WO2016153300A1 - 2-fucosyllactose producing mutant microorganisms and method for producing 2-fucosyllactose using same - Google Patents

Abstract

The present invention relates to mutant microorganisms for producing 2-fucosyllactose and a method for producing 2-fucosyllactose using the same and, more specifically, to mutant microorganisms into which a lacZ-modified or removed operon is introduced and at least one gene selected from the group consisting of genes coding FucT2 or variants thereof and genes coding glucose-6-phosphate dehydrogenase (G6PDH) and guanosine-inosine kinase (GSK) is introduced or amplified, and to a method for producing 2-fucosyllactose using the same.

Description

2-fucosyllactose producing mutant microorganisms and a method for producing 2-fucosyllactose using the same

The present invention relates to a 2-fucosyllactose producing mutant microorganisms and a method for producing 2-fucosyllactose using the same, in particular lacZ modified or removed lac To the microorganism into which the operon was introduced, α-1,2 fucosyltransferase, a foreign gene encoding a variant thereof, G6PDH (glucose-6-phosphate dehydrogenase) and GSK (guanosine-inosine kinase) The present invention relates to a mutant microorganism in which one or more selected from the group consisting of coding genes are introduced or amplified and a method for producing 2-fucosyllactose using the same.

Human milk contains more than 200 unique oligosaccharides

milk oligosaccharides (HMO) are present at significantly higher concentrations (5-15 g / L) than milk from other mammals. HMOs have the ability to provide a variety of biological activities that affect human health, such as prebiotic effects that help the growth of intestinal lactic acid bacteria, prevention of pathogen infections, and immune system regulation.

HMO consists of D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and sialic acid (Sia; N-acetyl neuraminic acid [Neu5Ac]). The structure of HMO is so diverse and complex that about 200 isomers with different residues and glycosyl bonds can have different degrees of polymerization (DP 3-20). Despite the structural complexity, HMOs have some common structures. Most HMOs have lactose (Galβ1-4Glc) residues at the ends of the reduction. Gal of lactose is sialated in the form of 3-sialyllactose or 6-sialyllactose with α- (2,3)-and α- (2,6) -bonds, respectively Or by the α- (1,2)-and α- (1,3) -bonds of 2-fucosyllactose (2-FL) or 3-fucosyllactose, respectively. It can be fucosylation in form. About 200 different complex oligosaccharides were found in breast milk, including 137 fucosylated, including the three most abundant oligosaccharides, with a ratio of almost 77%, and the remaining oligosaccharides mostly sialized (39). Corresponds to%. Among them, 2-fucosyllactose is reported to be the major HMO involved in the various biological activities mentioned above, and is known to be the most abundant among oligosaccharides contained in breast milk. Attention has been paid to the availability of. However, since 2-fucosyllactose is currently difficult to mass-produce industrially, galactooligosaccharides or fructooligosaccharides are added to baby food in place of 2-fucosyllactose to expect similar effects. to be.

Production methods of 2-fucosyllactose include direct extraction from breast milk and synthesis by chemical or enzymatic methods. However, the direct extraction method was problematic due to the limitation of breastfeeding and low productivity, and the chemical synthesis method had problems such as expensive substrate, low iso-selectivity and production yield, and use of toxic reagents. In addition, the enzymatic synthesis method has a problem that GDP-L-fucose, which is used as a donor of fucose, is very expensive and the purification cost of fucose transferase is high. Therefore, due to these problems, direct extraction, chemical or enzymatic production methods are not applicable to industrial mass production. On the other hand, biotechnological production methods using microorganisms are suitable for industrial mass production compared to other systems such as chemical synthesis or enzymatic synthesis in that 2-fucosyllactose can be produced in large quantities from a cheap substrate through a simple process. Because of this spotlight.

In previous studies involving microbial HMO production, colanic acid (colanic

Attempts to produce 2-fucosyllactose extracellularly of E. coli designed by overexpressing the positive regulator RcsA for biosynthesis and inactivating the enzyme WcaJ, which uses GDP-L-fucose for collagen acid biosynthesis there was. However, there has been a problem that the production concentration and productivity of 2-fucosyllactose are low.

Under this technical background, the inventors of the present application, unlike the prior art, lac is modified or eliminated lacZ Operon introduction; Mutant microorganisms in which a gene encoding α-1,2-fucosyltransferase or a variant thereof and a gene encoding G6PDH (glucose-6-phosphate dehydrogenase) and a gene encoding a GSK (guanosine-inosine kinase) are introduced or amplified By confirming that 2-fucosyllactose can be produced by a high yield by this, the present invention was completed.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

There have been two problems in producing 2-fucosyllactose (2-FL) using the recombinant E. coli previously developed. The first is E. coli

JM107 or JM109 is used. These strains overproduce biofilm and acetic acid, making it difficult to culture high concentrations of cells. Second, the Helicobacter used in previous studies. The pylori- derived 1,2-fucosyltransferase has a limited titer because it forms an inclusion body upon overexpression. An object of the present invention is to recognize the problems of the existing technology, to provide a mutant microorganism that can improve the final concentration, yield and productivity while efficiently producing HMO and a method for producing HMO using the same. In addition, through high concentration cell culture and improved expression level of fucose transferase to finally develop and provide a technology that can produce 2-fucosyllactose at a higher concentration than the existing technology.

In order to achieve the above object, the present invention is a microorganism having a metabolic circuit that produces 2-fucosyllactose (2-FL), (a) endogenous lac operon is removed, lacZ modified or removed lac Operon introduced; And (b) α-1,2-fucosyltransferase or variant thereof, (c) G6PDH (glucose-6-phosphate

It relates to a mutant microorganism characterized in that the gene encoding at least one selected from the group consisting of dehydrogenase) and (d) GSK (guanosine-inosine kinase) is introduced or amplified.

The variant may be a tag of a gene encoding the 1-7th amino acid of the aspartate repeat sequence or infB (translation initiation factor) at the N-terminus of α-1,2 fucosyltransferase.

The present invention relates to a method for producing 2-fucosyllactose, characterized in that 2-fucosyllactose is recovered from the culture by culturing the mutant microorganisms.

