WO2016153300A1 - Micro-organismes mutants produisant du 2-fucosyllactose et procédé de production de 2-fucosyllactose les utilisant - Google Patents

Micro-organismes mutants produisant du 2-fucosyllactose et procédé de production de 2-fucosyllactose les utilisant Download PDF

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WO2016153300A1
WO2016153300A1 PCT/KR2016/003013 KR2016003013W WO2016153300A1 WO 2016153300 A1 WO2016153300 A1 WO 2016153300A1 KR 2016003013 W KR2016003013 W KR 2016003013W WO 2016153300 A1 WO2016153300 A1 WO 2016153300A1
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fucosyllactose
lac
mutant microorganism
operon
gene encoding
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Korean (ko)
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서진호
진영욱
김지영
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서울대학교 산학협력단
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12R2001/19—Escherichia coli

Definitions

  • 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, ⁇ -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
  • HMO milk oligosaccharides
  • 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).
  • HMOs have some common structures. Most HMOs have lactose (Gal ⁇ 1-4Glc) residues at the ends of the reduction.
  • Gal of lactose is sialated in the form of 3-sialyllactose or 6-sialyllactose with ⁇ - (2,3)-and ⁇ - (2,6) -bonds, respectively Or by the ⁇ - (1,2)-and ⁇ - (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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • lac is modified or eliminated lacZ Operon introduction; Mutant microorganisms in which a gene encoding ⁇ -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.
  • G6PDH glycose-6-phosphate dehydrogenase
  • GSK guanosine-inosine kinase
  • JM107 or JM109 is used. These strains overproduce biofilm and acetic acid, making it difficult to culture high concentrations of cells.
  • 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.
  • 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.
  • 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) ⁇ -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 ⁇ -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.
  • 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).
  • lacZ ⁇ M15 shredded la c operon
  • 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.
  • FIG. 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
  • 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 ⁇ L M15 BCGW-F recombinant E. coli
  • Figure 5b shows the fed-batch culture of ⁇ L M15 BCGW-Fz recombinant E. coli
  • Figure 5c shows the results of fed-batch culture of ⁇ L M15 BCGW-Fg recombinant E. coli
  • Figure 5d shows the fed-batch culture of ⁇ L M15 BCGW-D3F recombinant E. coli
  • Figure 5e shows the results of fed-batch culture of ⁇ L M15 BCGW-infBF recombinant E. coli
  • Figure 5f shows the results of fed-batch culture of ⁇ L M15 BCGW-W recombinant E. coli
  • Figure 5g shows the fed-batch culture results of ⁇ L YA BCGW-W recombinant E. coli.
  • Figure 6 is the result of confirming the production of 2-fucosyllactose in the culture medium of ⁇ L M15 / pmBCGW + pHwcfB through LC-MS / MS analysis.
  • 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) ⁇ -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.
  • the BL21star (DE3) strain having reduced or eliminated the titer of lactosease ( ⁇ -galactosidase, LacZ) was used.
  • 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 ⁇ -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. .
  • (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)
  • G6PDH glucose-6-phosphate dehydrogenase
  • Gsk guanosine-inosine kinase
  • Bacteroides which are more active than the conventional H-1 pylori- derived ⁇ -1,2-fucose transferase, are expressed.
  • fragilis Recombinant Escherichia coli can be constructed by discovering and introducing a gene ( wcfB ) encoding a derived ⁇ -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.
  • 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.
  • 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 ⁇ -galactosidase of wild type BL21star (DE3) was considered to be too high.
  • lactose degrading enzyme ⁇ -galactosidase
  • 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.
