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
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
  • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • 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
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • 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
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
  • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
  • C12Y104/01021—Aspartate dehydrogenase (1.4.1.21)
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
  • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)

Definitions

• the present invention relates to the field of microorganisms, and in particular to engineering bacteria expressing aspartate dehydrogenase and methods for producing vitamin B5 through fermentation.
• Vitamin B5 also known as D-Pantothenic acid, is a water-soluble vitamin. It is a component of coenzyme A and acyl carrier protein. It serves as a cofactor for more than 70 enzymes and participates in the synthesis of sugar and fat. , protein and energy metabolism, and plays an important role in regulating physiological metabolism.
• VB5 is mainly used in animal feed additives, food additives and pharmaceutical raw materials. With the discovery of new functions of VB5 and the expansion of application fields, its market demand will still show a steady growth trend.
• the industrial production method of VB5 is chemical synthesis. Enterprises basically use the isobutyraldehyde-formaldehyde-hydrocyanic acid method to synthesize DL-pantolactone. DL-pantolactone further L-pantolactone is obtained through chemical or enzymatic separation, and finally L-pantolactone is synthesized with ⁇ -alanine produced from acrylonitrile to synthesize VB5.
• the main raw materials for chemical synthesis of VB5 are flammable, explosive, and highly toxic. The production process will produce cyanide-containing wastewater, which is difficult to treat, causing VB5 to become a heavily polluting industry.
• ⁇ -Alanine serves as a C3 substrate and D-pantoic acid to synthesize VB5.
• a large amount of ⁇ -alanine needs to be exogenously added to the fermentation medium (Sahm, H., et al., (1999) Appl Environ Microb, 65, 1973-1979; Dusch, N.
• beta-alanine It can be produced by decarboxylation of L-aspartic acid. Therefore, enhancing the biosynthesis of aspartic acid is expected to improve the yield of VB5 produced by fermentation.
• the present invention overexpressed the above three enzymes in E. coli that produces VB5 through fermentation, and found that AspDH is more conducive to improving the fermentation yield of VB5 than AspC and AspA.
• the present invention provides the application of enhancing the expression of aspartate dehydrogenase gene aspDH in the production of vitamin B5;
• the aspartate dehydrogenase gene aspDH is derived from Delftia sp. Csl-4.
• the aspartate dehydrogenase gene aspDH has:
• nucleotide sequence shown in (I) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), and having the same or similar function as the nucleotide sequence shown in (I) the nucleotide sequence; or
• the BCD2 has:
• nucleotide sequence shown in (A) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (A), and having the same or similar function as the nucleotide sequence shown in (A) the nucleotide sequence;
• (C) a nucleotide sequence that is at least 80% homologous to the nucleotide sequence shown in (A) or (B);
• the L-aspartate ⁇ -decarboxylase gene panD derived from Bacillus licheniformis has:
• nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (a), and having the same or similar function as the nucleotide sequence shown in (a) the nucleotide sequence; or
• panB, panC and/or panE genes Increase the copy number of panB, panC and/or panE genes.
• the present invention also provides an expression vector, comprising the aspartate dehydrogenase gene aspDH; preferably, the aspartate dehydrogenase gene aspDH is derived from Delftia sp.Csl-4 (Delftia sp.Csl -4);
• the aspartate dehydrogenase gene aspDH has:
• nucleotide sequence shown in (I) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), and having the same or similar function as the nucleotide sequence shown in (I) the nucleotide sequence; or