According to the mutant microorganism according to the present invention and a manufacturing method using the same, it is possible to produce 2-fucosyllactose from lactose in a very good yield to overcome the limitations of the prior art that the production yield is not applicable in the industrial aspect. This facilitates mass production for industrialization, it can be suitably applied to the food or pharmaceutical industry.

Figure 1 shows the structural composition of 2-fucosyllactose (2'-fucosyllactose, 2-FL).

2a is endogenous lac In recombinant E. coli it was disrupted by the lacZ operon and introduction of shredded la c operon (lacZΔM15) section, illustrating a de novo pathway for the biosynthesis of GDP-L-fucose, and 2-Foucault room lactose.

2b is endogenous lac In recombinant E. coli was disrupted the lacZ operon and completely crushing the c la introducing an operon (lacYA), it illustrates the de novo pathway for the biosynthesis of GDP-L-fucose, and 2-Foucault room lactose.

3 is lac Schematic diagram showing the process for constructing recombinant E. coli strains engineered with operon [(a) Wild type E. coli-BL21star (DE3), (b) lac Removal of the E. coli operon - △ L, (c) the introduction of E. coli M15 lac △ - △ L M15, (d) the introduction of E. coli lacYA - △ L YA].

Figure 4 shows the batch culture results of the recombinant E. coli strains, optical mill

When the degree (OD600) reached about 0.8, IPTG and lactose were added so that the final concentrations were 0.1 mM and 20 g / L (vertical), respectively: (a) BL21star (DE3) BCGW-F, (b) ΔLBCGW- F, (c) ΔL M15 BCGW-F. The symbols in the graph are as follows, and the symbols in FIG. 4 represent representative values of three independent batch cultures: ●: dry cell weight, ■: lactose, ▲: 2-fucosyllactose.

Figures 5a-5f shows the results of fed-batch culture of recombinant E. coli strains, after all of the initial 20 g / L glycerol was consumed, and began to supply glycerol to pH-stat, IPTG and lactose added simultaneously (Large arrow). An additional 200 g / L lactose solution was added after lactose depletion (small arrow) and the glycerol feed mode was changed to manual mode after the dry cell weight reached about 60 (vertical line). The symbols in Fig. 5 are as follows: ●: dry cell weight, ■: lactose, ▲: 2-fucosyllactose, ◆: glycerol.

Figure 5a shows the fed-batch culture of ΔL M15 BCGW-F recombinant E. coli,

Figure 5b shows the fed-batch culture of ΔL M15 BCGW-Fz recombinant E. coli,

Figure 5c shows the results of fed-batch culture of ΔL M15 BCGW-Fg recombinant E. coli,

Figure 5d shows the fed-batch culture of ΔL M15 BCGW-D3F recombinant E. coli,

Figure 5e shows the results of fed-batch culture of ΔL M15 BCGW-infBF recombinant E. coli,

Figure 5f shows the results of fed-batch culture of ΔL M15 BCGW-W recombinant E. coli,

Figure 5g shows the fed-batch culture results of ΔL YA BCGW-W recombinant E. coli.

Figure 6 is the result of confirming the production of 2-fucosyllactose in the culture medium of ΔL M15 / pmBCGW + pHwcfB through LC-MS / MS analysis.

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. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention, in a microorganism having a metabolic circuit producing 2-fucosyllactose (2-FL), (a) endogenous lac Lac with operon removed and lacZ modified or removed Operon introduced; And (b) α-1,2-fucosyltransferase or a variant thereof, (c) G6PDH (glucose-6-phosphate dehydrogenase) and (d) GSK (guanosine-inosine kinase). It relates to a mutant microorganism characterized in that the introduced or amplified.

In order to utilize the Escherichia coli BL21star (DE3), which is suitable for high concentration culture and foreign protein expression, as a strain for producing 2-fucosyllactose, the BL21star (DE3) strain having reduced or eliminated the titer of lactosease (β-galactosidase, LacZ) was used. Built. To this end, the crushed endogenous lac operon of (a) wild-type BL21star (DE3), and, lacZ is removed or modified mutant lac Incorporation of operons significantly reduced or eliminated the titer of β-galactosidase, shifting metabolic flow so that most of the lactose entering the cell was used for the production of 2-fucosyllactose rather than cell growth. .

(b) genes encoding GDP-L-fucose biosynthesis enzymes ( gmd , wcaG , manB , manC ) and genes encoding α-1,2 fucosyltransferase ( fucT2 Or wcfB ) or aspartate repeats or infB at the N-terminus to enhance the fucosylation activity of a variant thereof, eg, α-1,2-fucose transferase. The amino acids 1-7 of the translation initiation factor can be introduced in the form of a tag.

(c) overexpresses genes encoding GDP-L-fucose biosynthetic enzymes ( gmd , wcaG , manB , manC ) and genes encoding G6PDH (glucose-6-phosphate dehydrogenase) and / or Gsk (guanosine-inosine kinase) By introducing ( zwf , gsk ), the supply of GDP-L-fucose, a fucose donor, can be improved.

In addition, (d) Bacteroides, which are more active than the conventional H-1 pylori- derived α-1,2-fucose transferase, are expressed. fragilis Recombinant Escherichia coli can be constructed by discovering and introducing a gene ( wcfB ) encoding a derived α-1,2-fucose transferase.

The microorganism having a metabolic circuit producing 2-fucosyllactose may be a bacteria, preferably E. coli It may be BL21star (DE3). Conventional JM107 and JM109 ( E. coli JM strains, such as K-12 variants, have an F 'plasmid to produce excess biofilm, and when grown at high cell densities, cell growth may be inhibited to increase the rate of product formation. Cell growth inhibition is believed to be caused by high levels of acetate accumulation. In contrast to the JM strain, E. coli The BL21 strain does not have an F 'plasmid, and has fast cell growth, low acetate accumulation, and better glucose utilization due to vigorous sugar metabolism. In addition, the BL21 strain is less sensitive to metabolic stress caused in producing large amounts of recombinant protein. While attempting to produce 2-fucosyllactose in BL21star (DE3), GDP-L-fucose was accumulated in cells, but instead of producing 2-fucosyllactose, assimilate lactose to produce only trace amounts of 2-fucosyllactose. It was. This is because the activity of β-galactosidase of wild type BL21star (DE3) was considered to be too high. In the present invention, in order to allow the metabolic flow of lactose to be used for the production of 2-fucosyllactose rather than going to the cell growth lac By replacing operon, the activity of lactose degrading enzyme (β-galactosidase) was reduced or eliminated by 3% compared to the existing enzyme.