  • 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 ⁇ -galactosidase is deleted, and may be represented as lacZ ⁇ M15 .
  • the gene encoding the 11 th to 42 th amino acids in lacZ ⁇ M15 may be a nucleotide sequence of 93 bp in length, and a nucleotide sequence of the corresponding portion may be deleted.
  • 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.
  • a gene encoding ⁇ -1,2 fucosyltransferase is introduced or amplified.
  • 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.
  • 2- fucosyllactose preferably fucT2 from Helicobacter pylori Or Bacteroides fragilis
  • the derived wcfB gene can be introduced or amplified.
  • 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 ⁇ -1,2 fucosyltransferase Can be.
  • a gene encoding G6PDH (glucose-6-phosphate dehydrogenase) and / or GSK (guanosine-inosine kinase) is introduced or amplified.
  • G6PDH glucose-6-phosphate dehydrogenase
  • GSK guanosine-inosine kinase
  • 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 .
  • 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.
  • L-fucose undergoes a sugar-transferring reaction to the receptor lactose through the donor GDP-fucose.
  • Salvage synthesis is a method in which L-fucose consumes ATP intracellularly.
  • GDP-D-mannose 4,6-dehydratase 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).
  • 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 +
  • the recombinant vector may comprise a promoter and a polynucleotide operably linked to the promoter.
  • promoter refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular recombinant microorganism.
  • 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.
  • a promoter acts to control or regulate the transcription of a linked sequence
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • BL21star (DE3) (Invitrogen, Carlsbad, CA, USA) was used respectively.
  • pHfucT2zwf and pHfucT2gsk were prepared, and at the N-terminus of ⁇ -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 ⁇ -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 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 pGlacZ ⁇ M15 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.
  • 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 TGGATTTCCTGTGTGAAATTGTTATCC
  • LB Lia-Bertani
  • yeast extract 1% sodium chloride
  • antibiotic ampicillin 50 ⁇ g / mL and kanamycin 50 ⁇ g / mL
  • 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.
  • a feeding solution containing 800 g / L glycerol and 20 g / L MgSO 4 ⁇ 7H 2 O was supplied by the pH-stat method.
  • 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.
  • 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.
  • 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 ⁇ l 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.
  • RI refractive index
  • 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.
  • ⁇ -galactosidase encoded by the lacZ gene is an enzyme that degrades glucose and galactose so that lactose can be used for cell growth.
  • BL21star BL21star
  • 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.
  • 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.
  • lacA from 20 bp upstream of lacI Endogenous lac up to 40 bp downstream
  • the operon site was deleted by ⁇ -red recombination to prepare a ⁇ L strain ( lac operon deficient strain).
  • 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.
  • 2-FL biosynthetic enzymes ManB, ManC, Gmd, WcaG and FucT2
  • G6PDH glucose 6-phosphate dehydrogenase
  • GSK guanosine-inosine kinase
  • a fusion ⁇ -1,2-fucose transferase library was constructed in which amino acid sequences such as aspartate and arginine were attached at various lengths.
  • a strain in which the mutant ⁇ -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 ⁇ -1,2-fu bound to the 1st to 21st sequences (ATGACAGATGTAACGATTAAA) of infB were identified.
  • 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 ⁇ -1,2-fucose transferase due to the attachment of aspartate and infB tag.
  • Table 5 The results of the fed-batch culture are shown in Table 5.
  • H. pylori- derived ⁇ -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.
  • fucT2 from H. pylori
  • 11 genes estimated to be ⁇ -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.