• the expression vector further includes:
• the strong promoter is PgapA, and the strong RBS is BCD2;
• the BCD2 has:
• nucleotide sequence shown in (A) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (A), and having the same or similar function as the nucleotide sequence shown in (A) the nucleotide sequence;
• (C) a nucleotide sequence that is at least 80% homologous to the nucleotide sequence shown in (A) or (B);
• the L-aspartate ⁇ -decarboxylase gene panD derived from Bacillus licheniformis has:
• nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (a), and having the same or similar function as the nucleotide sequence shown in (a) the nucleotide sequence; or
• the present invention also provides a host expressing the aspartate dehydrogenase gene aspDH;
• the aspartate dehydrogenase gene aspDH is derived from Delftia sp.Csl-4 (Delftia sp.Csl-4);
• the aspartate dehydrogenase gene aspDH has:
• nucleotide sequence shown in (I) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (I), and having the same or similar function as the nucleotide sequence shown in (I) the nucleotide sequence; or
• the host further includes:
• the strong promoter is PgapA, and the strong RBS is BCD2;
• the BCD2 has:
• nucleotide sequence shown in (A) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (A), and having the same or similar function as the nucleotide sequence shown in (A) the nucleotide sequence;
• (C) a nucleotide sequence that is at least 80% homologous to the nucleotide sequence shown in (A) or (B);
• the L-aspartate ⁇ -decarboxylase gene panD derived from Bacillus licheniformis has:
• nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (a), and having the same or similar function as the nucleotide sequence shown in (a) the nucleotide sequence; or
• the host is transfected or transformed with the expression vector as described in claim 4 or 5;
• the host is derived from Escherichia coli, preferably Escherichia coli K12, more preferably Escherichia coli K12 MG1655 strain.
• the present invention also provides the use of the expression vector or the host in producing vitamin B5.
• the present invention also provides a method for producing vitamin B5.
• the host is used as a fermentation strain, fermentation is carried out, the fermentation liquid is collected, and the supernatant is centrifuged to obtain vitamin B5.
• the invention discloses an Escherichia coli expressing aspartate dehydrogenase gene aspDH and a method for fermenting and producing vitamin B5 (VB5).
• the present invention enhances three pathways for synthesizing L-aspartic acid in the VB5 engineering bacterium.
• the aspartate aminotransferase encoded by the aspC gene respectively transfers the amino group of glutamic acid to oxaloacetate to produce L-aspartate.
• Aspartic acid and ketoglutarate encoded by the aspA gene Aspartate ammonia lyase catalyzes the synthesis of aspartate from ammonium and fumarate; aspartate dehydrogenase encoded by the aspDH gene catalyzes the synthesis of aspartate from oxaloacetate and ammonium.
• Aspartic acid and ketoglutarate encoded by the aspA gene
• Aspartate ammonia lyase catalyzes the synthesis of aspartate from ammonium and fumarate
• aspartate dehydrogenase encoded by the aspDH gene catalyzes the synthesis of aspartate from oxaloacetate and ammonium.
• the invention discloses an engineering bacterium expressing aspartate dehydrogenase and a method for producing vitamin B5 through fermentation. Persons skilled in the art can learn from the contents of this article and appropriately improve the process parameters to achieve the goal. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
• Escherichia coli has two pathways to produce aspartate.
• Aspartate dehydrogenase (AspDH) which catalyzes the synthesis of aspartate from oxaloacetate and ammonium, has been found in some archaea. By overexpressing the above three enzymes respectively in E. coli that produces VB5 through fermentation, the present invention found that AspDH is more conducive to improving the fermentation yield of VB5 than AspC and AspA.
• the inventors compared three pathways to improve aspartate synthesis and found that the heterologous aspartate dehydrogenation (aspDH gene encoded) pathway is more suitable for their own aspartate conversion.
