WO2023169176A1 - 表达天冬氨酸脱氢酶的工程菌及发酵生产维生素b5的方法 - Google Patents

表达天冬氨酸脱氢酶的工程菌及发酵生产维生素b5的方法 Download PDF

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WO2023169176A1
WO2023169176A1 PCT/CN2023/076669 CN2023076669W WO2023169176A1 WO 2023169176 A1 WO2023169176 A1 WO 2023169176A1 CN 2023076669 W CN2023076669 W CN 2023076669W WO 2023169176 A1 WO2023169176 A1 WO 2023169176A1
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nucleotide sequence
sequence shown
gene
seq
aspartate
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French (fr)
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刘树文
温廷益
孙佳慧
李忠财
张芸
邓爱华
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中国科学院微生物研究所
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    • 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
    • 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
    • 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)

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.

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Abstract

一种表达天冬氨酸脱氢酶基因aspDH的大肠杆菌，及其用于发酵生产维生素B5（VB5）的方法。通过比较VB5的发酵产量，过表达aspDH基因的效果最好。与高污染的化学法生产维生素B5相比，生物法生产维生素B5，具有原料可再生，废渣、废水和废气易于处理和资源化利用等优点，从而在实践上可用于维生素B5的工业化生产，具有重要的应用价值。

Description

表达天冬氨酸脱氢酶的工程菌及发酵生产维生素B5的方法 技术领域
本发明涉及微生物领域，特别涉及表达天冬氨酸脱氢酶的工程菌及发酵生产维生素B5的方法。
背景技术
维生素B5(Vitamin B5，VB5)又称D-泛酸(D-Pantothenic acid)，是一种水溶性维生素，是辅酶A及酰基载体蛋白的组成部分，作为70多种酶的辅助因子参与糖、脂肪、蛋白质和能量代谢，具有重要的生理代谢调控作用。VB5主要用于动物饲料添加剂、食品添加剂和医药原料药，随着VB5新功能的发现和应用领域的拓展，其市场需求仍将呈现稳定增长的趋势。
中国是VB5生产和出口的第一大国，工业生产VB5方法为化学合成法，企业基本采用异丁醛-甲醛-氢氰酸法合成DL-泛解酸内酯，DL-泛解酸内酯进一步通过化学或酶法拆分获得L-泛解酸内酯，最后L-泛解酸内酯与以丙烯腈为原料生产的β-丙氨酸合成VB5。化学合成VB5的主要原料易燃、易爆、剧毒，生产过程中会产生含氰废水，处理难度大，导致VB5成为重污染产业。
近几年我国经济发展进入绿色环保新常态，环保对VB5产业的影响已经逐步显现。在大规模高强度的环保治理下，高污染的VB5企业限产甚至停产，市场供应短缺，价格暴涨，限制了下游饲料、食品和医药行业的健康发展。在高污染的VB5生产技术没有重大改进之前，这样的供需局面仍会长期持续，因此，VB5绿色制造技术的创新迫在眉睫！
微生物发酵法生产VB5，不仅以可再生的葡萄糖为原料，而且生产过程中形成的废渣、废水和废气易于处理和资源化利用，可有效解决VB5产业的高污染问题。微生物利用葡萄糖合成VB5的代谢途径的调控机制复杂，发酵产量极低。β-丙氨酸作为C3底物与D-泛解酸合成VB5。为了提高VB5的发酵产量，需要在发酵培养基中外源补加大量的β-丙氨酸(Sahm,H.,et al.,(1999)Appl Environ Microb,65,1973-1979；Dusch,N.,et al.,(1999)Appl Environ Microb,65,1530-1539；Zhang,B.,et al.,(2019)Food Chemistry,294,267-275.)。β-丙氨酸 可以通过L-天冬氨酸脱羧产生，因此，增强天冬氨酸的生物合成有望提高发酵法生产VB5的产量。
发明内容
有鉴于此，本发明通过在发酵法生产VB5的大肠杆菌中分别过表达上述三个酶，发现AspDH比AspC和AspA更有利于提高VB5的发酵产量。
为了实现上述发明目的，本发明提供以下技术方案：
第一方面，本发明提供了增强天冬氨酸脱氢酶基因aspDH的表达在生产维生素B5中的应用；
作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl-4(Delftia sp.Csl-4)。
在本发明的一些具体实施方案中，所述天冬氨酸脱氢酶基因aspDH具有：
(I)、如SEQ ID No.55所示的核苷酸序列；或
(II)、如(I)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(I)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(III)、与(I)或(II)所示的核苷酸序列至少有80％同源性的核苷酸序列。
在本发明的一些具体实施方案中，还包括：
