CN117568302A - Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade - Google Patents

Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade Download PDF

Info

Publication number
CN117568302A
CN117568302A CN202311425202.6A CN202311425202A CN117568302A CN 117568302 A CN117568302 A CN 117568302A CN 202311425202 A CN202311425202 A CN 202311425202A CN 117568302 A CN117568302 A CN 117568302A
Authority
CN
China
Prior art keywords
mannose
coli
pet
gdp
transferase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311425202.6A
Other languages
Chinese (zh)
Inventor
张荣珍
邹宇欣
徐岩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN202311425202.6A priority Critical patent/CN117568302A/en
Publication of CN117568302A publication Critical patent/CN117568302A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01132—GDP-mannose 6-dehydrogenase (1.1.1.132)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01271—GDP-L-fucose synthase (1.1.1.271)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01069—Galactoside 2-alpha-L-fucosyltransferase (2.4.1.69)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01063—Polyphosphate--glucose phosphotransferase (2.7.1.63)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07013—Mannose-1-phosphate guanylyltransferase (2.7.7.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/08—Transferases for other substituted phosphate groups (2.7.8)
    • C12Y207/08031—Undecaprenyl-phosphate glucose phosphotransferase (2.7.8.31)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/02—Phosphotransferases (phosphomutases) (5.4.2)
    • C12Y504/02008—Phosphomannomutase (5.4.2.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses efficient synthesis of 2' -fucosyllactose by multi-enzyme cascade catalysis of D-mannose, belonging to the technical field of biocatalysis. The invention successfully constructs a polyphosphate dependent kinase mutant which partially removes feedback inhibition of mannose-6-phosphate and improves enzyme catalytic activity. According to the invention, the vectors for coexpression of xcpc, rfbM, xref, 7LL6 and FutC genes and the vectors for expression of the mutants are simultaneously transformed into escherichia coli, so that the enzyme amount of each reaction step can be flexibly adjusted in one-pot reaction, so that sufficient transformation is realized, the yield of 2' -FL is improved, and a foundation is laid for industrial production of the escherichia coli. At present, the final product yield is 82.7% through optimization of bioconversion reaction conditions, and the yield can reach 201.97g/L, which is the highest level reported at home and abroad at present. The technology belongs to the field of biocatalysis engineering, and lays a foundation for industrial production of the technology.

Description

Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade
Technical Field
The invention relates to efficient synthesis of 2' -fucosyllactose by multi-enzyme cascade catalysis of D-mannose, belonging to the technical field of biocatalysis.
Background
2'-Fucosyllactose (2' -FL) is the highest component in human milk oligosaccharides and has many physiological functions. Besides maintaining the intestinal health of infants, the health care agent plays an important role in the establishment and regulation of the infant immune system and the development of the cerebral nervous system. 2' -FL is linear trisaccharide formed by connecting L-fucose, D-galactose and D-glucose through alpha-1, 2 glycosidic bond and beta-1, 4 glycosidic bond, and has molecular weight of 488.44 and molecular formula of C 18 H 32 O 15 . In recent years, a large number of reports indicate that breast milk oligosaccharides play an important role in promoting the health of infants. Besides the ecological balance of the intestinal canal and the probiotics effects of protecting the intestinal canal health of infants, some oligosaccharides have similar receptor structures with the surface of intestinal mucosa cells, can be used as soluble receptors of intestinal pathogenic bacteria, inhibit the adsorption of viruses, bacteria, fungi and the like on intestinal mucosa epithelial cells, and resist intestinal infection. In addition, breast milk oligosaccharides are also critical for the regulation and establishment of the neonatal immune system and the development of the cerebral nervous system.
2' -fucosyllactose, which is the most abundant component (30%) in human milk oligosaccharides, also has a variety of physiological functions: (1) 2' -FL can be metabolized by intestinal probiotics (such as bifidobacteria) of infants, and the generated metabolites are beneficial to maintenance of the healthy environment of the intestinal tract and promote growth and reproduction of other symbiotic bacteria; (2) 2' -FL can be used as a structural analogue of glycolipid, glycoprotein and the like on intestinal epithelial cells, can be competitively combined with some intestinal pathogenic bacteria, and reduces the infection risk caused by pathogenic bacteria; in addition, 2' -FL has been reported to have a role in preventing diarrhea in infants; (3) 2' -FL can be used as an immune factor, exerting an immunomodulatory effect in the intestinal system; part of 2' -FL can enter blood circulation through internal transportation, and plays an immunoregulatory role in the whole body; (4) 2' -FL also plays a positive role in the brain development process of infants. Due to the application value of the 2'-FL in the aspects of medicines, infant formula milk powder and the like, the 2' -FL rapidly becomes a research hot spot.
Currently, 2 '-fucosyllactose on the market is mainly produced by fermentation, and the final product 2' -FL obtained by biological fermentation has also passed the safety certification by the us Food and Drug Administration (FDA) and the european union food safety agency (EFSA). The known milk powder enterprises nest and Yaban have put the infant formula milk powder added with 2'-FL into the market, and some countries have also proposed the addition limit of 2' -FL in infant milk powder. At present, china is allowed to add 2'-FL as a nutrition enhancer to infant formulas, and how to better realize large-scale commercial production of 2' -FL is the focus of attention.
The 2' -FL can be separated from the breast milk, but the yield is lower due to more oligosaccharide types in the breast milk and complex structure; thus, the production of 2' -FL is mainly dependent on chemical synthesis, enzymatic synthesis, and whole cell synthesis. The chemical synthesis method has more limitations on the problems of complicated steps, severe reaction, high-pressure and high-temperature conditions and the like due to the use of a large amount of organic reagents in the reaction process; in addition, fucose is used as one of the starting materials, and is expensive, so that the cost is high, and the advantage of industrial application is lacking. In general, enzymatic production of 2' -FL requires synthesis by glycosylation reactions in the presence of fucosyltransferase or alpha-L-fucosidase. However, the production of 2' -FL is greatly limited due to the cumbersome steps involved in the acquisition of the precursor GDP-fucose and the high cost. Overall, enzymatic synthesis of 2' -FL still lacks certain application advantages due to the lack of a low cost, safe and mass-available fucosyl donor. Currently, whole cell fermentation methods are widely used to produce more valuable target compounds from inexpensive raw materials, and cell factory methods have also proven to be the most efficient strategy to produce 2' -FL. In general, the production of GDP-fucose by the de novo synthesis pathway mainly involves reactions catalyzed by enzymes such as phosphomannose mutase (xcpc), mannose-1-guanosine transferase (rfbM), GDP-D-mannose-4, 6-dehydratase (Xref), and GDP-L-fucose synthase (7 LL 6). Under the action of heterologously expressed alpha-1, 2-fucosyltransferase (FucT 2), the fucosyl molecule of the donor GDP-fucose is transferred to the lactose reducing end of the acceptor to generate 2' -FL. Efficient accumulation of intracellular GDP-fucose is a key factor in the production of 2' -FL using cell factories.
However, the introduction of enzymes from different organisms into a target host strain faces a number of challenges, including lower or even inactivation of the enzyme for heterologous expression and an imbalance in metabolic flux resulting from the increased metabolic burden on the host. Furthermore, the target pathway may also interfere with complex intracellular environmental metabolites. These factors all affect the improvement of 2' -FL yield. Therefore, how to successfully and heterologously express a target enzyme and make it function, and at the same time, solving the interference between the target pathway and the host is still a problem to be solved.