Mutant microorganism according to the present invention is (a) endogenous lac Lac with operon removed and lacZ modified or removed Operon is introduced. Mutant microorganism produced results of lactose assimilation speed shifted to the invention, endogenous lac Of the lacZ operon is modified or removed lac Replacement with operon was found to be effective for 2-fucosyllactose production.

In one embodiment, the lacZ is modified lac The operon may be one in which the gene encoding the 11 th to 42 th amino acids of the amino acid encoding β-galactosidase is deleted, and may be represented as lacZΔM15 . The gene encoding the 11 th to 42 th amino acids in lacZΔM15 may be a nucleotide sequence of 93 bp in length, and a nucleotide sequence of the corresponding portion may be deleted.

Lac including lacZΔM15 Operon E. coli From K-12 strains, for example E. coli JM109 derived, the inventors of the present application is E. coli JM109 strain does not check the moving image as to absorb only the lactose, and the endogenous lac operon in the microorganism E. coli Lac including lacZΔM15 from JM109 As a result of replacing with operon, it was confirmed that the yield of 2-fucosyllactose can be improved by about 3 times or more, compared to the wild type strain. Mutant microorganism produced results of lactose assimilation speed shifted to the invention, endogenous lac Operon lac containing lacZΔM15 Replacement with operon was found to be effective for 2-fucosyllactose production.

Further, if there is BL21star (DE3) Won Kyun (lysogen) for such DE3 as, by acting with lacZΔM15, because the activity of the LacZ up partially revive the α-complementation phenomenon ΔL YA strain was completely removed lacZΔM15 Fig. It was confirmed that the remaining 3% LacZ activity was completely removed.

Based on this, in another embodiment, the lacZ is removed lac The operon was constructed. The lacZ is removed lac Operon lacY And lacA , and may be represented by lacYA . lacYA is to remove the lacZ completely lac Means operon.

As a result of the production of the mutant microorganism according to the present invention which slowed down the lactose assimilation rate, endogenous lac LacZΔM15 operon Or lac containing lacYA Replacement with operon was found to be effective for 2-fucosyllactose production.

In the mutant microorganism according to the present invention, (b) a gene encoding α-1,2 fucosyltransferase is introduced or amplified. Such genes include, for example, Helicobacter pylori, Bacteroides fragilis , Dyadobacter fermentans , Enterococcus faecium DO, Escherichia coli O128: B12 , Helicobacter hepaticus, Lactococcus lactis subsp . Cremoris , Silicibacter pomeroyi , Pedobacter heparinus Or Pedobacter saltans It may be derived from, but is not limited thereto. In view of the appropriate activity for the production of the final product 2- fucosyllactose , preferably fucT2 from Helicobacter pylori Or Bacteroides fragilis The derived wcfB gene can be introduced or amplified.

In the previous study, there was a problem of low activity due to insoluble expression of Candida antarctica- derived lipase in Escherichia coli. It has been reported to be effective [Jung et al . (2011) Bioprocess and Biosystems Engineering. In addition, according to another group of previous studies, infB encoding translation initiation factor II. Activity has been reported to be improved when the 1st to 21st sequences of the gene are attached to the N-terminus of Green fluorescent protein (GFP) [Hansted et al . (2011) Journal of Biotechnology 155 (3): 275-283]. In the present invention, a method of attaching an amino acid tag to α-1,2-fucose transferase derived from H. pylori, which has a problem in expression in previous studies on the production of 2-fucosyllactose, was applied. In one embodiment, the variant is a tag combining the gene encoding the aspartate repeat sequence or the 1-7 amino acids of infB (translation initiation factor) at the N-terminus of α-1,2 fucosyltransferase Can be.

In the mutant microorganism according to the present invention, (c) a gene encoding G6PDH (glucose-6-phosphate dehydrogenase) and / or GSK (guanosine-inosine kinase) is introduced or amplified. The inventors of the present application zwf which is a gene encoding the G6PDH And / or the mutant microorganism into which the gene gsk encoding GSK is introduced or amplified is effective in producing 2-fucosyllactose.

Zwf Genes, for example Escherichia coli (strain K-12), Escherichia coli O157: H7, Escherichia coli O6: H1, Leuconostoc mesenteroides, Saccharomyces cerevisiae , Dictyostelium discoideum , Pseudomonas aeruginosa , Nostoc sp ., Mycobacterium smegmatis , Rhizobium meliloti, Streptococcus pneumoniae serotype 4, Zymomonas mobilis subsp . Mobilis, Bacillus subtilis , Chlamydia pneumonia, Mycobacterium bovis , Mycobacterium tuberculosis, Mycobacterium bovis , Arabidopsis thaliana , Synechococcus elongates, Dickeya dadantii , Aggregatibacter actinomycetemcomitans, Borrelia burgdorferi , Buchnera aphidicola subsp . Acyrthosiphon pisum , Buchnera aphidicola subsp . Schizaphis graminum , Buchnera aphidicola subsp . Baizongia pistaciae , Chlamydia muridarum , Chlamydia trachomatis , Haemophilus influenza, Nostoc punctiforme , Synechocystis sp ., Thermotoga maritime, Treponema pallidum , Helicobacter pylori, Encephalitozoon cuniculi , Kluyveromyces lactis , Schizosaccharomyces pombe , Bacillus thuringiensis , Solanum tuberosum , Takifugu rubripes , or Macropus robustus It may be derived from, but is not limited thereto, preferably may be derived from Escherichia coli K-12.

Gsk Genes, for example Escherichia coli (strain K12), Escherichia coli O157: H7, Escherichia coli O6: H1, may be derived from Exiguobacterium acetylicum , but is not limited thereto, and may preferably be derived from Escherichia coli K-12.