Abstract

La présente invention concerne des micro-organismes mutants pour la production de 2-fucosyllactose et un procédé de production de 2-fucosyllactose les utilisant et, plus particulièrement, des micro-organismes mutants dans lesquels un opéron à lacZ modifié ou supprimé est introduit et au moins un gène choisi dans le groupe constitué par les gènes codant pour FucT2 ou des variants de celui-ci et des gènes codant pour la glucose-6-phosphate déshydrogénase (G6PDH) et la guanosine-inosine kinase (GSK) sont introduits ou amplifiés et un procédé de production de 2-fucosyllactose les utilisant.
PCT/KR2016/003013 2015-03-24 2016-03-24 Micro-organismes mutants produisant du 2-fucosyllactose et procédé de production de 2-fucosyllactose les utilisant WO2016153300A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3315610A1 (fr) * 2016-10-29 2018-05-02 Jennewein Biotechnologie GmbH Procédé amélioré de production d'oligosaccharides fucosylés
CN109735479A (zh) * 2019-01-30 2019-05-10 光明乳业股份有限公司 一种合成2’-岩藻糖基乳糖的重组枯草芽孢杆菌及其构建方法与应用
CN110637091A (zh) * 2018-04-04 2019-12-31 高级蛋白质技术公司 利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基乳糖的生产方法
CN111471637A (zh) * 2020-05-08 2020-07-31 江苏华燕集团有限公司 2`-岩藻糖基乳糖高产菌株及其制备方法和用途
CN112625987A (zh) * 2020-12-21 2021-04-09 南通励成生物工程有限公司 一种同时生产2`-岩藻糖基乳糖和d-阿洛酮糖的方法
CN112625990A (zh) * 2020-12-29 2021-04-09 量子高科(广东)生物有限公司 一种合成2`-岩藻糖基乳糖的重组大肠杆菌及其构建方法
CN114317384A (zh) * 2020-09-30 2022-04-12 中国科学院上海高等研究院 生产2’-岩藻糖基乳糖的重组枯草芽孢杆菌及其构建方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110061520A (ko) * 2011-04-15 2011-06-09 서울대학교산학협력단 구아노신-이노신 키나아제의 추가 발현에 의한 재조합 대장균에서 ｇｄｐ-푸코즈의 생산방법
US20130122553A1 (en) * 2010-07-12 2013-05-16 Universiteit Gent Metabolically engineered organisms for the production of added value bio-products
US20140031541A1 (en) * 2012-07-25 2014-01-30 Glycosyn LLC Alpha (1,2) Fucosyltransferases Suitable for Use in the Production of Fucosylated Oligosaccharides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130122553A1 (en) * 2010-07-12 2013-05-16 Universiteit Gent Metabolically engineered organisms for the production of added value bio-products
KR20110061520A (ko) * 2011-04-15 2011-06-09 서울대학교산학협력단 구아노신-이노신 키나아제의 추가 발현에 의한 재조합 대장균에서 ｇｄｐ-푸코즈의 생산방법
US20140031541A1 (en) * 2012-07-25 2014-01-30 Glycosyn LLC Alpha (1,2) Fucosyltransferases Suitable for Use in the Production of Fucosylated Oligosaccharides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEE, WON - HEONG ET AL.: "Whole Cell Biosynthesis of a Functional Oligosaccharide, 2' -Fucosyltactose, using Engineered Escherichia Coli", MICROBIAL CELL FACTORIES, vol. 11, no. e48, 2012, pages 1 - 9, XP021115889 *
ROWLEY, DANIEL L. ET AL.: "Molecular Characterization of the Escherichia Coli K-1 2 Zwf Gene Encoding Glucose 6-Phosphate Dehydrogenase", JOURNAL OF BACTERIOLOGY, vol. 173, no. 3, 1991, pages 968 - 977, XP055316226 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3315610A1 (fr) * 2016-10-29 2018-05-02 Jennewein Biotechnologie GmbH Procédé amélioré de production d'oligosaccharides fucosylés
WO2018077892A1 (fr) * 2016-10-29 2018-05-03 Jennewein Biotechnologie Gmbh Procédé amélioré pour la production d'oligosaccharides fucosylés
US11898185B2 (en) 2016-10-29 2024-02-13 Chr Hansen Hmo Gmbh Process for the production of fucosylated oligosaccharides
CN110637091A (zh) * 2018-04-04 2019-12-31 高级蛋白质技术公司 利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基乳糖的生产方法
JP7075494B2 (ja) 2018-04-04 2022-05-25 アドヴァンスド プロテイン テクノロジーズ コーポレーション シュードペドバクターサルタンス由来フコース転移酵素を用いた２’－フコシルラクトースの生産方法
JP2021514633A (ja) * 2018-04-04 2021-06-17 アドヴァンスド プロテイン テクノロジーズ コーポレーションＡｄｖａｎｃｅｄ Ｐｒｏｔｅｉｎ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｃｏｒｐ． シュードペドバクターサルタンス由来フコース転移酵素を用いた２’−フコシルラクトースの生産方法
EP3736336A4 (fr) * 2018-04-04 2021-11-24 Advanced Protein Technologies Corp. Procédé de production de 2'-fucosyllactose à l'aide de fucosyltransférase dérivée de pseudopedobater saltans
CN109735479A (zh) * 2019-01-30 2019-05-10 光明乳业股份有限公司 一种合成2’-岩藻糖基乳糖的重组枯草芽孢杆菌及其构建方法与应用
CN111471637A (zh) * 2020-05-08 2020-07-31 江苏华燕集团有限公司 2`-岩藻糖基乳糖高产菌株及其制备方法和用途
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