Enzyme cascades are effective tools for multi-step synthesis in a green sustainable manner in one pot, and natural or non-natural high value chemicals can be synthesized from inexpensive and readily available raw materials by artificially constructing cascades of two or more enzymes. In recent years, developments in synthetic biology, protein engineering, metabolic engineering and DNA sequencing technology have led to rapid developments in multi-step artificial enzyme cascade reactions. Most of the cascade reactions reported so far consist of two to three enzymes. The method is also considered as a value-added process, and materials such as amino acid, saccharides and the like are adopted by the method, which are cheap and easily obtained. The whole cell in-vivo enzyme cascade reaction is used, the catalyst preparation process is simple and low in cost, and a cofactor regeneration system can be provided by using a cell metabolic pathway for the oxidation-reduction reaction step. Due to the unique potential of the multienzyme cascade in green sustainable chemicals production, it has become one of the research directions of great interest in the biocatalysis field.
Disclosure of Invention
In order to solve the problems that the enzyme activity of a heterologously expressed target enzyme is low and the host metabolism is affected to cause low 2' -FL yield at present, the invention improves the metabolic pathway of mannose-2 ' -FL by knocking out genes related to the metabolic degradation of a key intermediate GDP-L-fucose in escherichia coli, strengthens the genes related to 2' -FL synthesis, screens hundreds of genes by database mining comparison, selects a polyphosphate dependent kinase from Arthrobacter sp.I3, combines the reported escherichia coli whole genome sequence with site-directed mutagenesis by homologous modeling, molecular docking and multiple sequence comparison and the like, successfully constructs an enzyme mutant which partially removes the feedback inhibition of mannose-6-phosphate and improves the enzyme catalytic activity, realizes the in vitro production of mannose-6-phosphate, and improves the conversion rate of the enzyme catalytic reaction by a single factor variable.
According to the invention, the recombinant plasmid pET-CMXLF is obtained by coexpression of xcpc, rfbM, xref, 7LL6 and FutC genes on the E.coli expression vector pET21a, and the enzyme amount of each reaction step can be flexibly regulated in one-pot reaction, so that sufficient conversion is realized, the yield of 2' -FL is improved, and a foundation is laid for industrial production of the recombinant plasmid pET-CMXLF. At present, the yield of the final product 2' -FL is 82.7% by optimizing the bioconversion reaction conditions, and the yield can reach 201.97g/L.
The invention provides a polyphosphate dependent kinase mutant, which is obtained by mutating leucine at position 169 of the polyphosphate dependent kinase with an amino acid sequence shown as SEQ ID NO.8 into isoleucine; or the leucine is mutated from isoleucine at position 174 of polyphosphate dependent kinase with the amino acid sequence shown in SEQ ID NO. 8; or by mutating leucine at position 169 of polyphosphate dependent kinase having the amino acid sequence shown in SEQ ID NO.8 to isoleucine and mutating isoleucine at position 174 to leucine.
The method for efficiently preparing mannose-6-phosphate by using the constructed recombinant bacteria can ensure that the yield of the mannose-6-phosphate reaches 98.6 percent at maximum.
The invention provides engineering bacteria for producing 2' -fucosyllactose, which knocks out UDP-glucose lipid carrier transferase gene wcaJ, and overexpresses phosphomannose mutase xcpc, mannose-1-phosphoguanyl transferase rfbM, GDP-mannose-6-dehydrogenase xref, GDP-fucose synthase 7LL6, alpha-1, 2 fucosyl transferase futC from escherichia coli sp.I3 or polyphosphate dependent kinase mutant.
In one embodiment of the invention, the nucleotide sequence of the UDP-glucose lipid carrier transferase gene wcaJ is shown as SEQ ID NO. 1; the amino acid sequences of the phosphomannose mutase xcpc, mannose-1-phosphoguanyl transferase rfbM and GDP-mannose-6-dehydrogenase xref and GDP-fucose synthetase 7LL6 and alpha-1, 2 fucosyl transferase futC are respectively shown as SEQ ID NO. 2-6.
In one embodiment of the present invention, the recombinant vector is a pET-series vector, a PRSF-series vector or a pCDF-series vector as an expression vector.
In one embodiment of the present invention, the recombinant vector uses pET28a plasmid, PRSFDuet1 plasmid or pCDF plasmid as expression vector.
In one embodiment of the invention, the pET28a vector is used to express a polyphosphate-dependent kinase; the pET21a vector was used to express phosphomannose mutase xcpc, mannose-1-phosphoguanyl transferase rfbM, GDP-mannose-6-dehydrogenase xref, GDP-fucose synthase 7LL6, alpha-1, 2 fucosyl transferase futC.
In one embodiment of the invention, the amino acid sequence of the polyphosphate dependent kinase mutant from Arthrobacter sp.I3 is shown in SEQ ID NO. 7.
In one embodiment of the present invention, E.coli is used as the expression host.
In one embodiment of the present invention, E.coli pET-21a, pET-28a or pRSF-Duet1 is used as an expression host.
The invention also provides a recombinant escherichia coli which expresses the parent enzyme polyphosphate-dependent mannose kinase, wherein the recombinant escherichia coli is as follows: escherichia coli BL21/pET-ppgmk.
The invention also provides a recombinant escherichia coli which co-expresses the xcpc, rfbM, xref and 7LL6 genes, wherein the recombinant escherichia coli is as follows: escherichia coli BL21/pET-CMXL.
The invention also provides a recombinant escherichia coli which expresses 1, 2-fucosyltransferase, wherein the recombinant escherichia coli is as follows: escherichia coli BL21/pET-FutC.
The invention also provides a recombinant escherichia coli which co-expresses the xcpc, rfbM, xref, 7LL6 and FutC genes, wherein the recombinant escherichia coli is as follows: escherichia coli BL21/pET-CMXLF.
In one embodiment of the invention, the recombinant strains E.coli BL21/pET-ppgmk, E.coli BL21/pET-CMXL, E.coli BL21/pET-FutC and E.coli BL21/pET-CMXLF are constructed,
The polyphosphate dependent mannose kinase gene ppgmk and the 1, 2-fucosyltransferase FutC are respectively inserted into a vector pET28a to construct recombinant plasmids pET-ppgmk and pET-FutC, the recombinant plasmids pET-ppgmk and pET-FutC are used for transforming competent cells of E.coli BL21 (DE 3), the recombinant strains E.coli BL21/pET-ppgmk and E.coli BL21/pET-FutC are obtained by screening LB plates containing 100 mug/mL kanamycin, the method comprises the steps of constructing a recombinant plasmid pET-CMXL by inserting four enzyme genes into a vector pET21a by taking SD-AS AS a linker through phosphomannose mutant enzyme (xcpc), mannose-1-guanosine transferase (rfbM), GDP-D-mannose-4, 6-dehydratase (Xref) and GDP-L-fucose synthetase (7 LL 6), transforming the recombinant plasmid pET-CMXL into competent cells of E.coli BL21 (DE 3) through LB plate screening containing 100 mu g/mL of ampicillin to obtain a recombinant strain E.coli BL21/pET-CMXL, and constructing the recombinant strain E.coli BL21/pET-CMXLF by the same method, wherein the steps are AS follows:
1) Acquisition of the polyphosphate-dependent mannose kinase gene ppgmk the strain used to call the ppgmk gene (deposited in this laboratory) was Arthrobacter sp. Cloning of polyphosphate-dependent mannose kinase gene ppgmk: the ppgmk gene is obtained by PCR amplification reaction by taking the arthrobacter genome as a template. PCR amplification conditions: pre-denatured at 98 ℃ for 30s, then the following cycle was performed: denaturation at 98℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; extending at 72 ℃ for 10min, and preserving heat at 4 ℃.
The genes for phosphomannose mutase (xcpc), mannose-1-guanosine transferase (rfbM), GDP-D-mannose-4, 6-dehydratase (Xref), and GDP-L-fucose synthase (7 LL 6) were obtained, and the strain for gene transfer (this laboratory deposit) was Escherichia coli BL. Cloning of genes xcpc, rfbM, xref, 7LL 6: the 4 genes are obtained by PCR amplification reaction by taking the escherichia coli genome as a template. PCR amplification conditions: pre-denatured at 98 ℃ for 30s, then the following cycle was performed: denaturation at 98℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; extending at 72 ℃ for 10min, and preserving heat at 4 ℃.