(1)、在cadA基因中插入强启动子和/或强RBS，其中强启动子为PgapA，强RBS为BCD2；
作为优选，所述BCD2具有：
(A)、如SEQ ID No.2所示的核苷酸序列；或
(B)、如(A)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(A)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(C)、与(A)或(B)所示的核苷酸序列至少有80％同源性的核苷酸序列；
和/或
(2)、表达了来源于大肠杆菌BL21的ilvGM基因；和/或
(3)、表达了来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD；和/或
作为优选，所述来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD具有：
(a)、如SEQ ID No.1所示的核苷酸序列；或
(b)、如(a)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(a)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(c)、与(a)或(b)所示的核苷酸序列至少有80％同源性的核苷酸序列；和/或
(4)、增加了panB、panC和/或panE基因的拷贝数。
第二方面，本发明还提供了表达载体，包含天冬氨酸脱氢酶基因aspDH；作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl-4(Delftia sp.Csl-4)；
作为优选，所述天冬氨酸脱氢酶基因aspDH具有：
(I)、如SEQ ID No.56所示的核苷酸序列；或
(II)、如(I)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(I)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(III)、与(I)或(II)所示的核苷酸序列至少有80％同源性的核苷酸序列。
在本发明的一些具体实施方案中，所述表达载体还包括：
(i)、强启动子和/或强RBS；
其中强启动子为PgapA，强RBS为BCD2；
作为优选，所述BCD2具有：
(A)、如SEQ ID No.2所示的核苷酸序列；或
(B)、如(A)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(A)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(C)、与(A)或(B)所示的核苷酸序列至少有80％同源性的核苷酸序列；
和/或
(ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
(iii)、来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD；和/或
作为优选，所述来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD具有：
(a)、如SEQ ID No.1所示的核苷酸序列；或
(b)、如(a)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(a)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(c)、与(a)或(b)所示的核苷酸序列至少有80％同源性的核苷酸序列；和/或
(iv)、增加了拷贝数的panB、panC和/或panE基因。
第三方面，本发明还提供了宿主，表达了天冬氨酸脱氢酶基因aspDH；
作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl-4(Delftia sp.Csl-4)；
作为优选，所述天冬氨酸脱氢酶基因aspDH具有：
(I)、如SEQ ID No.56所示的核苷酸序列；或
(II)、如(I)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(I)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(III)、与(I)或(II)所示的核苷酸序列至少有80％同源性的核苷酸序列。
在本发明的一些具体实施方案中，所述宿主还包括：
(i)、强启动子和/或强RBS；
其中强启动子为PgapA，强RBS为BCD2；
作为优选，所述BCD2具有：
(A)、如SEQ ID No.2所示的核苷酸序列；或
(B)、如(A)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(A)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(C)、与(A)或(B)所示的核苷酸序列至少有80％同源性的核苷酸序列；
和/或
(ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
(iii)、来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD；和/或
作为优选，所述来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD具有：
(a)、如SEQ ID No.1所示的核苷酸序列；或
(b)、如(a)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(a)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(c)、与(a)或(b)所示的核苷酸序列至少有80％同源性的核苷酸序列；和/或
(iv)、增加了拷贝数的panB、panC和/或panE基因。
在本发明的一些具体实施方案中，所述宿主转染或转化如权利要求4或5所述的表达载体；
作为优选，所述宿主源自大肠杆菌，优选为大肠杆菌K12，更优选为大肠杆菌K12 MG1655株。