L-fucose is a sugar present in glycoproteinated residues of glycoproteins and glycolipids of HMO and eukaryotes and plays an important role in various types of biochemical recognition processes. To biosynthesize fucosylactose, L-fucose undergoes a sugar-transferring reaction to the receptor lactose through the donor GDP-fucose. There are two pathways for biosynthesis of such fucosyllactose, salvage biosynthesis pathway and de novo biosynthetic pathway.

Salvage synthesis is a method in which L-fucose consumes ATP intracellularly.

It is phosphorylated by kinase (fkp) to form L-fucose-1-phosphate, and it is combined with guanosine triphosphate (GTP) by L-fucose-1-phosphate guanylyl-transferase to form GDP-fucose. Via to produce fucosyllactose from lactose.

De novo synthesis showed that the intermediate of glycolysis, fructose-6-phosphate, was synthesized by mannose-6-phosphate isomerase (ManA) and phosphomannomutase (ManB).

It is metabolized to phosphate and combined with GTP by mannose-1-phosphate guanyltransferase (ManC) to form GDP-D-mannose. Subsequently, GDP-D-mannose 4,6-dehydratase (Gmd) removes water molecules from GDP-D-mannose and GDP-L-fucose synthase (WcaG) is located at the C4 position of GDP-4-keto-6-deoxymannose. Promoting the reduction of the keto group in which the reduced NADPH provides reducing power to produce fucosylactose from lactose via fucosyltransferase after GDP-L-fucose is produced (FIG. 2).

In this regard, the mutant microorganism according to the present invention can synthesize fucosilactose via the de novo biosynthetic pathway. Accordingly, as shown in FIG. 2, ManA

(mannose-6-phosphate isomerase), ManB (phosphomannomutase), ManC (mannose-1-phosphate guanyltransferase), Gmd (GDP-D-mannose dehydratase) and WcaG (GDPfucose synthase) Genes may be further introduced or amplified.

Recombinant vector (plasmid) to be introduced into the mutant microorganism according to the present invention refers to DNA fragment (s), nucleic acid molecules to be delivered into the cell, can be mixed with the plasmid. The plasmid is, for example, the genes of (a) to (c) and manA , manB , manC, gmd , wcaG Includes a gene, for example lacZ pGRG36 + △ M15, pGRG36 + lacYA, pETDuet-1 + manC - manB + gmd - wcaG , pCOLADuet-1 + fucT2 , pCOLADuet-1 + fucT2 + zwf , pCOLADuet-1 + fucT2 + gsk , pCOLADuet-1 + D3 fucT2 , pCOLADuet-1 + infBfucT2 or pCOLADuet-1 + wcfB vector.

The recombinant vector (plasmid) may comprise a promoter and a polynucleotide operably linked to the promoter. As used herein, "promoter" refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular recombinant microorganism. As a promoter, a constitutive promoter or a promoter that induces the expression of a target gene at a specific position or time can be used without limitation. The promoter may be natural, homologous, foreign or heterologous to the recombinant microorganism.

"Operably linked" refers to the parallel of two or more components, where the components are in a relationship such that they act in the intended manner. For example, when a promoter acts to control or regulate the transcription of a linked sequence, it is operably linked to a coding sequence. The operably linked DNA sequence can be contiguous, wherein the two or more coding for secretory leader / signal sequences and polynucleotides that are contiguous and within the reading frame can be conjugated.

As used herein, "introduction" may be carried out by a process related to the protoplast transformation followed by the protoplast preparation and cell wall reproduction. DNA inserted into the protoplasts is inserted into the chromosome of the host cell. The inserted DNA sequence remains stable in the cell. DNA can typically be inserted into homologous, nonhomologous sites in the chromosome. In addition, as used herein, "amplification" refers to a process for increasing the copy number of a DNA sequence, for example, amplification may be performed through Polymerase Chain Reaction (PCR).

In another aspect, the present invention relates to a method for producing 2-fucosyllactose, wherein the mutant microorganism is cultured to produce 2-fucosyllactose, and then 2-fucosyllactose is recovered from the culture medium.

In the present invention, the culturing and recovery of the mutant microorganism can be carried out using a conventionally known culture method, in addition to the specific medium and the specific culture method used in the embodiment of the present invention, the medium of other components can be used, Various methods can be used, such as fed-batch fermentation, batch fermentation, or continuous culture using glycerol and lactose together. Glycerol and lactose can be used simultaneously because the two sugars are transported into the cell through a system that catalyzes non-saccharide phosphorylation. In addition to the biosynthesis of GDP-L-fucose, lactose may be added for cell growth and glycerol fed-batch culture may be performed. This prevents the accumulation of acetate, but also improves the productivity of 2-fucosyllactose while maintaining cell growth through simultaneous assimilation of glycerol and lactose.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Recombinant Strains and Plasmids

E. coli for genetic engineering and 2-fucosyllactose production TOP10 and E. coli

BL21star (DE3) (Invitrogen, Carlsbad, CA, USA) was used respectively. Use of plasmids pmBCGW and pHfucT2 for overexpression of GDP-L-fucose biosynthetic enzymes (ManB, ManC, Gmd and WcaG) and Helicobacter pylori- derived α-1,2 fucosyltransferase (FucT2), respectively It was. In order to further express G6PDH (glucose-6-phosphate dehydrogenase) or GSK (guanosine-inosine kinase), pHfucT2zwf and pHfucT2gsk were prepared, and at the N-terminus of α-1,2-fucose transferase derived from H. pylori Aspartate repeat sequence or infB pHD3fucT2 and pHinfBfucT2 were prepared to bind the 1-7th amino acid of the translation initiation factor in the form of a tag. Bacteroides fragilis , a novel α-1,2-fucose transferase Plasmid pHwcfB was prepared for overexpression of derived WcfB. Gene sequences used in the examples are shown in Table 1 below.