Acquisition of the 1, 2-fucosyltransferase gene FutC the strain used to call the FutC gene (deposited in this laboratory) was Helicobacter pylori. Cloning of the 1, 2-fucosyltransferase Gene FutC: the helicobacter pylori genome is used as a template, and the FutC gene is obtained through PCR amplification reaction. PCR amplification conditions: pre-denatured at 98 ℃ for 30s, then the following cycle was performed: denaturation at 98℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; extending at 72 ℃ for 10min, and preserving heat at 4 ℃.
2) Construction of recombinant plasmid pET-ppgmk: and (3) respectively carrying out double enzyme digestion on the target gene ppgmk and the expression vector pET28a by using restriction enzyme, and connecting the treated DNA fragments through a sticky end to obtain a recombinant plasmid pET-ppgmk with the polyphosphate dependent mannose kinase gene ppgmk.
Construction of recombinant plasmid pET-CBGW: and (3) respectively carrying out double enzyme digestion on the target gene and the expression vector pET21a by using restriction enzyme, and connecting the treated DNA fragments through the sticky ends to obtain a recombinant plasmid pET-CMXL with the target gene.
Construction of recombinant plasmid pET-FutC: and (3) respectively carrying out double enzyme digestion on the target gene FutC and the expression vector pET28a by using restriction enzyme, and connecting the treated DNA fragments through a sticky end to obtain a recombinant plasmid pET-FutC with the polyphosphate dependent mannose kinase gene FutC.
3) E.coli transformed with the recombinant plasmid: 0.5 mu L of recombinant plasmid is taken, competent cells of E.coli BL21 (DE 3) are transformed, the transformation solution is coated on an LB plate containing 100 mu g/mL kanamycin, and the plates are cultured overnight at 37 ℃ in an inverted mode, so that positive clones E.coli BL21/pET-ppgmk and E.coli BL21/pET-FutC are obtained. The transformation solution is coated on an LB plate containing 100 mug/mL of ampicillin, and the transformation solution is cultured at 37 ℃ reversely for overnight to obtain positive clones E.coli BL21/pET-CMXL and E.coli BL21/pET-CMXLF.
The invention also provides a method for producing the 2 '-fucosyllactose, which comprises the step of producing the 2' -fucosyllactose in a fermentation system with mannose as a substrate by taking the engineering bacteria as a fermentation strain.
In one embodiment of the invention, the substrate mannose concentration is between 10mM and 500mM.
In one embodiment of the invention, the substrate mannose concentration is between 10mM and 500mM.
In one embodiment of the invention, the whole cell concentration of the microbial cells or recombinant E.coli is 10-50 mg/ml.
In one embodiment of the invention, the method comprises the steps of:
1) Firstly, culturing microbial cells or recombinant escherichia coli, wherein a culture medium is an LB liquid culture medium, picking single bacterial colonies of the microbial cells or the recombinant escherichia coli, inoculating the single bacterial colonies into 5mL of LB liquid culture medium containing 100 mug/mL kanamycin, and carrying out shaking culture at 200rpm at 37 ℃ for overnight; transferring 500. Mu.L of culture solution into 50mL of LB liquid medium containing 100. Mu.g/mL kanamycin, and shaking culturing at 37 ℃ and 200rpm until OD 600 Adding inducer isopropyl-beta-D-thiogalactoside (IPTG) 0.1mmol/L into the culture, and inducing culture at 30deg.C for 16 hr; centrifugation at 12,000Xg for 10min, collecting thalli, washing twice with normal saline, and collecting to obtain microbial cells or recombinant escherichia coli whole cells;
2) Taking microbial cells or recombinant escherichia coli whole cells as a catalyst, taking mannose as a substrate, wherein a reaction buffer solution is 1mL of 0.1mol/L acetic acid buffer solution with pH of 4.5-6.0, or 1mL of 0.1mol/L phosphoric acid buffer solution with pH of 6.5-7.5, or 1mL of 0.1mol/L Tris-HCl with pH of 8.0-9.0, wherein the mannose concentration of the substrate is 10-500 mM, and the reaction temperature is 20-40 ℃; the concentration of the microbial cells or the whole cells of the recombinant escherichia coli is 10-50 mg/ml, and the reaction is carried out for 16 hours.
3) After the reaction was completed, the reaction mixture was centrifuged to remove solid matters, and the 2' -FL content was measured using High Performance Liquid Chromatography (HPLC) to calculate the conversion. Detection was performed by means of an Aminex HPX-87H organic acid column. The mobile phase was 5mM dilute sulfuric acid, the flow rate was 0.6mL/min, and the column temperature was 65 ℃. The signal was detected by a differential detector at a temperature of 40 ℃.
4) Product 2' -FL conversion calculation method: conversion (%) =cs/c0×100%.
Wherein C0 is the concentration of mannose before the reaction, and CS is the concentration of 2' -FL after the reaction.
The invention also provides a method for improving the yield of 2' -fucosyllactose produced by escherichia coli, which comprises the steps of knocking out a UDP-glucose lipid carrier transferase gene wcaJ in escherichia coli, and overexpressing phosphomannomutase xcpc, mannose-1-phosphoguanyl transferase rfbM, GDP-mannose-6-dehydrogenase xref, GDP-fucose synthase 7LL6, alpha-1, 2 fucosyl transferase futC and polyphosphate dependent kinase from Arthrobacter sp.I3.
In one embodiment of the invention, the nucleotide sequence of the UDP-glucose lipid carrier transferase gene wcaJ is shown as SEQ ID NO. 1; the amino acid sequences of the phosphomannose mutase xcpc, mannose-1-phosphoguanyl transferase rfbM and GDP-mannose-6-dehydrogenase xref, GDP-fucose synthetase 7LL6 and alpha-1, 2 fucosyl transferase futC are respectively shown as SEQ ID NO. 2-6; the amino acid sequence of the polyphosphate dependent kinase from Arthrobacter sp.I3 is shown in SEQ ID NO. 7.
The invention also provides application of the engineering bacteria in producing 2 '-fucosyllactose and products containing the 2' -fucosyllactose.
Advantageous effects
(1) The amino acid sequence of polyphosphate-dependent mannose kinase from Arthrobacter sp.KM was obtained from NCBI, and the gene was codon-optimized according to the codon preference of E.coli. The method uses Asppgmk (NCBI database gene accession number WP_ 028275864.1) as a template, selects a protein structure with homology in PDB through a swiss-model, uses BLASTP to carry out multi-sequence alignment, and carries out saturation mutation and optimizes bioconversion reaction conditions, so that the yield of the final product mannose-6-phosphate is 92.1%, and the final yield is up to 479.17g/L, which is the highest yield reported at present after the inhibition of a substrate product is released.
(2) The invention utilizes genetic engineering technology to modify escherichia coli, and prevents the synthesis path of GDP-fucose flowing to capsular iso-polygluconic acid by knocking out wcaJ gene of UDP-glucose lipid carrier transferase. Thereby reducing the loss of metabolic intermediate products and improving the production efficiency of the products.
(3) According to the invention, the recombinant plasmid pET-CMXLF is obtained by coexpression of xcpc, rfbM, xref, 7LL6 and FutC genes on the E.coli expression vector pET21a, and the enzyme amount of each reaction step can be flexibly regulated in one-pot reaction, so that sufficient conversion is realized, and the yield of 2' -FL are obviously improved.
(4) The invention designs and constructs a multi-enzyme cascade path by taking low-cost mannose as a substrate, and can obtain 2' -FL with high conversion efficiency and stereoselectivity. The whole-cell biocatalysis reaction does not need complicated steps, generates no redundant byproducts, has mild reaction conditions and is environment-friendly. The invention obtains the target product through a biocatalysis one-step method, and is a green and efficient method for biosynthesis of 2' -FL. By optimizing bioconversion reaction conditions, 500mM substrate mannose (94.08 g/L in total) was catalytically converted at 30℃for 16h in 50mg/mL of recombinant cells expressing six enzymes of the de novo synthesis route in 0.1mol/L Tris-HCl buffer pH 7.5, resulting in a yield of 82.7% of the final product 2' -FL of 201.97g/L.