• Ammonia pathway encoded by aspC gene
• aspartate ammonia cleavage pathway encoded by aspA gene
• An aspartate dehydrogenase catalyzes the reaction of oxaloacetate with ammonium radicals and NAD(P)H to generate a reversible reaction of aspartate, water and NAD(P)+, which is produced by one of the following microorganisms Species or species produced: Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia proteamaculans, Thermotoga maritima, Halobacterium halophilum (Chromohalobacter salexigens), Acinetobacter baumannii, Delftia sp.Csl-4, Ochrobactrum anthropi, Caulobacter sp., Methanophila mazei Methanohalophilus mahii, Dinoroseobacter shibae, Methanosphaerula palustris, Methanobrevibacter ruminantium, etc.
• the present invention uses a strong promoter to regulate the expression of aspDH gene.
• the promoter may be a strong promoter, which may be the following promoter or a mutant thereof: L promoter, trc promoter, T5 promoter, lac promoter, tac promoter, T7 promoter or gapA promoter.
• the present invention uses a highly active RBS sequence to regulate the translation initiation of the aspDH gene.
• the present invention integrates the above-mentioned aspDH gene with high intensity of translation initiation and transcription initiation regulation into the chromosome of E. coli used to produce VB5 by fermentation method to realize the expression of foreign genes.
• the integration site is the cadA gene, which encodes lysine decarboxylase and theoretically has no effect on the biosynthesis of VB5.
• the same cadA gene locus on the E. coli chromosome used to produce VB5 through fermentation was used to integrate aspA and aspC genes respectively, and the same promoter was used to regulate transcription.
• the effects of overexpressing aspA, aspC and aspDH genes on VB5 synthesis were compared.
• the E. coli that produces VB5 by fermentation method of the present invention also overexpresses the panB, panC and panE genes on the VB5 terminal synthesis pathway.
• the panB gene of Escherichia coli encodes ketopantoate hydroxymethyltransferase, which catalyzes the addition of a methyl group to the substrate a-ketoisovalerate to form ketopantoate.
• Ketopantoate is reduced to pantoate by ketopantoate reductase encoded by the panE gene.
• Pantothenate synthase encoded by the panC gene further catalyzes the condensation of pantoate and ⁇ -alanine to form VB5.
• the Escherichia coli used in the present invention is the K12 MG1655 strain, and its ilvG gene is mutated and inactivated. Therefore, the present invention introduces the active ilvG gene of E. coli BL21 to improve the synthesis supply of acetolactate, the precursor of VB5.
• the present invention inserts the ilvG + M gene derived from E. coli BL21 into the chromosome of E. coli K12 MG1655, uses the trc strong promoter to regulate the transcription initiation of ilvG + M, and uses the terminator Ter to regulate the transcription termination of ilvG + M.
• the insertion site of the ilvG + M gene in the chromosome is the coding sequence of the avtA gene, which causes the inactivation of AvtA and weakens the synthesis of valine, thus weakening the competition pathway of VB5 and favoring the biosynthesis of VB5.
• panD gene derived from Bacillus licheniformis was also integrated into the avtA gene of the engineered bacterium.
• the same strong promoters PPL and BCD2 are used to regulate transcription and translation initiation, respectively.
• the culture medium contains carbon sources, nitrogen sources, inorganic ions, antibiotics and other nutritional factors.
• carbon source sugars such as glucose, lactose, and galactose can be used.
• inorganic nitrogen source you can use ammonia, ammonium sulfate, ammonium phosphate, ammonium chloride and other inorganic nitrogen sources;
• organic nitrogen source you can use corn steep liquor, soybean meal hydrolyzate, hair powder, yeast extract, peptone and other organic nitrogen sources.
• Inorganic ions include one or more of iron, calcium, magnesium, manganese, molybdenum, cobalt, copper, potassium and other ions.