Gene name	SEQ ID NO:
gmd	SEQ ID NO: 1
wcaG	SEQ ID NO: 2
manC	SEQ ID NO: 3
manB	SEQ ID NO: 4
fucT2	SEQ ID NO: 5
D3fucT2	SEQ ID NO: 6
infBfucT2	SEQ ID NO: 7
zwf	SEQ ID NO: 8
gsk	SEQ ID NO: 9
wcfB	SEQ ID NO: 10
lacZΔM15 lac operon	SEQ ID NO: 28
lacYA lac operon	SEQ ID NO: 29

Gene zwf And gsk were amplified from genomic DNA of E. coli K-12 (ATCC10798) using oligonucleotides of Table 3 via PCR. The amplified genes were cloned into the pHfucT2 plasmid by treatment with the restriction enzymes and the ligase of Table 2, and pHfucT2zwf and pHfucT2gsk were prepared, respectively. To prepare pGlacZΔM15, two DNA fragments were amplified from E. coli K-12 genomic DNA using two pairs of primers P1_M15 lac / P2_M15 lac and P3_M15 lac / P4_M15 lac.

In addition, to prepare pGlacYA, two pairs of primers P1_M15 lac / P2_lacYA

Two DNA fragments were amplified using P3_lacYA / P4_M15 lac. The amplified DNA fragments were cloned into pGRG36 using In-Fusion HD Cloning Kit (TAKARA, Japan), respectively, resulting in pGlacZΔM15 and pGlacYA. All constructs were confirmed through restriction enzyme treatment and DNA sequencing.

Λ-red mediated deletion method using plasmids pKD46, pKD13 and pCP20 [Datsenko et al . (2000) Proceedings of the National Academy of Sciences 97 (12): 6640] endogenous lac on the E. coli chromosome through The operon was deleted. In addition, the process of inserting lacZ ΔM15 onto genomic chromosomes using transposons is described in McKenzie et al . (2006) BMC microbiology 6 (1): 39. All strains, plasmids and oligonucleotides used are listed in Tables 2 and 3.

Strains and Plasmids

Strain	Related Features
E. coli TOP10	F -, mcr A Δ (mrr - hsd RMS - mcr BC) φ80lacZM15 Δ lacX74 recA1 araD139 Δ ( ara-leu ) 7697 galU galK rpsL (Str R ) endA1 nupG
E. coli BL21star (DE3)	F -, ompT, hsdSB (r B - m B -), gal, dcm rne131 (DE3)
ΔL	BL21star (DE3) Δ lacZYA
ΔL M15	BL21star (DE3) Δ lacZYA Tn7 :: lacZM15
ΔL YA	BL21star (DE3) Δ lacZYA Tn7 :: lacYA
pETDuet-1	Two T7 promoters, pBR322 replicon, Amp R
pCOLADuet-1	Two T7 promoters, ColA replicon, Kan R
pGRG36	Tn7 insertion vector, pSC101 replicon, Amp R
pmBCGW	pETDuet-1 + manC - manB ( Nco I / Sac I) + gmd - wcaG ( Nde I / Kpn I)
pHfucT2	pCOLADuet-1 + fucT2 ( Nco I / Sac I)
pGlacZM15	pGRG36 + lacZ Δ M15 ( Sma I)
pGlacYA	pGRG36 + lacYA ( Sma I)
pHfucT2zwf	pCOLADuet-1 + fucT2 + zwf ( Nde I / Pac I)
pHfucT2gsk	pCOLADuet-1 + fucT2 + gsk ( Nde I / Pac I)
pHD3fucT2	pCOLADuet-1 + D3 fucT2 ( Nde I / Kpn I)
pHinfBfucT2	pCOLADuet-1 + infB fucT2 (Nde I / Kpn I)
pHwcfB	pCOLADuet-1 + wcfB ( Nde I / Kpn I)

Primer used

primer  name	order( 5'3 ')	SEQ ID NO:
F_del_lac	CGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGA GTGTAGGCTGGAGCTGCTTCG	SEQ ID NO: 11
R_del_lac	TCCTGCGCTTTGTTCATGCCGGATGCGGCTAATGTAGATCGCTGAACTTG ATTCCGGGGATCCGTCGACC	SEQ ID NO: 12
P1_M15 lac	AATTAATCAGATCCCGGGACCATCGAATGGCGCAAAACCTTTC	SEQ ID NO: 13
P2_M15 lac	GGTGCGGGCCACGACGGCCAGTGAATCCGTAATCA	SEQ ID NO: 14
P3_M15 lac	TGGCCGTCGTGGCCCGCACCGATCGCC	SEQ ID NO: 15
P4_M15 lac	GGCCGCTATTGACCCGGGGCTGTGGGTCAAAGAGGCATGATG	SEQ ID NO: 16
P2_lacYA	TGGATTTCCTGTGTGAAATTGTTATCCGCTCACAATTCC	SEQ ID NO: 26
P3_lacYA	AATTTCACACAGGAAATCCATTATGTACTATTTAAAAAACACAAACTTTTGG	SEQ ID NO: 27
F_NdeI_gsk	GGAATTC CATATG AAATTTCCCGGTAAACGTAA	SEQ ID NO: 17
R_PacI_gsk	C TTAATTAA TTAACGATCCCAGTAAGACTC	SEQ ID NO: 18
F_NdeI_zwf	GGAATTC CATATG GCGGTAACGCAAACAGC	SEQ ID NO: 19
R_PacI_zwf	C TTAATTAA TTACTCAAACTCATTCCAGGAACG	SEQ ID NO: 20
F_ NdeI_D3fucT2	GGAATTC CATATG GATGATGAT GCTTTTAA	SEQ ID NO: 21
F_ NdeI_infBfucT2	GGAATT CATATG ACAGATGTAACGATTAAA GCTTTTAAGGTGGTGCAAATTTGC	SEQ ID NO: 22
R_KpnI_fucT2	G GGTACC ATTAAGCGTTATACTTTTGGGATTTTACCT	SEQ ID NO: 23
F_ NdeI_wcfB	GGAATT CATATG TTATATGTAATTTTACGTGGACGATTAGG	SEQ ID NO: 24
R_KpnI_wcfB	G GGTACC TCACATATTCTTCTTTCTTTTCCATATTAATCGC	SEQ ID NO: 25

* Sequences shown in italics are E. coli Lac on chromosome Means homologous recombination site of operon

* Underlined sequences indicate recognition sites for specific restriction enzymes

