-mannose-dehydratase gene;
GDP-L-fucose synthase gene; and
A lactose permease gene is introduced,
A microorganism for producing 3-fucosyllactose, wherein each of the genes is operably linked to an expression control sequence.
According to paragraph 1,
The α-1,3-fucosyltransferase gene, the GDP-D-mannose-dehydratase gene, the GDP-L-fucose synthase gene and the lactose permease gene are one or more non-integral A microorganism for producing 3-fucosyllactose, characterized in that it is introduced and included on a (non-integrating) vector.
According to paragraph 1,
The α-1,3-fucosyltransferase gene is on a vector different from the GDP-D-mannose-dehydratase gene, the GDP-L-fucose synthase gene, and the lactose permease gene. Included,
The vector containing the α-1,3-fucosyltransferase gene is a high copy number vector, and the GDP-D-mannose-dehydratase gene and the GDP-L-fucose A microorganism for producing 3-fucosyllactose, wherein the vector containing the synthase gene and the lactose permease gene is one or more low copy number vectors.
According to paragraph 3,
The microorganism for producing 3-fucosyllactose, wherein the low copy number vector is a centromere vector.
According to paragraph 1,
A microorganism for producing 3-foucault lactose, wherein the α-1,3-fucosyltransferase gene is derived from Parabacteroides johnsonii .
According to paragraph 1,
The α-1,3-fucosyltransferase gene is a microorganism for producing 3-fucosyllactose, characterized in that it contains a base sequence encoding the amino acid sequence of SEQ ID NO: 1.
According to paragraph 1,
The α-1,3-fucosyltransferase gene is a microorganism for producing 3-fucosyllactose, characterized in that it contains the base sequence of SEQ ID NO: 2.
According to paragraph 1,
A microorganism for producing 3-fucosyllactose, wherein the GDP-D-mannose-dehydratase and GDP-L-fucose synthase genes are derived from E. coli.
According to paragraph 1,
A microorganism for producing 3-fucosyllactose, wherein the lactose permease gene is derived from Kluyveromyces lactis .
According to paragraph 1,
A microorganism for producing 3-foucault room lactose, wherein the microorganism is yeast.
A host cell into which an α-1,3-fucosyltransferase gene derived from Parabacteroides johnsonii has been introduced.
3-Foucault room lactose produced using the microorganism according to claim 1.