第四方面，本发明还提供了所述表达载体或所述宿主在生产维生素B5中的应用。
第五方面，本发明还提供了生产维生素B5的方法，以所述宿主为发酵菌株，发酵，收集发酵液，离心取上清液，获得维生素B5。
本发明公开了一种表达天冬氨酸脱氢酶基因aspDH的大肠杆菌，及其用于发酵生产维生素B5(VB5)的方法。本发明在VB5工程菌中分别增强了三种合成L-天冬氨酸的途径，分别为aspC基因编码的天冬氨酸氨基转移酶，将谷氨酸的氨基转移到草酰乙酸产生L-天冬氨酸和酮戊二酸；aspA基因编码的 天冬氨酸氨裂解酶，催化铵和延胡索酸产生天冬氨酸；aspDH基因编码的天冬氨酸脱氢酶，催化草酰乙酸和铵合成天冬氨酸。通过比较VB5的发酵产量，过表达aspDH基因的效果最好。与高污染的化学法生产维生素B5相比，本发明生物法生产维生素B5，具有原料可再生，废渣、废水和废气易于处理和资源化利用等优点，从而在实践上可用于维生素B5的工业化生产，具有重要的应用价值。
具体实施方式
本发明公开了表达天冬氨酸脱氢酶的工程菌及发酵生产维生素B5的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
大肠杆菌有两条产生天冬氨酸的途径，一条可以通过aspC基因编码的天冬氨酸氨基转移酶，将谷氨酸的氨基转移到草酰乙酸产生L-天冬氨酸和酮戊二酸；另一条通过aspA基因编码的天冬氨酸氨裂解酶，催化铵和延胡索酸产生天冬氨酸。此外，在一些古菌中还发现了催化草酰乙酸和铵合成天冬氨酸的天冬氨酸脱氢酶(Aspartate dehydrogenase，AspDH)。本发明通过在发酵法生产VB5的大肠杆菌中分别过表达上述三个酶，发现AspDH比AspC和AspA更有利于提高VB5的发酵产量。
为了突破高效合成维生素B5的代谢瓶颈，发明人比较了3种提高天冬氨酸合成的途径，发现异源的天冬氨酸脱氢(aspDH基因编码)途径更适合自身的天冬氨酸转氨途径(aspC基因编码)和天冬氨酸氨裂解途径(aspA基因编码)。
一种天冬氨酸脱氢酶(AspDH)，催化草酰乙酸与铵根以及NAD(P)H反应生成天冬氨酸与水以及NAD(P)+的可逆反应，由以下微生物中的一种或几种产生：绿脓杆菌(Pseudomonas aeruginosa)、克雷伯氏肺炎菌(Klebsiella pneumoniae)、变形斑沙雷氏菌(Serratia proteamaculans)、极端嗜热菌海栖热袍菌(Thermotoga maritima)、需盐色盐杆菌 (Chromohalobacter salexigens)、鲍氏不动杆菌(Acinetobacter baumannii)、戴尔福特菌Csl-4(Delftia sp.Csl-4)、人苍白杆菌(Ochrobactrum anthropi)、柄杆菌(Caulobacter sp.)、马氏甲烷嗜盐菌(Methanohalophilus mahii)、柴氏海洋玫瑰杆菌(Dinoroseobacter shibae)、嗜酸产甲烷菌(Methanosphaerula palustris)、瘤胃甲烷短杆菌(Methanobrevibacter ruminantium)等。
在表达的天冬氨酸脱氢酶方面，本发明使用强启动子调控aspDH基因的表达。所述启动子可以是强启动子可以是以下启动子或其突变体：L启动子、trc启动子、T5启动子、lac启动子、tac启动子、T7启动子或gapA启动子。此外，本发明使用了活性较强RBS序列调控aspDH基因的翻译起始。
本发明将上述高强度的翻译起始和转录起始调控的aspDH基因整合到发酵法生产VB5的大肠杆菌染色体上，实现外源基因的表达。整合位点为cadA基因，该基因编码赖氨酸脱羧酶，理论上对VB5的生物合成没有影响。
在发酵法生产VB5的大肠杆菌染色体上相同的cadA基因位点，分别整合aspA和aspC基因，并使用相同的启动子调控转录，比较过表达aspA、aspC和aspDH基因的VB5合成的影响。
本发明所述的发酵法生产VB5的大肠杆菌，还过表达了VB5终端合成途径上的panB、panC和panE基因。大肠杆菌的panB基因编码酮泛解酸羟甲基转移酶，催化底物a-酮异戊酸增加一个甲基形成酮泛解酸。酮泛解酸由panE基因编码的酮泛解酸还原酶还原为泛解酸。由panC基因编码的泛酸合成酶进一步催化泛解酸和β-丙氨酸缩合形成VB5。
本发明所用的大肠杆菌为K12 MG1655株，其ilvG基因突变失活。因此本发明引入了大肠杆菌BL21的具有活性的ilvG基因，提高了VB5的前体乙酰乳酸合成供应。本发明在大肠杆菌K12 MG1655的染色体上插入了来源于大肠杆菌BL21的ilvG+M基因，且使用trc强启动子调控ilvG+M的转录起始，使用终止子Ter调控ilvG+M的转录终止。ilvG+M基因在染色体的插入位点为avtA基因的编码序列，导致AvtA失活，弱化了缬氨酸的合成，从而弱化了VB5的竞争途径，有利于VB5的生物合成。
在工程菌avtA基因上还整合了源于地衣芽孢杆菌(Bacillus licheniformis)的panD基因。使用相同的强启动子PPL和BCD2分别调控转录和翻译起始。
发酵生产VB5的方法，培养基包含碳源、氮源、无机离子、抗生素和其它的营养因子。作为碳源，可以使用葡萄糖、乳糖、半乳糖等糖类。作为无机氮源，可以使用氨水、硫酸铵、磷酸铵、氯化铵等无机氮源；作为有机氮源可以使用玉米浆、豆粕水解液、毛发粉、酵母提取物、蛋白胨等有机氮源。无机离子包含铁、钙、镁、锰、钼、钴、铜、钾等离子中的一种或多种。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器、试剂，可参见《分子克隆实验指南(第3版)》(科学出版社)、《微生物学实验(第4版)》(高等教育出版社)以及相应仪器和试剂的厂商说明书等参考。
若说明书中记载的序列与序列表中不一致，则以说明书中记载的序列为准。
大肠杆菌K12 MG1655：ATCC编号为700926。pACYC184质粒：NEB公司，产品目录号E4152S。质粒pcas9购自Addgene公司，货号62225；质粒pTargetF购自Addgene公司，货号62226。
下面结合实施例，进一步阐述本发明：
实施例1检测方法
使用HPLC法定量测定发酵液VB5的产量，具体方法如下。取发酵液上清，加入纯净水稀释到适当浓度，用0.22μm滤膜过滤。使用色谱柱为Agilent ZORBAX SB-Aq，4.6ⅹ250mm，柱温为30℃，检测波长为210nm，流动相流速为1mL/min。流动相为3.12g/L NaH2PO4·2H2O，使用磷酸调节pH至2.2。以从sigma公司购买的泛酸钙(VB5)为标准品，测定0.1-0.5g/L泛酸钙的浓度与光吸收值的标准曲线。
实施例2发酵生产VB5的工程菌的构建
以P1和P2为引物，以野生型大肠杆菌K12 MG1655菌株的基因组DNA为模板，使用高保真聚合酶KAPA HiFiTM HotStar，PCR扩增的核苷酸序列如SEQ ID No.3所示，其中10nt-45nt为启动子trc，74nt-868nt为panB基因的编 码序列，880nt-1731nt为panC基因的编码序列。引物P1上设计引入强启动子trc，P1和P2引物5’端分别设计BamHI和SphI限制性核酸内切酶位点。PCR程序为：98℃变性30秒，65℃退火15秒，72℃延伸90秒，26个循环，获得约1800bp的Ptrc-panBC基因片段。
P1：
(如SEQ ID No.10所示，下划线所示序列为BamHI酶切识别位点，斜体为启动子trc的序列)
P2：5’-ACATGCATGC CCTGTGTTAT GACAGATGAC-3’
(如SEQ ID No.11所示，下划线所示序列为SphI酶切识别位点)
PCR扩增得到的Ptrc-panBC产物，凝胶电泳鉴定回收后，使用BamHI和SphI双酶切，同时双酶切pACYC184质粒。切胶回收上述PCR电泳条带,使用限制性内切酶BamHI和SphI双酶切上述扩增的Ptrc-panBC基因的DNA片段和pACYC184质粒。凝胶电泳回收双酶切的Ptrc-panBC和pACYC184质粒，使用T4连接酶连接后，连接产物化学转化至大肠杆菌DH5α感受态细胞，复苏1小时后涂布氯霉素平板。涂布后的平板置于37℃培养箱12小时，挑取单菌落传代，提取重组质粒后进行测序，获得正确的重组质粒pACYC184-panBC。
以大肠杆菌K12 MG1655的基因组为模板，以P3和P4为引物，PCR扩增得到的序列如SEQ ID No.4所示，其中11nt-45nt为PJ23119启动子，66nt-977nt为panE基因的编码序列，988nt-1731nt为终止子序列。扩增引物P3上设计启动子PJ23119，引物P4上设计终止子L3S2P56序列，P3和P4引物5’端分别设计SphI和BsaBI限制性核酸内切酶位点。使用上述的PCR反应条件，扩增得到的PJ23119-panE产物，凝胶电泳鉴定回收后，使用SphI和BsaBI双酶切，同时双酶切pACYC184-Ptrc-panBC质粒。凝胶电泳回收双酶切的PJ23119-panE和pACYC184-panBC质粒，使用T4连接酶连接后，连接产物化学转化至大肠杆菌DH5α感受态细胞，复苏1小时后涂布氯霉素平板。涂布后的平板置于37℃培养箱12小时，挑取单菌落传代，提取重组质粒后进 行测序，获得正确的重组质粒pACYC184-panBCE，从而获得了过表达维生素B5终端合成途径基因的重组质粒。
P3：
(如SEQ ID No.12所示，下划线所示序列为SphI酶切识别位点，斜体为启动子J23119的序列)
P4：
(如SEQ ID No.13所示，下划线所示序列为BsaBI酶切识别位点，斜体为L3S2P56终止子序列)
应用已报道的包含pCas9和pTargetF载体的CRISPR-Cas9基因编辑系统(Jiang,Y.,Chen,B.,Duan,C.L.,Sun,B.B.,Yang,J.J.,and Yang,S.(2015)Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System,Appl Environ Microb 81,2506-2514.)。
使用NEB公司的基因突变试剂盒(Site-Directed Mutagenesis Kit，货号E0552S)，按照试剂盒说明书设计引物P5和P6突变pTargetF载体。突变后的N20序列为CTTTCCAAGC TGGGTCTACC，靶向avtA基因。突变后的pTargetF命名为pTargetFavtA。
P5：TGGGTCTACCG TTTTAGAGCT AGAAATAGC(如SEQ ID NO.14所示)；
P6：GCTTGGAAAG GACTAGTATT ATACCTAGG(如SEQ ID NO.15所示)；
P7:CG GACTGGAAGA AGATCTG(如SEQ ID NO.16所示)；
P8:TTTCTTAGAC GTCGGAATTG AGACTCATGC ACAGCACGA(如SEQ ID NO.17所示)；
P9:TCGTGCTGT GCATGAGTCTCAATTCCGACGTCTAAGAAAC(如SEQ ID NO.18所示)；
P10:GATCTCCTTT TTAAGTGAAC TTGGGGTCAG TGCGTCCTGCTGAT(如SEQ ID NO.19所示)；
P11:ATCAGCAGGACGCACTGACCCCAAGTTCACTTAAAAAGGAGATC(如SEQ ID NO.20所示)；
P12:TGCCGTTCAT ATTGGTGATG CAAAAAACCC CTCAAGACC(如SEQ ID NO.21所示)；
P13:GGTCTTGAGGGGTTTTTTGCATC ACCAATATGAACGGCA(如SEQ ID NO.22所示)；
P14:GCTGATAGAG CTGCTTGGT(如SEQ ID NO.23所示)；
P15:GGAGCTACTC ACACTGCTTG(如SEQ ID NO.24所示)；
P16:CGCATACATT GATGCGTATG(如SEQ ID NO.25所示)。