Drawings
FIG. 1 is a metabolic pathway diagram of the synthesis of 2' -FL from the de novo synthesis pathway.
FIG. 2 is a flow chart for recombinant plasmid construction: wherein, (1): pET-ppgmk; (2): pET-CMXL; (3): pET-FutC; (4): pET-CMXLF.
FIG. 3 is a gel electrophoresis diagram of nucleic acid related to gene knockout; wherein Lane 1: a homologous fragment upstream of the gene; lane 2: a downstream homologous fragment of the gene; lane 3, 4: fusion fragments of genes at upstream and downstream; lane 5: a template DNA fragment; lane 6: a PCR product after successful gene knockout; lane 7: negative control without knockdown of gene.
FIG. 4 shows E.coli BL 21. Delta. W/pET-ppgmk and its mutants expressed in E.coli; wherein Lane M: protein MW Marker (Low); WT: wild type E.coli BL21- ΔW/pET28a-ppgmk protein; 169I: e. supernatant of fermentation broth of the coli BL 21-DeltaW/pET-L169I transformant; 172I: e. supernatant of fermentation broth of the coli BL 21-DeltaW/pET-L169I transformant; 174L: e. supernatant of fermentation broth of the coli BL 21-DeltaW/pET-I174L transformant; 174V: e. supernatant of fermentation broth of the coli BL 21-. DELTA.W/pET-I174V transformant.
FIG. 5 shows E.coli BL 21-. DELTA.W/pET-CMXL expression in E.coli; wherein Lane M: protein MW Marker (Low); lane 1: e. supernatant of fermentation broth of the coli BL 21-DeltaW/pET-CMXL transformant; lane 2: protein of empty competent cells E.coli BL21/pET21 a.
FIG. 6 is a diagram showing a liquid phase assay for the synthesis of GDP-L-fucose from D-mannose catalyzed by a multi-enzyme cascade; wherein a in fig. 6 is whole cell transformation solution; b in fig. 6 is a blank fermentation broth without cells; c in FIG. 6 is a GDP-L-fucose label.
FIG. 7 shows E.coli BL 21. Delta. W/pET-FutC expression in E.coli; wherein Lane M: protein MW Marker (Low); lane 1: protein of empty competent cells E.coli BL21- ΔW/pET28 a; lane 2: supernatant of fermentation broth of the coli BL 21-. DELTA.W/pET-FutC transformant.
FIG. 8 shows E.coli BL 21-. DELTA.W/pET-CMXLF expression in E.coli; wherein Lane M: protein MW Marker (Low); lane 1: e. supernatant of fermentation broth of the coli BL 21-DeltaW/pET-CMXLF transformant; lane 2: protein of empty competent cells E.coli BL21/pET21 a.
FIG. 9 is a diagram showing the liquid phase detection of 2' -fucosyllactose synthesized by the multienzyme cascade catalysis of D-mannose.
FIG. 10 is a nuclear magnetic resonance hydrogen spectrum of a multienzyme cascade catalyzed D-mannose synthesis of 2' -fucosyllactose.
FIG. 11 is an optimization of the biocatalytic conditions of whole-cell mannose synthesis of 2' -fucosyllactose.
Detailed Description
The following examples relate to the following media:
LB liquid medium: tryptone 1%, yeast extract 0.5%, naCl 1%, pH 7.0. Kanamycin (100. Mu.g/mL) was added before use, and 1.5% agar powder was added to the solid medium.
The detection method involved in the following examples is as follows:
the detection method of the product 2' -FL comprises the following steps:
to measure the yield of 2'-FL in the reaction solution after the reaction, 1mL of the supernatant of the fermentation broth was collected by centrifugation (12,000Xg, 10 minutes) and the 2' -FL content was measured using High Performance Liquid Chromatography (HPLC) to calculate the conversion. Detection was performed by means of an Aminex HPX-87H organic acid column. The mobile phase was 5mM dilute sulfuric acid, the flow rate was 0.6mL/min, and the column temperature was 65 ℃. The signal was detected by a differential detector at a temperature of 40 ℃.
Detection of polyphosphate-dependent mannose kinase enzyme activity:
the enzymatic activity of ppgmk is mediated by NADPH NA 4 And (5) measuring an absorbance value. The reaction system was 100mM mannose, 10mM sodium hexametaphosphate, 5mM MgCl2, tris-HCl buffer was added and reacted at 30℃for 10 minutes. The enzyme was inactivated by boiling water treatment at 100deg.C for 3 min. Then 0.5mM NADPH 2 And 1U of glucose-6-phosphate dehydrogenase, 1U of mannose-6-phosphate isomerase, 1U of glucose-6-phosphate isomerase treatment NADPH aa at 340nm (ε=6220/M/cm) was monitored at 30℃using a multifunctional microplate reader (BioTek, vermont, USA) 4 Absorbance values. All experiments were repeated three times; on the basis, the enzyme activity of ppgmk is measured by taking mannose as a substrateSex.
The enzyme activity was quantitatively determined by High Performance Liquid Chromatography (HPLC) and, according to the literature, by using an Aminex (Hercules, calif., USA) organic acid column HPX-87H. Mobile phase 5mM dilute H 2 SO 4 The flow rate was 0.6mL/min and the column temperature was 65 ℃. The temperature was measured by a differential detector and found to be 40 ℃. The reaction substrate is directly mannose, and the catalytic activity of mannose can be detected simultaneously.
Firstly, a standard curve is prepared, mannose aqueous solutions of 1g/L, 4g/L, 10g/L, 15g/L and 20g/L are configured, and the standard curve is prepared by taking the mannose peak area as an ordinate and the concentration as an abscissa through HPLC detection, so that the content of residual mannose can be reflected from the peak area of a sample. The substrate system is 5g/L mannose, 5g/L sodium hexametaphosphate and 10mM MgCl 2 The preparation method comprises the steps of preparing a Tris-HCl buffer solution with the pH value of 8.5 and 0.1mol/L, fixing the volume, detecting the mannose peak area for 11-12 min by HPLC after membrane passing, and calibrating the actual concentration on a standard curve. 1mL of substrate system is added with a certain amount of pure enzyme, reacted for 10min at 30 ℃, heated and deactivated by boiling water for 3min, centrifugated to obtain supernatant, and subjected to HPLC (high performance liquid chromatography) through a membrane.
The procedure for the transformation of E.coli with the recombinant plasmids described in the examples below was as follows:
(1) To 50. Mu.L of E.coli BL21 (DE 3) or E.coli BL21 (DE 3) - ΔwcaJ competent cell suspension per tube, 0.5. Mu.L of the ligation product was added, and after gentle mixing, the mixture was subjected to ice bath for 30min, heat shock at 42℃for 90s, and rapidly transferred to ice bath and cooled for 2min. mu.L of LB liquid medium was added to each tube, and the culture was performed by shaking at 200rpm at 37℃for 1 hour. After culturing, the bacterial liquid was centrifuged at 3,000Xg for 2min, 600. Mu.L of the supernatant was discarded, and the remaining bacterial liquid was mixed uniformly and then applied to LB plates containing 100. Mu.g/mL of ampicillin, and the mixture was cultured at 37℃overnight in an inverted manner.