* Sequences in bold indicate amino acid tags

Example 2 Culture Conditions and Methods

For spawn, preculture and batch cultures, LB (Luria-Bertani) medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride) containing the appropriate antibiotic (ampicillin 50 μg / mL and kanamycin 50 μg / mL) was used. The test tube was used for the spawn culture, and the preculture and the batch culture were incubated in a 500 mL baffled flask containing 100 mL of LB medium at 25 ° C. and maintained at 250 rpm. In batch culture, when the optical density (OD600) reached 0.8, IPTG (isopropyl-β-Dthiogalactopyranoside) and lactose final concentrations were added to 0.1 mM and 20 g / L, respectively. Fed-batch cultivation for high concentration cell culture was performed with a 1.0 L minimum medium containing 20 g / L of glycerol and appropriate antibiotics [13.5 g / L KH2PO4, 4.0 g / L (NH4) 2HPO4, 1.7 g / L citric acid, 1.4 g / L MgSO4 · 7H2O, 10 ml / L trace element solution (10 g / L Fe (III) citrate, 2.25 g / L ZnSO4 · 7H2O, 1.0 g / L CuSO4 · 5H2O, 0.35 g / L MnSO4 · H2O, 0.23 g / L Na2B4O7.10H2O, 0.11 g / L (NH4) 6Mo7O24, 2.0 g / L CaCl2.2H2O), pH6.8], in a 2.5 L bioreactor (Kbiobiotech, Incheon, Korea). After the initially added glycerol was completely depleted, a feeding solution containing 800 g / L glycerol and 20 g / L MgSO 4 · 7H 2 O was supplied by the pH-stat method. At the same time, 2-fucosyllactose was produced by adding IPTG and lactose to final concentrations of 0.1 mM and 20 g / L to induce T7 promoter-mediated gene expression. For the pH-stat method, when the pH was higher than the set-point due to glycerol depletion, an appropriate amount of influent solution was automatically supplied in the reactor. Also, when the pH of the medium was lower than the set-point, 28% NH 4 OH was automatically added to maintain the pH within a certain range (pH6.78 ~ 6.82). The pH of the medium was measured in real time using a pH electrode (Mettler Toledo, USA). After the dry cell weight (DW) reached about 60, the glycerol feed rate was manually adjusted. Stirring speed was increased from initial 1000 rpm up to 1,200 rpm to prevent dissolved oxygen deficiency and aeration rate was maintained at 2 vvm for the entire incubation time.

Example 3 Determination of Cell and Extracellular Metabolite Concentration

Dry cell weight was measured through optical density and 0.36 conversion constant. Optical density was measured at 600 nm absorbance using a spectrophotometer (Ultrospec 2000, Amersham Pharmacia Biotech, USA) after diluting the sample to maintain the optical density between 0.1-0.5. Concentrations of 2-fucosyllactose, lactose, glycerol, galactose and acetic acid were measured using a Carbohydrate Analysis column (Rezex ROA-organic acid, Phenomenex, USA) and high performance liquid chromatography (RI) equipped with a refractive index (RI) detector (Agilent 1100LC). , USA). A column heated at 60 ° C. was applied to analyze 20 μl of culture medium diluted 10-fold. 5 mM H 2 SO 4 solution at 0.6 mL / min flow rate was used as mobile phase.

[Test Example 1] Evaluation of the effect of lac operon modification on 2-fucosyllactose production in recombinant E. coli

The lactose supplied into the cells is 2-fucosyllactose (2'-) in recombinant E. coli.

fucosyllactose (2-FL) is one of the essential elements for efficient biosynthesis. As shown in FIG. 2A, β-galactosidase encoded by the lacZ gene is an enzyme that degrades glucose and galactose so that lactose can be used for cell growth. In wild-type Escherichia coli, most attempts were made to delete part of lacZ in BL21star (DE3) in order to convert the metabolic flux of lactose used for cell growth into the biosynthetic pathway of 2-fucosyllactose. No production of fucosyllactose was observed. This is considered to not be lacY expression by a frame shift (frame-shift) since the lacY carrying the lactose into the cells form the lacZ and one operon.

On the other hand, the JM109 strain only absorbs lactose into the cell but cannot use it for cell growth, so the endogenous lac of BL21star (DE3) The lac operon comprising only the lac operon or lacYA containing lacZ △ M15 of JM109 derived Replacement with operon was attempted (biosynthetic pathway in FIG. 2B).

Figure 3 shows endogenous lac in BL21star (DE3) strain Fruiting operon, new

lac It is a schematic diagram showing the process of introducing each operon. First, lacA from 20 bp upstream of lacI Endogenous lac up to 40 bp downstream The operon site was deleted by λ-red recombination to prepare a ΔL strain ( lac operon deficient strain). Secondly, lacZ △ M15 or the new operon fragment containing the lacYA was inserted into the glmS gene region by Tn7 substrate transposon, through which △ L M15 strain (lacZ △ M15 knock-in strains) and △ L YA strain (lacYA knock-in strain) was produced.

lac The effect of operon variation on the production of 2-fucosyllactose was determined by pmBCGW and

Three strains of wild type BL21star (DE3), ΔL and ΔL M15 containing pHfucT2 plasmids were evaluated by batch culture. The control strain wild type BL21star (DE3) consumed most of the initial lactose within 75 hours and produced 0.58 g / L 2-FL with a yield of 0.03 g 2-FL / g lactose (FIG. 4A). Since the final dry cell mass was the highest compared to other strains, it was thought that most of the lactose consumed was used for cell growth. In contrast, the ΔL strain consumed only a small amount of lactose (0.06 g / L) and produced 0.04 g / L of 2-fucosyllactose (FIG. 4B), because a small amount of lactose migrated into the cells through simple diffusion. It is considered to be. ΔL M15 strain consumed 1.65 g / L lactose and accumulated 0.15 g / L 2-FL. However, the yield of 0.09 g 2-FL / g lactose in the ΔL M15 strain increased 3.3 fold compared to the control strain (FIG. 4C). As a result, the rate of lactose assimilation of the strain BL21star (DE3) was significantly lowered, which was due to endogenous lac. Replacing operons with modified operons indicates that they are effective for 2-fucosyllactose production. Batch culture results are summarized in Table 4.