• the experimental methods in the following examples are all conventional methods unless otherwise specified.
• the test materials used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified.
• the quantitative experiments in the following examples were repeated three times, and the results were averaged.
• the technical means used in the examples are conventional means well known to those skilled in the art and commercially available commonly used instruments and reagents. Please refer to "Molecular Cloning Experiment Guide (3rd Edition)" ( Science Press), “Microbiology Experiments (4th Edition)” (Higher Education Press) and manufacturers' instructions for corresponding instruments and reagents.
• E. coli K12 MG1655 ATCC number is 700926.
• pACYC184 plasmid NEB Company, catalog number E4152S. Plasmid pcas9 was purchased from Addgene Company, product number 62225; plasmid pTargetF was purchased from Addgene Company, product number 62226.
• Primer P1 was designed to introduce a strong promoter trc, and BamHI and SphI restriction endonuclease sites were designed at the 5' ends of primers P1 and P2 respectively.
• the PCR program was: denaturation at 98°C for 30 seconds, annealing at 65°C for 15 seconds, extension at 72°C for 90 seconds, 26 cycles to obtain a P trc -panBC gene fragment of approximately 1800 bp.
• the Ptrc -panBC product obtained by PCR amplification was identified and recovered by gel electrophoresis, and then double-digested with BamHI and SphI, and simultaneously double-digested the pACYC184 plasmid.
• the above-mentioned PCR electrophoresis band was recovered by cutting the gel, and the amplified DNA fragment of the P trc -panBC gene and the pACYC184 plasmid were double-digested using restriction endonucleases BamHI and SphI.
• the double-digested Ptrc-panBC and pACYC184 plasmids were recovered by gel electrophoresis.
• the ligation products were chemically transformed into Escherichia coli DH5 ⁇ competent cells. After recovery for 1 hour, the plasmids were spread on chloramphenicol plates. The coated plate was placed in a 37°C incubator for 12 hours, a single colony was picked for passage, and the recombinant plasmid was extracted and sequenced to obtain the correct recombinant plasmid pACYC184-panBC.
• the sequence obtained by PCR amplification is shown in SEQ ID No. 4, in which 11nt-45nt is the PJ23119 promoter and 66nt-977nt is the coding sequence of the panE gene. , 988nt-1731nt is the terminator sequence.
• the promoter PJ23119 was designed on the amplification primer P3, the terminator L3S2P56 sequence was designed on the primer P4, and SphI and BsaBI restriction endonuclease sites were designed on the 5' ends of the primers P3 and P4 respectively.
• the PJ23119-panE product was amplified using the above PCR reaction conditions.
• the mutated N20 sequence is CTTTCCAAGC TGGGTCTACC, targeting the avtA gene.
• the mutated pTargetF was named pTargetFavtA.
• P16 CGCATACATT GATGCGTATG (as shown in SEQ ID NO. 25).
• the aspartate ⁇ -decarboxylase gene panD (shown in SEQ ID No. 1) derived from Bacillus licheniformis was synthesized in a gene synthesis company. When the above panD gene sequence was customized and synthesized, it was removed through synonymous codon substitution. XbaI and HindIII restriction enzyme sequences. When custom-synthesizing the above panD gene sequence, the same BCD2 sequence (as shown in SEQ ID No. 2) was synthesized before each panD sequence, and XbaI and HindIII restriction enzymes were added to both ends of the BCD2-panD sequence. site. The synthesized sequence is ligated into the vector.
• the above synthesized BCD2-panD vector and pET28a(+) plasmid were double digested using restriction endonucleases XbaI and HindIII, and the digested BCD2-panD gene fragment and linearized vector segment were recovered by gel electrophoresis, and further used T4 Ligase connects the two fragments, and the ligation product is transformed into E. coli DH5 ⁇ competent cells, and screened on LB plates containing 50 mg/L kanamycin to obtain transformants containing the recombinant plasmid. After the transformants were expanded, the plasmid was extracted and sent for sequencing to verify that the correct plasmid pET28a-BCD2-panDBl was obtained.