(2) 4 clones were picked, transferred into LB liquid medium containing 5mL of 100. Mu.g/mL of ampicillin, cultured at 37℃for 12 hours, and plasmids were extracted from the cultured bacterial liquid using plasmid extraction kit Mini-Plasmid Rapid I solation Kit (Bobolothac Biotechnology Co., ltd.). The verification is carried out by the following enzyme digestion system: 10 XBuffer 2. Mu.L, plasmid DNA 5. Mu.L, bamH I0.5. Mu.L, xho I0.5. Mu.L, ddH 2 O makes up 20. Mu.L of the system; wherein E.coli BL21 (DE 3) - ΔwcaJ LB liquid medium containing 100. Mu.g/mL ampicillin and 100. Mu.g/mL kanamycin was used.
Example 1: polyphosphate dependent mannose kinase gene ppgmk and acquisition of recombinant bacteria containing mutant ppgmk
The method comprises the following specific steps:
1. preparation of mutants
(1) The amino acid sequence of polyphosphate-dependent mannose kinase from Arthrobacter sp (NCBI database accession number WP_028275864.1, amino acid sequence shown as SEQ ID NO.8, nucleotide sequence shown as SEQ ID NO. 9) was obtained from NCBI, and the gene was codon-optimized according to the codon preference of E.coli.
Sequence alignment of 3 ppgmk of different origin, including mipgmk from Micrococcaceae, asppgmk from archibacter sp.I3 and Psppgmk from Pseudarthrobac ter sp, showed that the three polyphosphate mannose kinases have similar functional properties, indicating that these enzymes convert mannose to mannose-6-phosphate.
Polyphosphate-dependent mannose kinase (NCBI database accession number WP_028275864.1, amino acid sequence shown in SEQ ID NO.8, nucleotide sequence shown in SEQ ID NO. 9) from Arthrobacter sp. Was crossed to a gene synthesis company to synthesize a gene and ligated to pET-28a vector. Through homologous modeling, molecular docking and multiple sequence comparison and equivalent methods, specific amino acid sites of polyphosphate dependent kinase are mutated, full-plasmid PCR is carried out by taking pET-ppgmk containing a wild mannose kinase sequence as a template, template digestion and product purification are carried out on the obtained PCR product, and plasmid pET-L169I-I174L, pET-L169I, pET-I174L, pET-L172I containing the polyphosphate dependent mannose kinase mutant is prepared; the primer sequences involved are shown in Table 1.
Table 1: primer for polyphosphate dependent mannose kinase mutation
The PCR amplification system is as follows: prime STAR 25. Mu.L, template 1. Mu.L, upstream and downstream primer 1. Mu.L.times.2, ddH 2 O 22μL。
PCR amplification conditions: pre-denatured at 98 ℃ for 30s, then the following cycle was performed: denaturation at 98℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; extending at 72 ℃ for 10min, and preserving heat at 4 ℃.
The plasmids pET-L169I-I174L, pET-L169I and p ET-I174L, pET-L172I containing the polyphosphate dependent mannose kinase mutant are respectively transformed into competent cells of E.coli BL21 (DE 3) to obtain recombinant strains E.coli BL21/pET-L169I-I174L, E.coli BL21/pET-L169I, E.coli BL21/pET-I174L and E.coli BL21/p ET-L172I.
According to the method, recombinant bacteria expressing wild polyphosphate dependent mannose kinase are prepared: e.coli B L/pET-ppgmk.
(2) Culturing recombinant bacteria:
e.coli BL21/pET-ppgmk, E.coli BL21/pET-L169I-I174L, E.coli BL21/pET-L169I, E.coli BL21/pET-I174L and E.coli BL21/pET-L172I in the step (1) are respectively picked up, inoculated in 5ml LB liquid culture medium containing 100 mug/ml kanamycin, and subjected to shaking culture at 37 ℃ and 200rpm for overnight to prepare culture solution;
Transferring 500. Mu.L of culture solution into 50mL of LB liquid medium containing 100. Mu.g/mL kanamycin, and shaking culturing at 37 ℃ and 200rpm until OD 600 After 0.6, 0.1mM IPTG is added, and induced culture is carried out for 12 hours at 30 ℃ to obtain fermentation liquor;
centrifuging the fermentation broth under 10,000Xg for 10min, collecting thallus, washing thallus twice with physiological saline, and collecting recombinant bacteria whole cells. E.coli BL21/pET-ppgmk, E.coli BL21/pET-L169I-I174L, E.coli BL21/pET-L169I, E.coli BL21/pET-I174L and E.coli BL21/pET-L172I whole cells are prepared.
(3) Specific enzyme activity of wild-type polyphosphate-dependent mannose kinase and mutant thereof
Preparation of pure enzyme solution: the induction conditions are as follows: culturing for 14-16 h at 30 ℃ with 0.1mM IPTG to obtain polyphosphate dependent mannose kinase whole cell, wherein the recombinant expression vector pET28a (+) contains His histidine tag, and histidine and Ni can be utilized + Can utilize Ni for ppgmk + The column was used for purification. A single band of protein was obtained. The construction, induced expression and purification of the strain are all conventional operations; respectively preparing the pure enzyme solutions. The results of the enzymatic activity detection of the polyphosphate-dependent mannose kinase and the mutant thereof obtained are shown in Table 2:
Table 2 specific enzyme activity of mutant enzyme purified enzyme solution
The results showed that the L169I-I174L mutant enzyme had the highest enzyme activity, and continued the study with it.
Example 2: multi-enzyme co-expression plasmid and strain construction
One plasmid is designed to carry one gene, the other plasmid carries four genes, and five enzymes are constructed into a co-expression strain in a double-plasmid co-expression mode.
The method comprises the following specific steps:
(1) The amino acid sequences of phosphomannose mutase (xcpc), mannose-1-guanosine phosphate transferase (rfbM), GDP-D-mannose-4, 6-dehydratase (Xref), GDP-L-fucose synthase (7 LL 6) from E.coli (the amino acid sequences are shown in SEQ ID NO. 2-5, respectively) were obtained from NCBI. The Escherichia coli BL genome is used as a template, and the target gene is obtained through PCR amplification reaction.
The PCR amplification system is as follows: prime STAR 25. Mu.L, template 1. Mu.L, upstream and downstream primer 1. Mu.L.times.2, ddH 2 O 22μL。
PCR amplification conditions: pre-denatured at 98 ℃ for 30s, then the following cycle was performed: denaturation at 98℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; extending at 72 ℃ for 10min, and preserving heat at 4 ℃.
(2) The four key enzymes phosphomannose mutase (xcpc), mannose-1-guanosine transferase (rfbM), GDP-D-mannose-4, 6-dehydratase (Xref), GDP-L-fucose synthase (7 LL 6) obtained in step (1) were constructed on one plasmid using SD-AS linker. Double enzyme digestion is respectively carried out on the target gene and the expression vector pET21a by utilizing restriction enzyme, and the treated DNA fragment is connected through an adhesive end to obtain a recombinant plasmid pET21a-xcpc-rfbM-Xref-7LL6 with a multienzyme gene, which is named as: pET-CMXL.
And (3) converting the recombinant plasmid pET-CBGW prepared in the step (2) into competent cells of E.coli BL21 (DE 3), and screening by an LB plate containing 100 mug/mL of ampicillin to obtain the recombinant strain E.coli BL21/pET-CBGW.
(4) Culturing recombinant bacteria:
picking a single colony of E.coli BL21/pET-CMXL in the step (3), inoculating the single colony into 5ml of LB liquid medium containing 100 mug/ml ampicillin, and carrying out shaking culture at 37 ℃ and 200rpm for overnight to prepare a culture solution; transferring 500. Mu.L of culture solution into 50mL of LB liquid medium containing 100. Mu.g/mL of ampicillin, and shaking culturing at 37 ℃ and 200rpm until OD 600 Adding 0.1mM IPTG after the fermentation time is 0.6, and performing induction culture at 30 ℃ for 12 hours to obtain fermentation liquor; centrifuging the fermentation liquor for 10min under the condition of 10,000Xg, collecting thalli, washing thalli twice with physiological saline, and collecting recombinant bacterium E.coli BL21/pET-CBGW whole cells.