Strain name	Dry cell mass (g / L)	Maximum 2-fucosyllactose concentration (g / L)	Lactose Consumption (g / L)	Yield (g 2-fucosyllactose / g lactose
BL21star (DE3) pmBCGWpHfucT2	4.0	0.58	20.5	0.028
ΔL pmBCGWpHfucT2	1.4	0.04	0.06	0.667
ΔLM15 pmBCGWpHfucT2	1.1	0.15	1.65	0.091

[Test Example 2] Evaluation of the effect of co-expression of G6PDH (glucose-6-phosphate dehydrogenase) or GSK (guanosine-inosine kinase) on the production of 2-fucosyllactose

To assess whether further overexpression of cofactor production-related enzymes, such as NADPH, also affects the production of 2-fucosyllactose, 2-FL biosynthetic enzymes (ManB, ManC, Gmd, WcaG and FucT2) in ΔL M15 ) Overexpressed strains and strains supplemented with G6PDH (glucose 6-phosphate dehydrogenase) or GSK (guanosine-inosine kinase), respectively, were incubated in a 2.5 L bioreactor. As shown in FIG. 5A, in the strain overexpressing only 2-FL biosynthetic enzyme in the control strain ΔL M15, the final dry cell mass was 73.1 g / L, and the final concentration of 2.56 g / L 2-fucosyllactose was 0.064. Obtained in g 2-fucosyllactose / g lactose. In addition, as can be seen in Figures 5b and 5c, the recombinant E. coli ΔL M15 strain overexpressing G6PDH or GSK with 2-FL biosynthetic enzymes showed a similar growth pattern compared to the control during the entire culture process. In addition, acetate accumulation was not observed in both fed batches. Yield of 0.095 g 2-FL / g lactose in yielded culture of recombinant E. coli overexpressing G6PDH yielded 3.45 g / L of 2-fucosyllactose, which was compared with the control strain and yield of 2-fucosyllactose. 35% and 48% respectively. In the recombinant Escherichia coli culture overexpressed GSK, yield of 0.02 2 g / L lactose was obtained at 3.52 g / L 2-fucosyllactose, which was compared with the control strain at the concentration and yield of 2-fucosyllactose, respectively. 38% and 33% improvement. In light of these findings, the additional expression of G6PDH or GSK may play an important role in the production of 2-fucosyllactose as well as intracellular GDP-L-fucose production. The results of the fed-batch culture are shown in Table 5.

[Test Example 3] Effect of Amino Acid Fragments Inserted at the N-Terminal of α-1,2-fucose Transfer Enzyme on Production of 2-fucosyllactose

In the previous studies on the production of 2-fucosyllactose, by applying an amino acid tag to the α-1,2-fucose transferase derived from H. pylori, which was problematic in expression, The purpose of this study was to determine the effect on the production of. First, aspartame between the initiation codon (ATG) of FucT2 and the following sequence

A fusion α-1,2-fucose transferase library was constructed in which amino acid sequences such as aspartate and arginine were attached at various lengths. In addition, a strain in which the mutant α-1,2-fucose transferase was attached to the end of the 1st to 21st sequences of infB was also constructed. The recombinant E. coli was constructed by batch-level batch culture. As a result, a combination of three aspartates (GATGATGAT) at the N-terminus and a variant α-1,2-fu bound to the 1st to 21st sequences (ATGACAGATGTAACGATTAAA) of infB were identified. It was confirmed that 2-fucosyllactose was produced in the strain in which the course transfer enzyme was introduced. 5D and 5E show the results of culturing ΔLM15 / pmBCGW + pHD3fucT2 and ΔL M15 / pmBCGW + pHinfBfucT2 by a fed-batch process, respectively. Cell growth and production of 2-fucosyllactose continued to increase as protein expression was induced after glycerol feeding was started with addition of IPTG and lactose at about 22 hours of fermentation. Dry cell mass increased to 71.1 g / L and 73.0 g / L, respectively, and 2-fucosyllactose increased to 6.4 g / L and 6.1 g / L, respectively (about 2.5 and 2.4 times than without tag). increase). This may be due to the enhanced activity of α-1,2-fucose transferase due to the attachment of aspartate and infB tag. The results of the fed-batch culture are shown in Table 5.

[Test Example 4] Effect of the introduction of a novel α-1,2-fucose transferase on the production of 2-fucosyllactose

H. pylori- derived α-1,2-fucose transferase (FucT2), which was used in previous studies, has a problem of showing very low levels of activity since most of them form an inclusion body. Thus, fucT2 from H. pylori To find genes that are more well-expressed, 11 genes estimated to be α-1,2-fucose transferases from different genes were introduced into the △ L M15 strain along with pmBCGW plasmid to build a gene library. Afterwards, the productivity of 2-fucosyllactose was tested through fed-batch culture. Only one of them is Bacteroides It was confirmed that 2- fucosyllactose was produced only in recombinant Escherichia coli introduced with wcfB derived from fragilis . As shown in FIG. 5F, in fed-batch culture, the final dry cell mass was grown to 62.0 g / L, and 2-fucosyllactose was obtained at a final concentration of 13.8 g / L. This is a 5.4-fold improvement over the use of fucT2 from H. pylori , which wcfB provides It is thought that the fucosylation of lactose introduced into the cells was faster because of higher fucosylation activity. The results of the fed-batch culture are shown in Table 5.

[Test Example 5] Effect of removal of residual activity of LacZ on the production of 2-fucosyllactose

In the case of strains with lysogen such as DE3, such as BL21star (DE3), α-complementation phenomenon was observed in which the activity of β-galactosidase partially revived when lacZΔM15 was introduced. This is thought to occur because α-peptide of β-galactosidase is produced in a part of lacZ included in DE3 and ω-peptide is produced in lacZΔM15 . Therefore, by removing lacZΔM15 from the ΔL M15 strain, ΔL YA strain was constructed which completely eliminated the remaining LacZ activity, and the effect of this on the production of 2-fucosyllactose was confirmed by fed-batch culture (FIG. 5g). As a result of fed-batch culture of ΔL YA / pmBCGW + pHwcfB strain, dry cell mass reached 57.6 g / L at 46 hours of culture, 2-fucosilactose concentration was 15.4 g / L, yield was 0.858 g 2-FL / g lactose, yielding a productivity of 0.530 g / L per hour. This is about 2.2-fold and 2.0-fold improvement in yield and productivity of 2-fucosyllactose, respectively, compared to the ΔLM15 / pmBCGW + pHwcfB strain, which retained some β-galactosidase activity. The results of the fed-batch culture are shown in Table 5.