• Use primers P7 and P8 to amplify the upstream sequence of avtA gene use primers P9 and P10 to amplify the PL promoter, use primers P11 and P12, and use pET28a-BCD2-panDBl as a template to amplify the BCD2-panDBl-Ter gene fragment, use primers P13 and P14 amplified the downstream sequence of avtA gene.
• the above four fragments are connected through overlapping PCR to obtain a combination of four DNA fragments DonorBl (shown as SEQ ID No. 5), which is used as a template for gene editing. Among them, 1nt-312nt of SEQ ID No.
• 5 is the upstream sequence of the target gene avtA gene
• 313nt-474nt is the PL promoter
• 475nt-560nt is the BCD2 sequence
• 560nt-943nt is the panDB1 sequence
• 944nt-995nt is the terminator sequence
• 996- 1261nt is the downstream sequence of avtA gene.
• the pCas9 plasmid was transformed into MG1655, spread on a plate containing 50 mg/L kanamycin resistance, and cultured at 30°C to obtain strain MG655/pCas9.
• the OD600 of the medium is 0.2, add arabinose with a final concentration of 10mM for induction.
• the OD600 is 0.45 Preparation of competent cells.
• the engineered bacterium E.coli MG1655 avtA:panDBl/pCas was inserted into a non-resistant LB liquid medium, cultured at 37°C for 12 hours, diluted and spread on an LB plate to obtain the engineered bacterium E.coli MG1655 avtA:panDBl that eliminated the pCas plasmid.
• the gene panD is inserted into the coding sequence of the chromosomal avtA gene, resulting in the inactivation of AvtA and weakening the valine competition metabolism pathway.
• the ilvG gene of wild-type E. coli K12 MG1655 is mutated, and the acetolactate synthase encoded by it is inactive.
• the present invention introduces the active ilvG gene of E. coli BL21 into the chromosome of E. coli MG1655, thereby improving the synthesis of acetolactate, the precursor of VB5.
• the present invention inserts the ilvG + M gene derived from E. coli BL21 into the chromosome of E. coli K12 MG1655, uses the trc strong promoter to regulate the transcription initiation of ilvG + M, and uses the terminator Ter to regulate the transcription termination of ilvG + M.
• the ilvG + M gene is integrated into another N20 target sequence of the avtA gene.
• the mutation kit and primers P17 and P18 mutate the pTargetF vector, and the mutated pTargetF is named pTargetFavtA1.
• P21 TTGA CAATTAATCATCCGGCTCGTATAATGTGTGGACAAGATT CAGGACGGGG AAC (as shown in SEQ ID NO.30);
• P25 CACGTTCGGA TATGAACTG (as shown in SEQ ID NO.34);
• P26 CGTCAAGCTT CAGCAACTC (as shown in SEQ ID NO.35).
• Primers P19 and P20 were used to amplify the avtA gene upstream sequence
• primers P21 and P22 were used to amplify the ilvG + M sequence of E.coli BL21
• primers P23 and P24 were used to amplify the avtA gene downstream sequence.
• the trc promoter TTGACAATTAATCATCCGGCTCGTATAATGTGTGGA was introduced through primers P20 and P21
• the terminator sequence CCAGAAAAGAGACGCT TTTAG AGCGTCTTTTTTCGTTTT was introduced through primers P22 and P23.
• Use overlapping PCR to connect the above three fragments to obtain the combination DonorilvGM (shown as SEQ ID No.
• 1-305nt of SEQ ID No. 6 is the upstream sequence of the target gene avtA gene
• 306nt-341nt is the trc promoter
• 367nt-2013nt is the coding sequence of the ilvG + gene derived from E.coli BL21
• 2010nt-2273nt is the ilvM gene Coding sequence
• 2274-2328 is the terminator sequence
• 2329-2629 is the downstream sequence of avtA gene.
• non-resistant LB liquid culture medium culture at 37°C for 12 hours, dilute and spread on LB plates, and obtain the engineered bacteria E.coli MG1655 avtA:panDBl-ilvG + M that eliminates the pCas plasmid.
• active ilvG + M By integrating active ilvG + M on the chromosome, the synthesis of the VB5 precursor acetolactate is increased.
• pTargetFcadA The mutation kit and primers P27 and P28 mutate the N20 sequence of pTargetF vector. After mutation, pTargetF is named pTargetFcadA.
• P33 A CAGGCAACCTTTTATTCACCATCTTAATCATGCTAAGGAG (as shown in SEQ ID NO. 42);
• primers P29 and P30 were used to amplify the upstream sequence of the cadA gene
• primers P31 and P32 were used to amplify the gapA promoter
• primers P35 and P36 were used to amplify the downstream sequence of the cadA gene.