Example 3: functional verification of GDP-L-fucose synthesized by multienzyme cascade
In industrial production, GDP-L-fucose is an important intermediate for synthesizing breast milk oligosaccharides, and plays an important role in the synthesis of breast milk oligosaccharides. To confirm the feasibility of the designed cascade, the following experiments were performed:
the initial reaction conditions were:
20mM mannose, 2mM MgCl 2 50mg/mL whole cells (E.coli BL21/pET-L169I-I174L and E.coli BL21/pET-CMXL wet cells, ratio 1:1), 20. Mu.M NADP and 2mM NADPH. The reaction buffer was 0.1mol/L Tris-HCl (pH 7.0).
The reaction is carried out in a shaking table at 200rpm and 30 ℃ for 16 hours at the temperature, after the reaction is finished, whole cells are removed by high-speed centrifugation, liquid phase sample loading detection is carried out after supernatant is properly diluted, and all experiments are repeated for three times to obtain the average value.
HPLC detection conditions, ultraviolet detector; the chromatographic column is Rezex ROA-organic acid (Phenomenex, USA), the ultraviolet wavelength is 254nm, and the column temperature is 30 ℃; the mobile phase is 20mmol/L of triethylamine acetate and 14% of acetonitrile, the flow rate is 0.6mL/min, the sample injection amount is 10 mu L, and gradient elution is carried out.
The results show that: combining these five enzymes with 2mM mannose in a molar ratio of 1:1:1:1, after 12 hours, the formation of the final product GDP-L-fucose was confirmed using HPLC (a, b, c in FIG. 6), indicating that mannose could be converted to GDP-L-fucose using a cascade designed with ppgmk, xcpc, rfbM, xref and 7LL 6.
Example 4:1,2 fucosyltransferase gene FutC and recombinant bacterium thereof
The method comprises the following specific steps:
(1) The amino acid sequence of the 1, 2-fucosyltransferase from Helicobacter pylori (the amino acid sequence is shown as SEQ ID NO. 6) was obtained from NCBI, the gene was codon optimized according to the codon preference of E.coli, full plasmid PCR was performed using pET-FutC containing the 1, 2-fucosyltransferase sequence as a template, and the obtained PCR product was subjected to template digestion and product purification to prepare plasmid pET-FutC containing the polyphosphate-dependent mannose kinase mutant. The plasmid containing 1, 2-fucosyltransferase is transformed into competent cells of E.coli BL21 (DE 3) to obtain recombinant strain E.coli BL21/pET-FutC.
The PCR amplification system is as follows: prime STAR 25. Mu.L, template 1. Mu.L, upstream and downstream primer 1. Mu.L.times.2, ddH 2 O 22μL。
PCR amplification conditions: pre-denatured at 98 ℃ for 30s, then the following cycle was performed: denaturation at 98℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; extending at 72 ℃ for 10min, and preserving heat at 4 ℃.
(2) Culturing recombinant bacteria:
picking a single colony of E.coli BL21/pET-FutC in the step (1), inoculating the single colony into 5ml of LB liquid medium containing 100 mug/ml ampicillin, and shaking and culturing at 37 ℃ and 200rpm for overnight to prepare a culture solution;
transferring 500. Mu.L of culture solution into 50mL of LB liquid medium containing 100. Mu.g/mL of ampicillin, and shaking culturing at 37 ℃ and 200rpm until OD 600 Adding 0.1mmol/L IPTG after the concentration is 0.6, and performing induction culture at 30 ℃ for 12 hours to obtain fermentation liquor; centrifuging the fermentation broth under 10,000Xg for 10min, collecting thallus, washing thallus twice with physiological saline, and collecting recombinant bacteria whole cells. Ultrasonic crushing at 0 ℃ with power of 25% and cell breaking time of 15min. Centrifuging at 4deg.C and 12,000Xg for 20min, collecting supernatant, and preserving at-20deg.C.
The results showed (FIG. 7) that SDS-PAGE showed a single band with a molecular weight of about 30kDa, which is consistent with the theoretical molecular weight of the recombinant protein.
Example 5: construction of recombinant E.coli with knockout wcaJ
The method comprises the following specific steps:
(1) Carrying out PCR amplification on the existing pTargetF plasmid by using a primer F-delta wcaJ-F/F-delta wcaJ-R to obtain a pTargetF plasmid with a targeted wcaJ gene;
(2) The primers wcaJ-up-F/R and wcaJ-down-F/R are used for respectively amplifying upstream and downstream homologous fragments of wcaJ gene by PCR;
(3) Respectively taking upstream and downstream fragments of wacJ as templates, obtaining donor DNA templates of the gene wacJ by overlapping PCR by using primers wcaJ-up-F/wcaJ-down-R, and recovering the DNA fragments by glue; and (3) taking F-Deltawcaj-F/F-Deltawcaj-R as an upstream primer and a downstream primer, carrying out PCR amplification by taking pTargetF plasmid as a template, and carrying out restriction enzyme Dpn I digestion on an amplification product to remove redundant circular pTargetF plasmid. E.coli BL21 competent cells were transformed with the amplified product, miniplasmids were identified by sequencing with primer F-test/F-Deltawcaj-R, and the successfully constructed knockout plasmid was designated pTF-Deltawcaj. The primer sequences involved are as follows:
table 3: gRNA and primers
(4) Transferring pCas plasmid into E.coli BL21 (DE 3) by electrotransformation, and adding 20mM arabinose to induce the expression of the lambda-Red E.coli gene recombination system;
(5) The pTF-DeltawcaJ plasmid was electrotransformed into E.coli BL21 (DE 3) and applied to LB plates containing kanamycin and spectinomycin, and incubated at 30℃for 12 to 20 hours, and colonies on the plates were knocked out to verify (FIG. 3);
(6) Successful E.coli BL 21-DeltawcaJ strain was identified to eliminate pTargetF and pCas plasmid resistance, and cultured in LB liquid medium containing kanamycin until OD 600 After a value of about 0.2, 0.5mM IPTG was added, and after 12 hours of culture, LB solid plates containing kanamycin resistance were streaked, and then confirmation that the clones were sensitive to spectinomycin was confirmed that the recombinant plasmid had been eliminated. The knockdown recombinant was cultured overnight at 42 ℃ to eliminate pCas plasmid; e.coli BL 21-Deltawc aJ was prepared.
Example 6: multi-enzyme co-expression plasmid and strain construction
One plasmid is designed to carry one gene, the other plasmid carries five genes, and six enzymes are constructed into a co-expression strain in a double-plasmid co-expression mode.
The method comprises the following specific steps:
(1) Five key enzymes xcpc, rfbM, xref, 7LL6, futC (amino acid sequences shown in SEQ ID NOS.2-6, respectively)) verified in the above examples were constructed on a plasmid using SD-AS (SEQ ID NOS: GAAGGAGATATACC) AS linker in the order rfbM-xcpc-SD-AS-Xref-7LL6-SD-AS-FutC. Double enzyme digestion is respectively carried out on the target gene and the expression vector pET21a by utilizing restriction enzyme, and the treated DNA fragment is connected through a sticky end to obtain a recombinant plasmid with a multienzyme gene: pET21a-xcpc-rfbM-Xref-7LL6-FutC, designated pET-CMXLF.
(2) Simultaneously transforming the recombinant plasmid pET-CMXLF prepared in the step (1) and the plasmid pET-L169I-I174L prepared in the example 1 into competent cells of E.coli BL21 (DE 3) -DeltawcaJ, and screening by an LB plate containing 100 mug/mL of ampicillin and 100 mug/mL of kanamycin to obtain a recombinant strain E.coli BL 21-DeltawcaJ/pET-CMXLF/pET-L169I-I174L; named E.coli BL21- ΔwcaJ/pET-PCMXLF.