Strain name	Dry cell mass (g / L)	Maximum 2-fucosyllactose concentration (g / L)	Yield (g 2-fucosyllactose / g lactose)	Productivity (g / L / h)
ΔL M15pmBCGWpHfucT2	73.1	2.56	0.064	0.043
ΔL M15pmBCGWpHfucT2zwf	76.7	3.45	0.095	0.050
ΔL M15pmBCGWpHfucT2gsk	72.0	3.52	0.085	0.052
ΔL M15pmBCGWpHD3fucT2	71.1	6.40	0.225	0.118
ΔL M15pmBCGWpHinfBfucT2	73.0	6.10	0.190	0.105
ΔL M15pmBCGWpHwcfB	62.0	13.8	0.393	0.260
ΔL YApmBCGWpHwcfB	57.6	15.4	0.858	0.530

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

1. In microorganisms with metabolic circuits that produce 2-fucosyllactose (2-FL), (a) endogenous lac Lac with operon removed and lacZ modified or removed Operon introduced and

   a gene encoding one or more selected from the group consisting of (b) α-1,2-fucosyltransferase or a variant thereof, (c) G6PDH (glucose-6-phosphate dehydrogenase) and (d) GSK (guanosine-inosine kinase) Or mutant microorganisms, characterized in that amplified.
2. The method of claim 1,

   The lacZ is removed lac Operon lacY And lacA .
3. The method of claim 1,

   Lac lac is modified The operon is a mutant microorganism characterized in that it comprises lacZΔM15 from which a gene encoding the 11th to 42nd amino acids among the amino acids encoding β-galactosidase is deleted.
4. The method of claim 1,

   The microorganism having a metabolic circuit producing 2-fucosyllactose is E. coli Mutant microorganism, characterized in that BL21star (DE3).
5. The method of claim 1,

   The lac Operon E. coli Mutant microorganism, characterized in that derived from K-12 strain.
6. The method of claim 1,

   The gene encoding the G6PDH is Escherichia coli (strain K12), Escherichia coli O157: H7, Escherichia coli O6: H1, Leuconostoc mesenteroides, Saccharomyces cerevisiae , Dictyostelium discoideum , Pseudomonas aeruginosa , Nostoc sp ., Mycobacterium smegmatis , Rhizobium meliloti, Streptococcus pneumoniae serotype 4, Zymomonas mobilis subsp . Mobilis, Bacillus subtilis , Chlamydia pneumonia, Mycobacterium bovis , Mycobacterium tuberculosis, Mycobacterium bovis , Arabidopsis thaliana , Synechococcus elongates, Dickeyadadantii , Aggregatibacter actinomycetemcomitans, Borrelia burgdorferi , Buchnera aphidicola subsp . Acyrthosiphon pisum , Buchnera aphidicola subsp . Schizaphis graminum , Buchnera aphidicola subsp . Baizongia pistaciae , Chlamydia muridarum , Chlamydia trachomatis , Haemophilus influenza, Nostoc punctiforme , Synechocystis sp ., Thermotoga maritime, Treponema pallidum , Helicobacter pylori, Encephalitozoon cuniculi , Kluyveromyces lactis , Schizosaccharomyces pombe , Bacillus thuringiensis , Solanum tuberosum , Takifugu rubripes , or Macropus robustus The mutant microorganism characterized in that it is derived.
7. The method of claim 1,

   The gene encoding the G6PDH is a mutant microorganism, characterized in that zwf .
8. The method of claim 1,

   The gene encoding the GSK is Escherichia coli (strain K-12), Escherichia coli O157: H7, Escherichia coli O6: H1, a mutant microorganism characterized by being derived from Exiguobacterium acetylicum .
9. The method of claim 1,

   The gene encoding the GSK is a mutant microorganism, characterized in that gsk .
10. The method of claim 1,

    The gene encoding α-1,2 fucosyltransferase is Helicobacter pylori, Bacteroides fragilis , Dyadobacter fermentans , Enterococcus faecium DO, Escherichia coli O128: B12 , Helicobacter hepaticus , Lactococcus lactis subsp. Cremoris , Silicibacter pomeroyi , Pedobacter heparinus Or Pedobacter saltans The mutant microorganism characterized in that it is derived.
11. The method of claim 1,

    The gene encoding α-1,2 fucosyltransferase is fucT2 Or wcfB .
12. The method of claim 11,

    The fucT2 is a mutant microorganism, characterized in that from Helicobacter pylori .
13. The method of claim 11,

    The wcfB is Bacteroides fragilis The mutant microorganism characterized in that it is derived.
14. The method of claim 1,

    The variant microorganism characterized in that the tag is coupled to the gene encoding the 1 ~ 7th amino acid of the aspartate (aspartate) repeat sequence or InfB (translation initiation factor) at the N- terminal of α-1,2 fucosyltransferase.
15. The method of claim 1,

    The 2-fucosyllactose is a mutant microorganism, characterized in that synthesized via a de novo synthesis route.
16. The method of claim 13,

    1 selected from the group consisting of manA (mannose-6-phosphate isomerase), ManB (phosphomannomutase), ManC (mannose-1-phosphate guanyltransferase), Gmd (GDP-D-mannose dehydratase), and WcaG (Nucleoside-diphosphate-sugar epimerases) A mutant microorganism characterized in that a gene encoding at least one enzyme is further introduced or amplified.
17. The method for producing 2-fucosyllactose, characterized in that 2-fucosyllactose is recovered from the culture by culturing the mutant microorganism of any one of claims 1 to 16.
18. The method of claim 17,

    The culturing is a method for producing 2-fucosyllactose, characterized in that the value-added culture glycerol addition.

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