• the aspDH gene containing RBS and terminator was synthesized from a gene synthesis company, and primers P33 and P34 were used to amplify the RBS-aspDH-Ter sequence.
• the above four fragments were connected through overlapping PCR to obtain the combination DonoraspDH (shown as SEQ ID No. 7), which was used as a template for gene editing.
• 1-210nt of SEQ ID No. 7 is the upstream sequence of the target gene cadA gene
• 211nt-480nt is the gapA promoter
• 481nt-509nt is the RBS sequence
• 510nt-1307nt is derived from the coding sequence of the aspDH gene of Delftia sp.
• Csl-4 As shown in SEQ ID No. 56
• 1308nt-1360nt are the terminator sequence
• 1361-1535 are the downstream sequences of the cadA gene.
• primers P29 and P30 were used to amplify the upstream sequence of the cadA gene
• primers P31 and P39 were used to amplify the gapA promoter
• primers P40 and P41 were used to amplify the aspC gene
• primers P42 and P36 were used to amplify the cadA gene. downstream sequence.
• the above four fragments were connected by overlapping PCR to obtain the combination DonoraspC (shown as SEQ ID No. 8), which was used as a template for gene editing. 1-210nt of SEQ ID No.
• P42 CACTTTGCC AGAAATCCAG GAGCTGTG CGAAGAAATT AGC (as shown in SEQ ID NO. 51).
• primers P29 and P30 were used to amplify the upstream sequence of the cadA gene
• primers P31 and P43 were used to amplify the gapA promoter
• primers P44 and P45 were used to amplify the aspA gene
• primers P46 and P36 were used to amplify the cadA gene. downstream sequence.
• the above four fragments were connected by overlapping PCR to obtain the combination DonoraspA (shown as SEQ ID No. 9), which was used as a template for gene editing. 1-210nt of SEQ ID No.
• P44 A CAGGCAACCTTTTATTCACGCAGCTTGAAAAAGAAGGTTC (as shown in SEQ ID NO. 53);
• the vector pACYC184-panBCE constructed above was transformed into the above-mentioned engineering bacteria E.coli MG1655 avtA:panDBl-ilvG + M-aspDH, E.coli MG1655 avtA:panDBl-ilvG + M-aspC and E.coli MG1655 avtA:panDBl-ilvG + M-aspA, the engineering bacteria E.coli MG1655 avtA:panDBl-ilvG + M-aspDH/pACYC184-panBCE, E.coli MG1655 avtA:panDBl-ilvG + M-aspC/pACYC184-panBCE and E.coli MG1655 were obtained respectively.
• avtA:panDBl-ilvG + M-aspA/pACYC184-panBCE used for fermentation to produce VB5.
• E.coli MG1655 avtA:panDBl-ilvG + M-aspDH/pACYC184-panBCE Take the test strains engineering bacteria E.coli MG1655 avtA:panDBl-ilvG + M-aspDH/pACYC184-panBCE, E.coli MG1655 avtA:panDBl-ilvG + M-aspC/pACYC184-panBCE and E.coli MG1655 avtA:panDBl-ilvG + M-aspA/pACYC184-panBCE, streak inoculated on a solid LB medium plate containing 34 mg/L chloramphenicol, and incubate at 37°C for 12 hours.
• Fermentation medium MOPS 80g/L, glucose 20.0g/L, ammonium sulfate 10.0g/L, potassium dihydrogen phosphate 2.0g/L, magnesium sulfate heptahydrate 2.0g/L, yeast powder 5.0g/L, trace elements mixed
• the liquid is 5mL/L, and the balance is water.
• Trace element mixture FeSO 4 ⁇ 7H 2 O10g/L, CaCl 2 1.35g/L, ZnSO 4 ⁇ 7H 2 O2.25g/L, MnSO 4 ⁇ 4H 2 O0.5g/L, CuSO 4 ⁇ 5H 2 O1g/ L, (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O0.106g/L, Na 2 B 4 O 7 ⁇ 10H 2 O0.23g/L, CoCl 2 ⁇ 6H 2 O0.48g/L, 35% HCl10mL/ L, the balance is water.
• the present invention found that engineering bacteria that overexpress the aspDH gene are more conducive to improving the fermentation yield of VB5 than overexpressing aspC and aspA.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Tropical Medicine & Parasitology (AREA)
• Virology (AREA)
• Gastroenterology & Hepatology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (1)

Family

ID=84841728

Family Applications (1)

Country Status (5)

Families Citing this family (3)

Citations (8)

Family Cites Families (2)

• 2022
  • 2022-03-07 CN CN202210214588.5A patent/CN115595314B/zh active Active
• 2023
  • 2023-02-17 WO PCT/CN2023/076669 patent/WO2023169176A1/zh not_active Ceased
  • 2023-02-17 EP EP23765754.9A patent/EP4458960A1/en active Pending
  • 2023-02-17 JP JP2024553524A patent/JP2025516089A/ja active Pending
  • 2023-02-17 US US18/841,611 patent/US20260002182A1/en active Pending

Patent Citations (8)

Non-Patent Citations (8)

Also Published As

Similar Documents

Legal Events