在基因合成公司合成了来源于地衣芽孢杆菌Bacillus licheniformis天冬氨酸α-脱羧酶基因panD(如SEQ ID No.1所示)，在定制合成上述panD基因序列时，通过同义密码子替换去除XbaI和HindIII限制性内切酶序列。在定制合成上述panD基因序列时，在每个panD序列前同时合成了相同的BCD2序列(如SEQ ID No.2所示)，同时在BCD2-panD序列的两端加入XbaI和HindIII限制性酶切位点。合成后的序列连接到载体上。使用限制性内切酶XbaI和HindIII双酶切上述合成BCD2-panD的载体和pET28a(+)质粒，凝胶电泳回收得到酶切后的BCD2-panD的基因片段和线性化载体段，进一步使用T4连接酶连接这两个片段，连接产物转化至大肠杆菌DH5α感受态细胞，在含有50mg/L卡那霉素的LB平板上筛选，获得含有重组质粒的转化子。转化子扩培后提取质粒并将其送测序，验证获得正确的质粒pET28a-BCD2-panDBl。
使用引物P7和P8扩增avtA基因上游序列，使用引物P9和P10扩增PL启动子，使用引物P11和P12，以pET28a-BCD2-panDBl为模板扩增获得BCD2-panDBl-Ter基因片段，使用引物P13和P14扩增avtA基因下游游序列。通过重叠PCR连接上述4个片段，获得4个DNA片段的组合体DonorBl(如SEQ ID No.5所示)，作为基因编辑的模板。其中SEQ ID No.5的1nt-312nt为靶基因avtA基因上游序列，313nt-474nt为PL启动子，475nt-560nt为BCD2序列，560nt-943nt为panDBl序列，944nt-995nt为终止子序列，996-1261nt为avtA基因下游游序列。
将pCas9质粒转化入MG1655涂布含有50mg/L卡那霉素抗性平板，30℃培养，获得菌株MG655/pCas9。挑取MG1655/pCas9菌苔于50mL含卡那霉素的LB的500mL摇瓶中，30℃，220rpm培养，当培养基OD600为0.2时加入终浓度为10mM的阿拉伯糖进行诱导，OD600为0.45时制备感受态细胞。取2微升pTargetFavtA质粒和10微升DonorBs模板DNA，电转化至MG655/pCas9感受态细胞，涂布含有50mg/L卡那霉素和50mg/L壮观霉素的双抗性平板，30℃培养。使用引物P15和P16鉴定在avtA基因上整合PPL-BCD2-panD-Ter的单菌落，测序验证大小正确的PCR产物。挑选测序正确的单菌落，加入0.2mM的IPTG培养，消除pTargetFavtA质粒，分别获得工程菌E.coli MG1655 avtA:panDBl/pCas仍按照上述方法制备感受态备用。
工程菌E.coli MG1655 avtA:panDBl/pCas接入无抗性的LB液体培养基，37℃培养12小时，稀释涂布LB平板，分别获得消除pCas质粒的工程菌E.coli MG1655 avtA:panDBl。基因panD插入染色体avtA基因的编码序列，导致AvtA失活，弱化了缬氨酸竞争代谢途径。
野生型大肠杆菌K12 MG1655的ilvG基因突变，其编码的乙酰乳酸合成酶没有活性。本发明在大肠杆菌MG1655的染色体上引入了大肠杆菌BL21的具有活性的ilvG基因，提高了VB5的前体乙酰乳酸的合成。本发明在大肠杆菌K12 MG1655的染色体上插入了来源于大肠杆菌BL21的ilvG+M基因，且使用trc强启动子调控ilvG+M的转录起始，使用终止子Ter调控ilvG+M的转录终止。ilvG+M基因整合到avtA基因的另外一个N20靶序列。使用上述突变试剂盒和引物P17和P18突变pTargetF载体，突变后pTargetF命名为pTargetFavtA1。
P17:ACGGTCCACAG TTTTAGAGCT AGAAATAGC(如SEQ ID NO.26所示)；
P18:CGTAGTTACA GACTAGTATT ATACCTAGG(如SEQ ID NO.27所示)；
P19:GGCAGAAAAT CAGCCAGTTC(如SEQ ID NO.28所示)；
P20:TCCACACATT ATACGAGCCG GATGATTAAT TGTCAAGAACTCTGTAGCAA GGAAGG(如SEQ ID NO.29所示)；
P21:TTGA CAATTAATCATCCGGCTCGTATAATGTGTGGACAAGATT CAGGACGGGG AAC(如SEQ ID NO.30所示)；
P22:CGAAAAAAGA CGCTCTAAAA GCGTCTCTTT TCTGGTATATTCCTTTTGCG CTCAG(如SEQ ID NO.31所示)；
P23:CAGAAAAGAGACGCTTTTAGAGCGTCTTTTTTCGTTTTGGAGCTACTC ACACTGCTTG(如SEQ ID NO.32所示)；
P24:GCCAATATGC AGATGCTCATGAGCATCTGCATATTGG C(如SEQ ID NO.33所示)；
P25:CACGTTCGGA TATGAACTG(如SEQ ID NO.34所示)；
P26:CGTCAAGCTT CAGCAACTC(如SEQ ID NO.35所示)。
使用引物P19和P20扩增avtA基因上游序列，使用引物P21和P22扩增E.coli BL21的ilvG+M序列，使用引物P23和P24扩增avtA基因下游序列。通过引物P20和P21引入trc启动子TTGACAATTAATCATCCGGCTCGTATAATGTGTGGA，通过引物P22和P23引入终止子序列CCAGAAAAGAGACGCTTTTAGAGCGTCTTTTTTCGTTTT。使用重叠PCR连接上述3个片段，获得组合体DonorilvGM(如SEQ ID No.6所示)，作为基因编辑的模板。SEQ ID No.6的1-305nt为靶基因avtA基因上游序列，306nt-341nt为trc启动子，367nt-2013nt为源于E.coli BL21的ilvG+基因的编码序列，2010nt-2273nt为ilvM基因的编码序列，2274-2328为终止子序列，2329-2629为avtA基因下游游序列。