(3) Culturing recombinant bacteria:
picking the E.coli BL 21-DeltawcaJ/pET-PCMXLF single colony in the step (2), inoculating the single colony into 5mL of LB liquid medium containing 100 mug/mL ampicillin and 100 mug/mL kanamycin, and carrying out shaking culture at 37 ℃ and 200rpm for overnight to prepare a culture solution; mu.L of the culture medium was transferred to 50mL of LB liquid medium containing 100. Mu.g/mL of ampicillin and 100. Mu.g/mL of kanamycin, and cultured at 37℃under shaking at 200rpm to OD 600 Adding 0.1mmol/L IPTG after the concentration is 0.6, and performing induction culture at 30 ℃ for 12 hours to obtain fermentation liquor; centrifuging the fermentation liquor for 10min under the condition of 10,000Xg, collecting thalli, washing thalli twice with physiological saline, and collecting recombinant bacterium E.coli BL 21-DeltawcaJ/pET-PCMXLF whole cells.
Example 7: condition optimization for synthesizing 2' -fucosyl lactose by catalyzing D-mannose through multienzyme cascade
The method comprises the following specific steps:
(1) The initial reaction conditions were:
20mM mannose, 10mM sodium hexametaphosphate, 2mM MgCl 2 10mg/mL of the wet cells E.coli BL 21-. DELTA.wcaJ/pET-PCMXLF obtained in example 7, 40. Mu.M NADP and 2mM NADPH. The reaction buffer was 0.1M Tris-HCl (pH 8.5).
The reaction is carried out in a shaking table at 200rpm and 30 ℃ for 16 hours at the temperature, after the reaction is finished, whole cells are removed by high-speed centrifugation, liquid phase sample loading detection is carried out after supernatant is properly diluted, and all experiments are repeated for three times to obtain the average value.
The chromatographic analysis uses an Agilent-1260HPLC system (Agilent Technologies inc., palo Alto, USA) and the signal is detected with a differential detector at a temperature of 40 ℃. Chromatographic column: organic acid column Aminex HPX-87H, mobile phase: 5mM dilute sulfuric acid, flow rate: 0.6mL/min, temperature: 65 ℃.
(2) Concentration of whole cells of the reaction is optimized
The reaction system was 1mL and contained 10mM mannose, 10mM sodium hexametaphosphate, 2mM MgCl 2 40. Mu.M NADP, 2mM NADPH and 10-30 mg/mL of the wet cells E.coli BL 21-. DELTA.wcaJ/pET-PCMXLF obtained in example 7 (10 mg/mL, 15mg/mL, 20 mg)Each of the above-mentioned reaction buffers was 0.1mol/L Tris-HCl (pH 8.5), 25mg/ml and 30 mg/ml.
The reaction is carried out in a shaking table at 200rpm and at 35 ℃ for 16 hours, after the reaction is finished, whole cells are removed by high-speed centrifugation, liquid phase sample loading detection is carried out after supernatant is properly diluted, and all experiments are repeated for three times to obtain the average value.
The results show that: as shown in FIG. 11, the wet cell concentration was positively correlated with the substrate conversion rate in a certain range, and when the wet cell concentration was 30mg/mL, the conversion rate was maximized, which means that the enzyme content of the whole cell had reached the maximum enzyme content at this time, therefore 30mg/mL was selected as the optimum amount for the whole cell reaction of catalyzing the synthesis of 2' -fucosyllactose from D-mannose by the multienzyme cascade, and the substrate conversion rate was 82.9%, and the yield was: 4.05g/L.
(3) pH of the reaction is optimized
The reaction system was 1mL and contained 10mM mannose, 10mM sodium hexametaphosphate, 2mM MgCl 2 40. Mu.M NADP, 2mM NADPH and 30mg/mL of the wet cells E.coli BL 21-. DELTA.wcaJ/pET-PCMXLF obtained in example 7, the reaction buffer was 0.1mol/L Tris-HCl (pH 6.5-8.5).
The reaction is carried out in a shaking table at 200rpm and at 35 ℃ for 16 hours, after the reaction is finished, whole cells are removed by high-speed centrifugation, liquid phase sample loading detection is carried out after supernatant is properly diluted, and all experiments are repeated for three times to obtain the average value.
The results show that: as shown in FIG. 11, the reaction pH also has a large effect on the efficiency of the conversion reaction. Under the peracid or overbase environment, the spatial structure of the enzyme may be destroyed, affecting the binding of the enzyme molecule to the substrate and thus the efficiency of the enzymatic reaction. The 2' -FL yield was highest at a reaction pH of 7.5, at which time the substrate conversion was 87.8% and the yield was: 4.29g/L.
(4) Optimizing the temperature of the reaction
The reaction system was 1mL and contained 10mM mannose, 10mM sodium hexametaphosphate, 2mM MgCl 2 40. Mu.M NADP, 2mM NADPH and 30mg/mL wet cells E.coli BL 21-. DELTA.wcaJ/pET-PCMXLF obtained in example 7. The reaction buffer was 0.1mol/L Tris-HCl (pH 7.5).
The reaction is carried out in a shaking table at 200rpm, the temperature is set to be 20-40 ℃, the reaction time is 16h, the whole cells are removed by high-speed centrifugation after the reaction is finished, the supernatant is properly diluted and then liquid phase sample loading detection is carried out, and all experiments are repeated for three times to obtain the average value.
The results show that: as shown in FIG. 11, the increase of the temperature has an effect of promoting the conversion rate of 2' -fucosyllactose synthesized from D-mannose by the multi-enzyme cascade, and when the environmental conditions are extreme, such as low temperature (20 ℃) or high temperature (40 ℃), the enzyme catalytic reaction is inhibited, and the catalytic activity of the enzyme is low, and according to the experimental results, we speculate that the extreme temperature may partially inactivate the enzyme. The optimum reaction temperature was chosen at 30℃at which the substrate conversion was 88.3% with the yield: 4.31g/L.
(5) Optimized substrate addition amount of reaction
The reaction system is 1mL, and comprises 10-250 mM mannose, 10mM sodium hexametaphosphate and 2mM MgCl 2 40. Mu.M NADP, 2mM NADPH and 30mg/mL of the wet cells E.coli BL 21-. DELTA.wcaJ/pET-PCMXLF obtained in example 7, the reaction buffer was 0.1mol/L Tris-HCl (pH 7.5).
The reaction is carried out in a shaking table at 200rpm and 30 ℃ for 16 hours at the temperature, after the reaction is finished, whole cells are removed by high-speed centrifugation, liquid phase sample loading detection is carried out after supernatant is properly diluted, and all experiments are repeated for three times to obtain the average value.
The results show that: as shown in FIG. 11, the conversion rate was decreased with increasing substrate addition, and the conversion rate was as high as 96.5%, and the conversion rate was 92.01% when the substrate addition concentration was 250mM, and the yield was: 112.34g/L.
(6) Comparison of wild-type Strain and mutant Strain conversion under the same conditions
1) Preparation of wild-type Strain WT
The specific procedure is the same as in example 7, except that pET-L169I-I174L plasmid is adjusted to be the vector pET-ppgmk containing the wild-type polyphosphate-dependent mannose kinase gene prepared in example 1, and E.coli BL 21-DeltawcaJ/pET-CMXLF/pET-ppgmk is prepared; named E.coli BL 21-DeltawcaJ/pET-WT-CMXLF; and wet cells were prepared as in example 7
2) ReactionThe system was 1mL containing 250mM mannose, 10mM sodium hexametaphosphate, 2mM MgCl 2 40. Mu.M NADP, 2mM NADPH and 30mg/mL of the wet cells E.coli BL 21-. DELTA.wcaJ/pET-PCMXLF obtained in example 7 or 30mg/mL E.coli BL 21-. DELTA.wcaJ/pET-WT-CMXLF, the reaction buffer was 0.1mol/L Tris-HCl (pH 7.5). The reaction was carried out in a shaker at 200rpm and 30℃for 16h at a high speed to remove whole cells after the completion of the reaction, and the supernatant was suitably diluted and subjected to liquid phase loading detection, and the results were obtained by repeating all the above experiments three times and the average value is shown in Table 4.