取2微升pTargetFavtA1质粒和10微升DonorilvGM模板DNA，电转化至E.coli MG1655 avtA:panDBl/pCas感受态细胞，涂布含有50mg/L卡那霉素和50mg/L壮观霉素的双抗性平板，30℃培养。使用引物P25和P26鉴定在avtA基因上整合Ptrc-ilvG+M-Ter的单菌落，测序验证大小正确的PCR产物。挑选测序正确的单菌落，加入0.2mM的IPTG培养，消除pTargetFavtA1质粒。进一步接入无抗性的LB液体培养基，37℃培养12小时，稀释涂布LB平板，分别获得消除pCas质粒的工程菌E.coli MG1655 avtA:panDBl-ilvG+M。通过在染色体上整合有活性的ilvG+M，提高了VB5前体乙酰乳酸的合成。
使用上述突变试剂盒和引物P27和P28突变pTargetF载体的N20序列，突变后pTargetF命名为pTargetFcadA。
P27:TCATATCTCCG TTTTAGAGCT AGAAATAGC(如SEQ ID NO.36所示)；
P28:CTATGAACGT GACTAGTATT ATACCTAGG(如SEQ ID NO.37所示)；
P29:GTTGCGT GTTCTGCTTC ATC(如SEQ ID NO.38所示)；
P30:CCAGTTGGTG TTAATGTTTT GCTCCCAACA CATGGGACA(如SEQ ID NO.39所示)；
P31:TGTCC CATGTGTTGG GAGCA AAACATTAACACCAACTGG(如SEQ ID NO.40所示)；
P32:CTCCTTAGCA TGATTAAGAT GGTGAATAAA AGGTTGCCTG T(如SEQ ID NO.41所示)；
P33:ACAGGCAACCTT TTATTCACCATCTTAATCATGCTAAGGAG(如SEQ ID NO.42所示)；
P34:GCTAATTTCT TCGCACAGCT GGACCAAAAC GAAAAAAGAC G(如SEQ ID NO.43所示)；
P35:CGTCTTTTTTCGTTTTGGTCCAGCTGTG CGAAGAAATT AGC(如SEQ ID NO.44所示)；
P36:TCGTCAGTGG TCTGCTTGA(如SEQ ID NO.45所示)；
P37:CTAC TCTTGCGTTG ACCTGA(如SEQ ID NO.46所示)；
P38:GTGACCAGGA GTACAGAAAG(如SEQ ID NO.47所示)。
以大肠杆菌MG1655基因组为模板，使用引物P29和P30扩增cadA基因上游序列，使用引物P31和P32扩增gapA启动子，使用引物P35和P36扩增cadA基因下游序列。从基因合成公司合成了含RBS和终止子的aspDH基因，使用引物P33和P34扩增RBS-aspDH-Ter序列。通过重叠PCR连接上述4个片段，获得组合体DonoraspDH(如SEQ ID No.7所示)，作为基因编辑的模板。SEQ ID No.7的1-210nt为靶基因cadA基因上游序列，211nt-480nt为gapA启动子，481nt-509nt为RBS序列，510nt-1307nt源于戴尔福特菌Csl-4的aspDH基因的编码序列(如SEQ ID No.56所示)，1308nt-1360nt为终止子序列，1361-1535为cadA基因下游序列。
取2微升pTargetFcadA质粒和10微升DonoraspDH模板DNA，电转化至E.coli MG1655 avtA:panDBl-ilvG+M/pCas感受态细胞，涂布含有50mg/L卡那霉素和50mg/L壮观霉素的双抗性平板，30℃培养。使用引物P37和P38鉴定在cadA基因上整合PgapA-aspDH-Ter的单菌落，测序验证大小正确的PCR产物。挑选测序正确的单菌落，加入0.2mM的IPTG培养，消除pTargetFcadA质粒。进一步接入无抗性的LB液体培养基，37℃培养12小时，稀释涂布LB平板，获得消除pCas质粒的工程菌E.coli MG1655avtA:panDBl-ilvG+M-aspDH。
以大肠杆菌MG1655基因组为模板，使用引物P29和P30扩增cadA基因上游序列，使用引物P31和P39扩增gapA启动子，使用引物P40和P41扩增aspC基因，使用引物P42和P36扩增cadA基因下游序列。通过重叠PCR连接上述4个片段，获得组合体DonoraspC(如SEQ ID No.8所示)，作为基因编辑的模板。SEQ ID No.8的1-210nt为靶基因cadA基因上游序列，211nt-480nt为gapA启动子，611nt-1801nt为aspC基因的编码序列，1992-2166为cadA基因下游序列。
取2微升pTargetFcadA质粒和10微升DonoraspC模板DNA，电转化至E.coli MG1655 avtA:panDBl-ilvG+M/pCas感受态细胞，涂布含有50mg/L卡那霉素和50mg/L壮观霉素的双抗性平板，30℃培养。使用引物P37和P38鉴定在cadA基因上整合PgapA-aspC的单菌落，测序验证大小正确的PCR产物。挑选测序正确的单菌落，加入0.2mM的IPTG培养，消除pTargetFcadA质粒。进一步接入无抗性的LB液体培养基，37℃培养12小时，稀释涂布LB平板，获得消除pCas质粒的工程菌E.coli MG1655 avtA:panDBl-ilvG+M-aspC。
P39:GAGATTGCTC TGGAAGGTAT AGTGAATAAA AGGTTGCCTGT(如SEQ ID NO.48所示)；
P40:ACAGGCAACCTT TTATTCACTATACCTTCC AGAGCAATCT C(如SEQ ID NO.49所示)；
P41:GCTAATTTCT TCGCACAGCT CCTGGATTTC TGGCAAAGTG(如SEQ ID NO.50所示)；
P42:CACTTTGCC AGAAATCCAG GAGCTGTG CGAAGAAATT AGC(如SEQ ID NO.51所示)。
以大肠杆菌MG1655基因组为模板，使用引物P29和P30扩增cadA基因上游序列，使用引物P31和P43扩增gapA启动子，使用引物P44和P45扩增aspA基因，使用引物P46和P36扩增cadA基因下游序列。通过重叠PCR连接上述4个片段，获得组合体DonoraspA(如SEQ ID No.9所示)，作为基因编辑的模板。SEQ ID No.9的1-210nt为靶基因cadA基因上游序列，211nt-480nt为gapA启动子，504nt-1940nt为aspA基因的编码序列，2004-2178为cadA基因下游序列。
取2微升pTargetFcadA质粒和10微升DonoraspA模板DNA，电转化至E.coli MG1655 avtA:panDBl-ilvG+M/pCas感受态细胞，涂布含有50mg/L卡那霉素和50mg/L壮观霉素的双抗性平板，30℃培养。使用引物P37和P38鉴定在cadA基因上整合PgapA-aspA的单菌落，测序验证大小正确的PCR产物。挑选测序正确的单菌落，加入0.2mM的IPTG培养，消除pTargetFcadA质粒。进一步接入无抗性的LB液体培养基，37℃培养12小时，稀释涂布LB平板，获得消除pCas质粒的工程菌E.coli MG1655 avtA:panDBl-ilvG+M-aspA。
P43:GAACCTTCTT TTTCAAGCTG CGTGAATAAA AGGTTGCCTG T(如SEQ ID NO.52所示)；
P44:ACAGGCAACCTT TTATTCACGCAGCTTGAAAAA GAAGGTTC(如SEQ ID NO.53所示)；
P45:GCTAATTTCT TCGCACAGCT CTGCTCACAA GAAAAAAGGC(如SEQ ID NO.54所示)；
P46:GCCTTTTTTC TTGTGAGCAGAGCTGTG CGAAGAAATT AGC(如SEQ ID NO.55所示)。
将上述构建的载体pACYC184-panBCE转化至上述工程菌E.coli MG1655 avtA:panDBl-ilvG+M-aspDH、E.coli MG1655 avtA:panDBl-ilvG+M-aspC和E.coli MG1655 avtA:panDBl-ilvG+M-aspA中，分别获得工程菌E.coli MG1655 avtA:panDBl-ilvG+M-aspDH/pACYC184-panBCE、E.coli MG1655 avtA:panDBl-ilvG+M-aspC/pACYC184-panBCE和E.coli MG1655 avtA:panDBl-ilvG+M-aspA/pACYC184-panBCE，用于发酵生产VB5。
















实施例3 VB5工程菌的发酵试验
取试验菌株工程菌E.coli MG1655 avtA:panDBl-ilvG+M-aspDH/pACYC184-panBCE、E.coli MG1655 avtA:panDBl-ilvG+M-aspC/pACYC184-panBCE和E.coli MG1655 avtA:panDBl-ilvG+M-aspA/pACYC184-panBCE，划线接种于含34mg/L氯霉素的固体LB培养基平板，37℃静置培养12小时。挑取平板上的菌苔，接种至LB培养基斜面中，37℃静置培养10-12h。挑取平板上的菌苔，接种至液体LB培养基中，37℃、220rpm振荡培养12h，得到种子液。将种子液按照3％的接种量接种至发酵培养基中，37℃、220rpm震荡培养。
发酵培养基：MOPS 80g/L，葡萄糖20.0g/L、硫酸铵10.0g/L、磷酸二氢钾2.0g/L、七水硫酸镁2.0g/L、酵母粉5.0g/L、微量元素混合液5mL/L，余量为水。微量元素混合液：FeSO4·7H2O10g/L、CaCl21.35g/L、ZnSO4·7H2O2.25g/L、MnSO4·4H2O0.5g/L、CuSO4·5H2O1g/L、(NH4)6Mo7O24·4H2O0.106g/L、Na2B4O7·10H2O0.23g/L、CoCl2·6H2O0.48g/L、35％HCl10mL/L，余量为水。
培养过程中，每隔4h取样一次，用氨水调节反应体系的pH值使其维持在6.8-7.0。使用生物传感分析仪SBA-40D检测葡萄糖含量，当体系中的葡萄糖含量低于5g/L时，补加葡萄糖并使体系中的葡萄糖浓度达到20g/L。培养24h后取样，12000g离心2分钟，取上清液，检测VB5含量(如下表)。
表1
本发明通过在发酵法生产VB5的大肠杆菌中增强三条不同的产生天冬氨酸的途径，发现过表达aspDH基因的工程菌，比过表达aspC和aspA更有利于提高VB5的发酵产量。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 增强天冬氨酸脱氢酶基因aspDH的表达在生产维生素B5中的应用；
    作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl-4(Delftia sp.Csl-4)。
  2. 如权利要求1所述的应用，其特征在于，所述天冬氨酸脱氢酶基因aspDH具有：
    (I)、如SEQ ID No.56所示的核苷酸序列；或
    (II)、如(I)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(I)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (III)、与(I)或(II)所示的核苷酸序列至少有80％同源性的核苷酸序列。
  3. 如权利要求1或2所述的应用，其特征在于，还包括：
    (1)、在cadA基因中插入强启动子和/或强RBS，其中强启动子为PgapA，强RBS为BCD2；
    作为优选，所述BCD2具有：
    (A)、如SEQ ID No.2所示的核苷酸序列；或
    (B)、如(A)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(A)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (C)、与(A)或(B)所示的核苷酸序列至少有80％同源性的核苷酸序列；
    和/或
    (2)、表达了来源于大肠杆菌BL21的ilvGM基因；和/或
    (3)、表达了来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD；和/或
    作为优选，所述来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD具有：
    (a)、如SEQ ID No.1所示的核苷酸序列；或
    (b)、如(a)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(a)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (c)、与(a)或(b)所示的核苷酸序列至少有80％同源性的核苷酸序列；和/或
    (4)、增加了panB、panC和/或panE基因的拷贝数。
  4. 表达载体，其特征在于，包含天冬氨酸脱氢酶基因aspDH；
    作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl-4(Delftia sp.Csl-4)；
    作为优选，所述天冬氨酸脱氢酶基因aspDH具有：
    (I)、如SEQ ID No.56所示的核苷酸序列；或
    (II)、如(I)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(I)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (III)、与(I)或(II)所示的核苷酸序列至少有80％同源性的核苷酸序列。
  5. 如权利要求4所述的表达载体，其特征在于，还包括：
    (i)、强启动子和/或强RBS；
    其中强启动子为PgapA，强RBS为BCD2；
    作为优选，所述BCD2具有：
    (A)、如SEQ ID No.2所示的核苷酸序列；或
    (B)、如(A)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(A)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (C)、与(A)或(B)所示的核苷酸序列至少有80％同源性的核苷酸序列；
    和/或
    (ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
    (iii)、来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD；和/或
    作为优选，所述来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD具有：
    (a)、如SEQ ID No.1所示的核苷酸序列；或
    (b)、如(a)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(a)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (c)、与(a)或(b)所示的核苷酸序列至少有80％同源性的核苷酸序列；和/或
    (iv)、增加了拷贝数的panB、panC和/或panE基因。
  6. 宿主，其特征在于，表达了天冬氨酸脱氢酶基因aspDH；
    作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl-4(Delftia sp.Csl-4)；
    作为优选，所述天冬氨酸脱氢酶基因aspDH具有：
    (I)、如SEQ ID No.56所示的核苷酸序列；或
    (II)、如(I)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(I)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (III)、与(I)或(II)所示的核苷酸序列至少有80％同源性的核苷酸序列。
  7. 如权利要求6所述的宿主，其特征在于，还包括：
    (i)、强启动子和/或强RBS；
    其中强启动子为PgapA，强RBS为BCD2；
    作为优选，所述BCD2具有：
    (A)、如SEQ ID No.2所示的核苷酸序列；或
    (B)、如(A)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(A)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (C)、与(A)或(B)所示的核苷酸序列至少有80％同源性的核苷酸序列；
    和/或
    (ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
    (iii)、来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD；和/或
    作为优选，所述来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD具有：
    (a)、如SEQ ID No.1所示的核苷酸序列；或
    (b)、如(a)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列，且与(a)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (c)、与(a)或(b)所示的核苷酸序列至少有80％同源性的核苷酸序列；和/或
    (iv)、增加了拷贝数的panB、panC和/或panE基因。
  8. 如权利要求6或7所述的宿主，其特征在于，转染或转化如权利要求4或5所述的表达载体；
    作为优选，所述宿主源自大肠杆菌，优选为大肠杆菌K12，更优选为大肠杆菌K12 MG1655株。
  9. 如权利要求4或5所述的表达载体、如权利要求6至8任一项所述的宿主在生产维生素B5中的应用。
  10. 生产维生素B5的方法，其特征在于，以权利要求6至8任一项所述的宿主为发酵菌株，发酵，收集发酵液，离心取上清液，获得维生素B5。
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