Table 4: comparison of wild-type Strain and mutant Strain conversion
Example 8: expansion reaction whole cell synthesis of 2' -fucosyllactose
The concentration of substrate mannose is enlarged to 500mM to test the catalytic conversion efficiency of the multi-enzyme cascade. The whole cell catalytic system comprises:
300mM, 400mM, 500mM mannose are used as substrate, 10mM sodium hexametaphosphate, 2mM MgCl, respectively 2 40. Mu.M NADP, 2mM NADPH and 30-50 mg/mL wet cells (wet cells E.coli BL 21-. DELTA.wcaJ/pET-PCMXLF obtained in example 7) were reacted in a buffer of 0.1mol/L Tris-HCl (pH 7.5).
The reaction is carried out in a shaking table at 200rpm and 30 ℃ for 16 hours at the temperature, after the reaction is finished, whole cells are removed by high-speed centrifugation, liquid phase sample loading detection is carried out after supernatant is properly diluted, and all experiments are repeated for three times to obtain the average value.
The conversion effect is shown in Table 5.
Table 5: expanding the reaction result
The results show that the synthesis route of 2' -fucosyllactose synthesized by whole cells catalyzed by multienzyme cascade is optimized by an orthogonal method to obtain the total cell addition of 50mg/mL, the substrate conversion rate is 82.7% when the substrate addition is 500mM, and the yield is as follows: 201.97g/L.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A polyphosphate dependent kinase mutant, which is characterized in that the polyphosphate dependent kinase mutant is obtained by mutating leucine at position 169 of the polyphosphate dependent kinase with an amino acid sequence shown as SEQ ID NO.8 into isoleucine; or the leucine is mutated from isoleucine at position 174 of polyphosphate dependent kinase with the amino acid sequence shown in SEQ ID NO. 8; or by mutating leucine at position 169 of polyphosphate dependent kinase having the amino acid sequence shown in SEQ ID NO.8 to isoleucine and mutating isoleucine at position 174 to leucine.
2. An engineered bacterium for producing 2' -fucosyllactose, wherein the engineered bacterium knocks out a UDP-glucose lipid carrier transferase gene wcaJ and overexpresses a phosphomannose mutase xcpc, a mannose-1-phosphoguanyl transferase, a GDP-mannose-6-dehydrogenase xref, a GDP-fucose synthase 7LL6, an alpha-1, 2 fucosyl transferase futC, and the polyphosphate-dependent kinase mutant of claim 1.
3. The engineering bacterium according to claim 1, wherein the nucleotide sequence of the UDP-glucose lipid carrier transferase gene wcaJ is shown as SEQ ID NO. 1; the amino acid sequences of the phosphomannose mutase xcpc, mannose-1-phosphoguanyl transferase rfbM and GDP-mannose-6-dehydrogenase xref and GDP-fucose synthetase 7LL6 and alpha-1, 2 fucosyl transferase futC are respectively shown as SEQ ID NO. 2-6.
4. The engineered bacterium of claim 3, wherein the expression vector pET-21a, pET-28a or pRSF-Duet1 is used for overexpression.
5. The engineered bacterium of claim 4, wherein the expression host is escherichia coli.
6. A method for producing 2 '-fucosyllactose, characterized in that the method comprises producing 2' -fucosyllactose in a fermentation system using mannose as a substrate by using the engineering bacterium according to any one of claims 2 to 5 as a fermentation strain.
7. The method of claim 6, wherein the substrate mannose concentration is between 10mM and 500mM.
8. A method for increasing the yield of 2' -fucosyllactose produced by E.coli, characterized in that the UDP-glucose lipid carrier transferase gene wcaJ in E.coli is knocked out and the phosphomannomutase xcpc, mannose-1-phosphoguanyl transferase rfbM, GDP-mannose-6-dehydrogenase xref, GDP-fucose synthase 7LL6, alpha-1, 2 fucosyl transferase futC and the polyphosphate dependent kinase mutant of claim 1 are overexpressed.
9. The method according to claim 8, wherein the nucleotide sequence of the UDP-glucose lipid carrier transferase gene wcaJ is shown in SEQ ID NO. 1; the amino acid sequences of the phosphomannose mutase xcpc, mannose-1-phosphoguanyl transferase rfbM and GDP-mannose-6-dehydrogenase xref and GDP-fucose synthetase 7LL6 and alpha-1, 2 fucosyl transferase futC are respectively shown as SEQ ID NO. 2-6.
10. Use of an engineered bacterium according to any one of claims 2 to 5 for the production of 2 '-fucosyllactose and products containing 2' -fucosyllactose.
CN202311425202.6A 2023-10-30 2023-10-30 Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade Pending CN117568302A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311425202.6A CN117568302A (en) 2023-10-30 2023-10-30 Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311425202.6A CN117568302A (en) 2023-10-30 2023-10-30 Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade

Publications (1)

Publication Number Publication Date
CN117568302A true CN117568302A (en) 2024-02-20

Family

ID=89890775

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311425202.6A Pending CN117568302A (en) 2023-10-30 2023-10-30 Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade

Country Status (1)

Country Link
CN (1) CN117568302A (en)

Similar Documents

Publication Publication Date Title
CN109402158B (en) Recombinant expression plasmid vector for producing fucosyllactose, metabolic engineering bacteria and production method
TWI695888B (en) A composition for producing tagatose and methods for producing tagatose using the same
CN111712570A (en) Engineering strain for producing allulose and derivatives thereof, and construction method and application thereof
CN110804577A (en) Escherichia coli engineering strain for producing 2' -fucosyllactose
CN114480240B (en) Genetic engineering bacterium for producing fucosyllactose and production method thereof
CN113528480B (en) Alpha-1, 2-fucosyltransferase mutant and construction method and application thereof
CN112625990A (en) Recombinant escherichia coli for synthesizing 2' -fucosyllactose and construction method thereof
CN114350727B (en) Method for synthesizing D-psicose by combining phosphorylation and ATP regeneration system
CN109679887A (en) A method of trehalose is produced using double enzymes fusion enzyme coupled fermentation of efficient secretory expression
CN114874964B (en) Construction method and application of recombinant escherichia coli for high yield of 2' -fucosyllactose
CN112852796A (en) Cellobiose epimerase mutant and application thereof in preparation of lactulose
CN111394292A (en) Multi-way composite neuraminic acid-producing bacillus subtilis and application thereof
CN112662604A (en) Recombinant escherichia coli for synthesizing 3-fucosyllactose and construction method thereof
CN114480465A (en) Bacillus subtilis for producing 2' -fucosyllactose and application thereof
CN111411066B (en) Double-way composite neuraminic acid-producing bacillus subtilis and construction method thereof
CN111394410B (en) High-catalytic-activity neuraminic acid synthase and application thereof
CN113832092B (en) Genetically engineered bacterium for improving lactoyl-N-fucose yield and production method thereof
CN107460152A (en) Produce recombinant bacterium, construction method and the purposes of rhodioside and the like
CN117568302A (en) Efficient synthesis of 2' -fucosyllactose by catalyzing D-mannose through multienzyme cascade
CN115948314A (en) Bacillus licheniformis engineering strain for efficiently producing 2' -fucosyllactose
CN113957027B (en) Genetically engineered bacterium for improving lactoyl-N-fucose yield and production method thereof
CN114806991A (en) Engineering escherichia coli for improving yield of fucosyllactose and production method
CN114672448A (en) Escherichia coli for synthesizing 2' -fucosyllactose and construction method and application thereof
CN113684163A (en) Genetically engineered bacterium for improving yield of lactoyl-N-tetrasaccharide and production method thereof
WO2023169200A1 (en) Recombinant yeast and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination