WO2023169168A1 - 天冬氨酸脱羧酶在发酵生产维生素b5中的应用 - Google Patents

天冬氨酸脱羧酶在发酵生产维生素b5中的应用 Download PDF

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WO2023169168A1
WO2023169168A1 PCT/CN2023/076309 CN2023076309W WO2023169168A1 WO 2023169168 A1 WO2023169168 A1 WO 2023169168A1 CN 2023076309 W CN2023076309 W CN 2023076309W WO 2023169168 A1 WO2023169168 A1 WO 2023169168A1
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coli
gene
vitamin
aspartate
pand
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French (fr)
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温廷益
刘树文
李忠财
孙佳慧
邓爱华
张芸
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中国科学院微生物研究所
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01011—Aspartate 1-decarboxylase (4.1.1.11)

Definitions

  • the present invention relates to the field of microorganisms, and in particular to the application of aspartate decarboxylase in fermentative production of vitamin B5.
  • 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.
  • ⁇ -Alanine is produced from aspartate by catalyzing L-aspartate ⁇ -decarboxylase encoded by the panD gene. The initially translated and synthesized PanD is a zymogen without catalytic activity.
  • the zymogen is spontaneously cleaved at the Gly-Ser bond to produce two subunits, of which the N-terminal subunit containing a pyruvyl group has a catalytic effect.
  • the mature PanD pyruvyl group has formed with the substrate When transitioning to an intermediate state, amino transfer occurs easily, leading to irreversible loss of enzyme activity.
  • the accumulation of ⁇ -alanine is also regulated by the concentration of coenzyme A, a downstream metabolite of VB5.
  • the protein complex formed by coenzyme A and PanD/PanZ feedback negatively regulates the expression of PanD.
  • PanD not only has a slow maturation process of post-translational modifications, but also has problems with catalytic inactivation and feedback inhibition, resulting in a very low synthesis efficiency of ⁇ -alanine, the C3 precursor of VB5, limiting the efficient synthesis of 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. , et al., (1999) Appl Environ Microb, 65, 1530-1539; Zhang, B., et al., (2019) Food Chemistry, 294, 267-275.). Therefore, the biosynthesis of ⁇ -alanine is the metabolic bottleneck for fermentation production of VB5.
  • L-aspartate ⁇ -decarboxylase 16 key enzymes, L-aspartate ⁇ -decarboxylase, originating from different bacterial genera and with large evolutionary differences.
  • L-aspartate ⁇ -decarboxylase encoded by the panD gene, catalyzes the decarboxylation of L-aspartate to produce ⁇ -alanine.
  • the present invention provides the application of enhancing the expression of L-aspartate ⁇ -decarboxylase gene panD in the production of vitamin B5;
  • the L-aspartate ⁇ -decarboxylase is derived from Bacillus licheniformis.
  • the L-aspartate alpha-decarboxylase gene panD 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
  • panB, panC and/or panE genes (3). Increase the copy number of panB, panC and/or panE genes.
  • the present invention also provides an expression vector comprising the L-aspartate ⁇ -decarboxylase gene panD;
  • the L-aspartate ⁇ -decarboxylase is derived from Bacillus licheniformis.
  • the expression vector further includes:
  • the present invention also provides a host expressing the L-aspartate ⁇ -decarboxylase gene panD derived from Bacillus licheniformis.
  • the host further includes:
  • 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 K12MG1655 strain.
  • the present invention also provides the use of the expression vector and 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 without adding ⁇ -alanine, the fermentation liquid is collected, and the supernatant is centrifuged to obtain vitamin B5.
  • the invention discloses a highly active aspartate decarboxylase and a method for producing vitamin B5.
  • the present invention screens out L-aspartate ⁇ -decarboxylase derived from Bacillus licheniformis, and its activity in catalyzing the production of ⁇ -alanine is significantly higher than that of PanD from other sources.
  • PanD derived from B.licheniformis was used to construct engineering bacteria for fermentation and production of vitamin B5, which relieved the ⁇ -alanine metabolism bottleneck in the biosynthesis of vitamin B5.
  • the biological method of the present invention produces vitamin B5, which has renewable raw materials, waste residue, waste water and waste gas. It has the advantages of easy processing and resource utilization, so it can be used in the industrial production of vitamin B5 in practice and has important application value.
  • the invention discloses the application of aspartate decarboxylase in the fermentation production of vitamin B5. , those skilled in the art can learn from the content of this article and appropriately improve the implementation of process parameters. 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.
  • panD genes of the present invention are respectively derived from Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Chlorobium phaeobacteroides, Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium marinum, Escherichia coli, Haloquadratum walsbyi, Hydrothermal vent metagenome, methane Methanocaldococcus jannaschii, Magnetospirillum magneticum, Metarhizium robertsii, Mine drainage metagenome, Rhodopirellula baltica and Thermotoga maritima).
  • the present invention uses the same bicistronic design element BCD2 (Nature Methods, 2013, 10 (4): 354-360) to regulate the above 16 different sources.
  • BCD2 bicistronic design element
  • the BCD element introduces a leading cistron sequence before the exogenous structural gene.
  • the translation intensity of BCD is highly related to the affinity of the RBS core sequence and the ribosomal subunit, and has little correlation with the gene coding sequence, thereby avoiding interference from 16 different panD gene sequences on the same translation initiation element.
  • the present invention connects the above 16 BCD2-panD sequences to plasmids to construct 16 recombinant plasmids pET28a-BCD2-panD, using the corresponding The same promoter regulates transcription.
  • the plasmid vector used in the present invention can be a pET series vector, such as pET28a, pET32a, pET3, etc.; it can also be a pQE series vector or other Escherichia coli expression vector.
  • the promoter of the present invention may be a T7 promoter or the like.
  • the present invention transforms the above recombinant plasmid into derivative strains of E. coli B, including BL21, BL21-Codonplus (RIL), BL21 (DE3), BL21 Star, C41 (DE3), BL21 (DE3) pLys S/E, BL21-CodonPlus (DE3) strain, Origami (DE3) strain, Rosetta-gammi (DE3) strain, etc., to obtain whole-cell catalytic engineering bacteria.
  • the obtained target gene and vector are usually digested with restriction endonucleases and ligated with T4 ligase to construct a recombinant vector.
  • the recombinant vector can be transformed into host cells through conventional calcium chloride chemical transformation or electroporation transformation methods in molecular biology experiments to obtain engineered bacteria that can be used for whole-cell catalysis.
  • the present invention uses a whole-cell catalysis method to screen efficient L-aspartate ⁇ -decarboxylase.
  • bacterial cells are first cultured in a liquid culture medium, and the expression of L-aspartate ⁇ -decarboxylase is induced at an appropriate time.
  • the medium used for the growth of engineered bacteria can be a rich medium or an inorganic salt medium.
  • the culture medium contains carbon sources, nitrogen sources, inorganic ions, antibiotics and other nutritional factors.
  • carbon source sugars such as glucose, lactose, and galactose may be used; alcohols such as glycerin and mannitol may be used; and organic acids such as gluconic acid, citric acid, and succinic acid may be used.
  • inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium phosphate, and ammonium chloride can be used; organic nitrogen sources such as corn steep liquor, soybean meal hydrolyzate, hair powder, yeast extract, and peptone can also be used.
  • Inorganic ions include one or more of iron, calcium, magnesium, manganese, molybdenum, cobalt, copper, potassium and other ions.
  • Other nutritional factors also include vitamins such as vitamin B1, pyridoxal, and biotin.
  • the culture process is preferably carried out under aerobic conditions for about 5-48 hours.
  • the culture temperature is usually controlled at 25-45°C, and the pH is usually controlled at 5-8.
  • After culturing for 3 to 40 hours start adding at least one inducer selected from IPTG, lactose, and allolactose.
  • the inducer can be added in a one-time, intermittent, or continuous manner.
  • the amount of inducer added is 0.01 to 1 mmol.
  • the substrate L-aspartic acid added at once is usually preferably L-aspartic acid, L-aspartic acid sodium salt, or L-aspartic acid potassium salt. , L-aspartate ammonium salt, etc.
  • the pH value of the catalytic solution continues to rise, and acid needs to be added to maintain the pH in a range conducive to whole-cell catalysis, usually above 4.0, preferably 5.0 or above, more preferably 5.5 or more, usually 8.0 or less, preferably 7.5 or less, more preferably 6.8 or less.
  • the acid used here is L-aspartic acid.
  • the temperature of the catalytic reaction is usually between 25°C and 60°C, preferably between 30°C and 45°C.
  • the temperature during the catalytic process can be set to a fixed value in the above range, or it can be a variable value from low to high.
  • the present invention further applies the screened high-efficiency L-aspartate ⁇ -decarboxylase to construct engineering bacteria for producing vitamin B5 through fermentation, thereby relieving the ⁇ -alanine metabolism bottleneck in biosynthesizing vitamin B5.
  • the E. coli for producing VB5 by fermentation method of the present invention expresses 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 panBC gene was amplified by PCR using the genome of E. coli K12MG1655 as a template.
  • the strong promoter Ptrc was designed to be introduced into the amplification primer, and BamHI and SphI restriction endonuclease sites were designed at both ends of the primer.
  • 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 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-Ptrc-panBC.
  • the panE gene was amplified by PCR using the genome of E. coli K12MG1655 as a template.
  • the strong promoter PJ23119 was designed to be introduced into the amplification primer, and SphI and BsaBI restriction endonuclease sites were designed at both ends of the primer.
  • the PJ23119-panE product obtained by PCR amplification was identified and recovered by gel electrophoresis, and then double-digested with BamHI and SphI to simultaneously double-digest the pACYC184-Ptrc-panBC plasmid.
  • the double-digested PJ23119-panE and pACYC184-Ptrc-panBC plasmids were recovered by gel electrophoresis.
  • the ligation products were chemically transformed into E. 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, the recombinant plasmid was extracted and sequenced, and the correct recombinant plasmid pACYC184-Ptrc-panBC-PJ23119-panE was obtained, thereby overexpressing the vitamin B5 terminal Recombinant plasmids for synthetic pathway genes.
  • the ilvG gene of Escherichia coli K12MG1655 is mutated and inactivated.
  • the present invention introduces the active ilvG gene of Escherichia coli BL21 to improve the synthesis supply of VB5 precursors.
  • the present invention inserts the ilvG + M gene derived from E. coli BL21 into the chromosome of E. coli K12MG1655, 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.
  • the engineered strain E.coli MG1655 avtA:ilvG + M was constructed to improve the synthesis pathway of VB5 precursor acetolactate and weaken the valine competition pathway.
  • the avtA gene of E. coli K12 MG1655 is integrated with the three panD genes with higher activity selected above, which are respectively derived from Bacillus subtilis, Bacillus licheniformis and Corynebacterium glutamicum. glutamicum). These three genes use the same strong promoter PPL and the same BCD2 to regulate transcription and translation initiation respectively.
  • the recombinant plasmid pACYC184-Ptrc-panBC-PJ23119-panE constructed above was transformed into the engineering strain E.coli MG1655 avtA:panDBs-ilvG + M, the engineering strain E.coli MG1655 avtA:panDBl-ilvG + M and the engineering strain E.coli From MG1655 avtA:panDCg-ilvG + M, an engineering strain for producing VB5 by fermentation was obtained. Compare the VB5 production of engineering bacteria through shake flask fermentation to verify the optimal PanD.
  • 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.
  • the HPLC method was used to quantitatively determine the accumulation of ⁇ -alanine in the fermentation broth.
  • the specific method is as follows. Take the supernatant of the fermentation broth, add purified water to dilute it to an appropriate concentration, and filter it with a 0.22 ⁇ m filter membrane.
  • the o-phthalaldehyde (OPA) online pre-column derivatization method was used to determine the ⁇ -alanine concentration.
  • the chromatographic column used was Agilent AdvanceBio AAA C18, 4.6x100mm, 2.7 ⁇ m, the column temperature was 40°C, the detection wavelength was 338nm, and the mobile phase flow rate was is 1mL/min.
  • ⁇ -alanine purchased from Sigma Company as a standard, determine the standard curve of alanine concentration and light absorption value under the chromatographic conditions.
  • the HPLC method was used to quantitatively determine the production of VB5 in the fermentation broth.
  • the specific method is as follows. Take the supernatant of the fermentation broth, add purified water to dilute it to an appropriate concentration, and filter it with a 0.22 ⁇ m filter membrane.
  • the chromatographic column used is Agilent ZORBAX SB-Aq, 4.6x250mm, the column temperature is 30°C, the detection wavelength is 210nm, and the mobile phase flow rate is 1mL/min.
  • the mobile phase is 3.12g/L NaH2PO4 ⁇ 2H2O, and phosphoric acid is used to adjust the pH to 2.2.
  • Calcium pantothenate purchased from sigma company was used as a standard to measure the standard curve of concentration and light absorption value of 0.1-0.5g/L calcium pantothenate.
  • Example 2 Construction of a vector for overexpression of L-aspartate ⁇ -decarboxylase and whole-cell catalytic ⁇ -alanine-producing engineering bacteria
  • 16 L-aspartate ⁇ -decarboxylase genes panD were synthesized, which were derived from Bacillus amyloliquefaciens (as shown in SEQ ID No. 1) and Bacillus subtilis (as shown in SEQ ID No. 1). No. 2), Bacillus licheniformis (SEQ ID No. 3), Chlorobium phaeobacteroides (SEQ ID No. 4), Corynebacterium efficiens (shown in SEQ ID No. 5), Corynebacterium glutamicum (shown in SEQ ID No. 6), Corynebacterium marinum (shown in SEQ ID No.
  • the present invention connects the above 16 BCD2-panD sequences to plasmids to construct 16 recombinant plasmids pET28a-BCD2-panD.
  • the same BCD2 sequence (as shown in SEQ ID No. 17) 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 plasmids were extracted and sent for sequencing.
  • 16 correct plasmids were obtained: pET28a-BCD2-panDBa, pET28a-BCD2-panDBs, pET28a-BCD2-panDBl, pET28a-BCD2-panDCp, pET28a-BCD2-panDCe, pET28a-BCD2-panDCg, pET28a-BCD2-panDCm, pET28a-BCD2-panDEc, pET28a-BCD2-panDHw, pET28a-BCD2-panDHv, pET28a-BCD2-panDMj, pET28a-BCD2-panDMm, pET28a-BCD2-panDMr, pET28a- BCD2-panDMd, pET28a-BCD2-panDRb, pET28a-BCD2-panDTm.
  • 16 panD genes from different sources use the same promoter (T7) to regulate transcription initiation and
  • the 16 extracted pET28a-BCD2-panD plasmids expressing panD genes from different sources were transformed into competent cells of E.coli BL21 (DE3), screened on LB plates containing 50 mg/L kanamycin, and 16 strains were obtained.
  • the overexpression is derived from B. subtilis (shown in SEQ ID No. 2), B. licheniformis (such as The ⁇ -alanine production of the engineered bacteria with the panD gene of Corynebacterium glutamicum C. glutamicum and the magnetotactic bacterium M. magneticum is about ten times higher than that of other engineered bacteria.
  • B. subtilis shown in SEQ ID No. 2
  • B. licheniformis such as The ⁇ -alanine production of the engineered bacteria with the panD gene of Corynebacterium glutamicum C. glutamicum and the magnetotactic bacterium M. magneticum is about ten times higher than that of other engineered bacteria.
  • the L-aspartate ⁇ -decarboxylase of Bacillus licheniformis has the highest catalytic efficiency.
  • a 5L fermentation tank was further used to verify the catalytic performance of the engineering strain E.coli BL21/pET28a-BCD2-panDBl.
  • DO is controlled above 30%, and the tank pressure is controlled at 0.02 ⁇ 0.10MPa.
  • Ammonia is added back to control the pH to be maintained at 6.9, and the concentration of glucose in the culture medium is maintained below 5g/L by feeding the feed solution.
  • the culture medium is When the bacterial cell OD 600 reaches 30, add 0.1mM inducer IPTG. After 4 hours, the bacterial culture medium OD 600 reaches about 80.
  • the components of the inorganic salt culture medium and the feeding solution are as follows: Inorganic salt culture medium: 2g/L (NH 4 ) 2 HPO 4, 4g/L KH 2 PO 4 , 0.85g/L Citric acid, 0.7g /L MgSO 4 ⁇ 7H 2 O, 10mg/L FeSO 4 ⁇ 7H 2 O, 2.25mg/L ZnSO 4 ⁇ 7H 2 O, 0.2mg/L CuSO 4 ⁇ 5H 2 O, 0.5mg/L MnSO 4 ⁇ 5H 2 O, 0.23mg/L NaB 4 O 7 ⁇ 10H 2 O, 2.0mg/L CaCl 2 ⁇ 2H 2 O, 0.1mg/L NH 4 Mo 7 O 24 , 0.15mg/L CoCl 2 ⁇ 6H 2 O, balance for water.
  • the feeding solution contains 700g/L glucose and 20g/L MgSO 4 ⁇ 7H 2 O, and the balance is water.
  • Example 4 Construction of engineering bacteria for fermentation and production of VB5.
  • 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. 19, 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 at the 5' end of the primers P3 and P4 respectively.
  • the PJ23119-panE product was amplified using the above PCR reaction conditions.
  • 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-panBCE, thereby obtaining a recombinant plasmid that overexpresses the vitamin B5 terminal synthesis pathway gene. .
  • the mutated N20 sequence is CTTTCCAAGC TGGGTCTACC, targeting the avtA gene.
  • the mutated pTargetF was named pTargetFavtA.
  • primers P7 and P8 to amplify the upstream sequence of avtA gene, use primers P9 and P10 to amplify the PL promoter, and use primers P11 and P12 to amplify pET28a-BCD2-panDBs, pET28a-BCD2-panDBl, and E.coli BL21/pET28a-BCD2 respectively.
  • -panDCg was used as a template to amplify the BCD2-panDBs-Ter, BCD2-panDBl-Ter, and BCD2-panDCg-Ter gene fragments
  • primers P13 and P14 were used to amplify the downstream sequence of the avtA gene.
  • DonorBs as shown in SEQ ID No. 20
  • DonorBl as shown in SEQ ID No. 21
  • DonorCg as shown in SEQ ID No. 22
  • 1nt-312nt of SEQ ID No. 20, 21 and 22 are the upstream sequence of the target gene avtA gene
  • 313nt-474nt are the PL promoter
  • 475nt-560nt are the BCD2 sequence. 560nt-943nt of SEQ ID No.
  • 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 ilvG gene of wild-type E. coli K12MG1655 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 K12MG1655, 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.
  • P25 CACGTTCGGA TATGAACTG (as shown in SEQ ID No. 48);
  • P26 CGTCAAGCTT CAGCAACTC (as shown in SEQ ID No. 49).
  • 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.
  • Introduction of trc promoter TTGA through primers P20 and P21 CAATTAATCATCCGGCTCGTATAATGTGTGGA, introduce the terminator sequence CCAGAAAAGAGACGCT TTTAG AGCGTCTTTTTTCGTTTT through primers P22 and P23.
  • Use overlapping PCR to connect the above three fragments to obtain the combination DonorilvGM (shown as SEQ ID No.
  • SEQ ID No. 23 which serves as a template for gene editing.
  • 1-305nt of SEQ ID No. 23 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.
  • E.coli MG1655 avtA:panDBs/pCas Take 2 ⁇ l of pTargetFavtA1 plasmid and 10 ⁇ l of DonorilvGM template DNA and electrotransform them into E.coli MG1655 avtA:panDBs/pCas, E.coli MG1655 avtA:panDBl/pCas, and E.coli MG1655 avtA:panDCg/pCas competent cells respectively.
  • panDBl-ilvG + M and E.coli MG1655 avtA:panDCg-ilvG + M By integrating active ilvG + M on the chromosome, the synthesis of the VB5 precursor acetolactate is increased.
  • the vector pACYC184-panBCE constructed above was transformed into the above-mentioned engineering bacteria E.coli MG1655 avtA:panDBs-ilvG + M, E.coli MG1655 avtA:panDBl-ilvG + M and E.coli MG1655 avtA:panDCg-ilvG + M, The engineering bacteria E.coli MG1655 avtA:panDBs-ilvG + M/pACYC184-panBCE, E.coli MG1655 avtA:panDBl-ilvG + M/pACYC184-panBCE and E.coli MG1655 avtA:panDCg-ilvG + M/pACYC184- were obtained respectively. panBCE, used for fermentation production of VB5.
  • E.coli MG1655 avtA:panDBs-ilvG + M/pACYC184-panBCE Take the test strains E.coli MG1655 avtA:panDBs-ilvG + M/pACYC184-panBCE, E.coli MG1655 avtA:panDBl-ilvG + M/pACYC184-panBCE and E.coli MG1655 avtA:panDCg-ilvG + M/pACYC184-panBCE , streak inoculated on a solid LB medium plate containing 34 mg/L chloramphenicol, and incubated at 37°C for 12 hours. Pick the bacterial lawn on the plate, inoculate it into the LB medium slant, and incubate at 37°C for 10-12 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.
  • the present invention verifies that the PanD derived from Bacillus licheniformis screened by the present invention has the highest activity and can significantly increase the fermentation yield of VB5.

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Abstract

涉及微生物领域，具体涉及高活性的天冬氨酸脱羧酶用于生产维生素B5的方法。筛选到了源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶，催化生产β-丙氨酸的活性明显高于其它来源的PanD。应用B.licheniformis来源的PanD构建了发酵生产维生素B5的工程菌，解除了生物合成维生素B5的β-丙氨酸代谢瓶颈。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

天冬氨酸脱羧酶在发酵生产维生素B5中的应用 技术领域
本发明涉及微生物领域，特别涉及天冬氨酸脱羧酶在发酵生产维生素B5中的应用。
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。β-丙氨酸由panD基因编码的L-天冬氨酸α-脱羧酶催化天冬氨酸产生。最初翻译合成的PanD是没有催化活性的酶原，酶原在Gly-Ser键上自发剪切产生两个亚基，其中含有丙酮酰基的N端亚基具有催化作用。成熟的PanD丙酮酰基与底物形成过 渡中间态，容易发生的氨基转移，导致不可逆的酶活性丧失。此外，β-丙氨酸的积累量还受VB5下游代谢产物辅酶A浓度的调控，辅酶A与PanD/PanZ形成的蛋白复合物，反馈负调控PanD的表达。PanD不仅翻译后修饰的成熟过程缓慢，还存在催化性失活和反馈抑制的问题，导致VB5的C3前体β-丙氨酸的合成效率非常低，限制了VB5高效合成。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.)。因此，β-丙氨酸的生物合成是发酵法生产VB5的代谢瓶颈。
发明内容
有鉴于此，为了突破高效合成β-丙氨酸的代谢瓶颈，发明人筛选了16种不同菌属来源且进化差异较大的关键酶L-天冬氨酸α-脱羧酶。L-天冬氨酸α-脱羧酶由panD基因编码，催化L-天冬氨酸脱羧产生β-丙氨酸。
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L-天冬氨酸α-脱羧酶基因panD的表达在生产维生素B5中的应用；
所述L-天冬氨酸α-脱羧酶来源于地衣芽孢杆菌(Bacillus licheniformis)。
在本发明的一些具体实施方案中，所述L-天冬氨酸α-脱羧酶基因panD具有：
(I)、如SEQ ID No.3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avtA基因中插入强启动子和/或强RBS，其中强启动子为PPL，强RBS为BCD2；和/或
(2)、表达了来源于大肠杆菌BL21的ilvGM基因；和/或
(3)、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L-天冬氨酸α-脱羧酶基因panD；
所述L-天冬氨酸α-脱羧酶来源于地衣芽孢杆菌(Bacillus licheniformis)。
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；和/或
(ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
(iii)、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，表达了来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD。
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；和/或
(ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
(iii)、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K12MG1655株。
第四方面，本发明还提供了所述表达载体、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，无需添加β-丙氨酸，发酵，收集发酵液，离心取上清液，获得维生素B5。
本发明公开了一种高活性的天冬氨酸脱羧酶，及其用于生产维生素B5的方法。本发明筛选到了源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶，催化生产β-丙氨酸的活性明显高于其它来源的PanD。应用B.licheniformis来源的PanD构建了发酵生产维生素B5的工程菌，解除了生物合成维生素B5的β-丙氨酸代谢瓶颈。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
本发明公开了天冬氨酸脱羧酶在发酵生产维生素B5中的应用。，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。
本发明的panD基因分别来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、暗棕绿菌(Chlorobium phaeobacteroides)、有效棒杆菌(Corynebacterium efficiens)、谷氨酸棒杆菌(Corynebacterium glutamicum)、海洋棒杆菌(Corynebacterium marinum)、大肠杆菌(Escherichia coli)、嗜盐古细菌甲烷暖球菌属(Haloquadratum walsbyi)、海底火山口宏基因组(Hydrothermal vent metagenome)、甲烷暖球菌(Methanocaldococcus jannaschii)、趋磁细菌(Magnetospirillum magneticum)、绿僵菌(Metarhizium robertsii)、矿山排水宏基因组(Mine drainage metagenome)、波罗的海红小梨形菌(Rhodopirellula baltica)和海栖热袍菌(Thermotoga maritima)。
在筛选高效的L-天冬氨酸α-脱羧酶方面，本发明使用了相同的双顺反子设计元件BCD2(Nature Methods,2013,10(4):354-360)调控上述16种不同来源的L-天冬氨酸α-脱羧酶的翻译起始水平。BCD元件在外源结构基因前引入一段前导顺反子序列，核糖体顺利通过该顺反子，占据下一个顺反子RBS序列时，可避免与基因前端编码序列形成茎环结构，从而避免部分panD基因的翻译阻遏。BCD的翻译强度与RBS核心序列与核糖体亚基的亲和力高度相关，与基因编码序列相关性小，从而避免16种不同的panD基因序列对于同一种翻译起始元件的干扰。
在筛选高效的L-天冬氨酸α-脱羧酶方面，本发明将上述16种BCD2-panD序列连接到质粒上，构建16个重组质粒pET28a-BCD2-panD，使用相 同的启动子调控转录。本发明所使用的质粒载体可以是pET系列载体，如pET28a，pET32a，pET3等；也可以是pQE系列载体或其它大肠杆菌表达载体。本发明的启动子可以是T7启动子等。
本发明将上述重组质粒转化至大肠杆菌B的衍生株，包括BL21，BL21-Codonplus(RIL)，BL21(DE3)、BL21 Star，C41(DE3)，BL21(DE3)pLys S/E，BL21-CodonPlus(DE3)株、Origami(DE3)株、Rosetta-gammi(DE3)株等，从而获得全细胞催化工程菌。通常将获得的目的基因和载体通过限制内切酶酶切和T4连接酶连接的方式构建重组载体。重组载体可以通过分子生物学实验中常规的氯化钙化学转化或电穿孔转化的方法转化至宿主细胞中，获得可用于全细胞催化的工程菌。
本发明使用全细胞催化的方法筛选高效的L-天冬氨酸α-脱羧酶。在本发明全细胞催化过程中，首先在液体培养基中培养菌体细胞，并在恰当的时间诱导L-天冬氨酸α-脱羧酶表达。用于工程菌生长的培养基可以是丰富培养基，也可以是是无机盐培养基。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；也可以是甘油、甘露醇等醇类；也可以使用葡萄糖酸、柠檬酸、丁二酸等有机酸类。作为碳源，可以使用氨水、硫酸铵、磷酸铵、氯化铵等无机氮源；也可以使用玉米浆、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。其它营养因子还包括维生素B1、吡哆醛、生物素等维生素。
培养过程以在有氧条件下实施5-48小时左右培养为宜，培养温度通常控制在25～45℃，pH通常控制在5～8。培养3～40小时开始加入选自IPTG、乳糖、别乳糖中的至少一种诱导剂，诱导剂的加入方式可以是一次性、间歇或连续补加，诱导剂的补加量为0.01～1mmol。
在加入诱导剂0.5～30小时后，一次性加入的底物L-天冬氨酸，通常优选为L-天冬氨酸、L-天冬氨酸钠盐、L-天冬氨酸钾盐、L-天冬氨酸铵盐等。
在L-天冬氨酸脱羧反应生产β-丙氨酸的过程中，催化液的pH值持续升高，需要加入酸使pH维持在利于全细胞催化的范围，通常为4.0以上、优选为5.0以上、更优选为5.5以上，通常为8.0以下，优选为7.5以下，更优选为6.8以下。此处所使用的酸为L-天冬氨酸。可以采用固体粉末、悬浊液或 溶液的形式补加；可以间歇或连续补加维持pH在上述范围，也可以通过发酵罐pH电极信号反馈补加使pH维持在恒定值。
催化反应的温度通常介于25℃和60℃之间，优选为30℃和45℃之间。催化过程中的温度可是设为上述范围中的固定值，也可以是由低到高变化值。
本发明进一步应用筛选到的高效的L-天冬氨酸α-脱羧酶，构建发酵法生产维生素B5的工程菌，解除了生物合成维生素B5的β-丙氨酸代谢瓶颈。
本发明所述的发酵法生产VB5的大肠杆菌，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。
以大肠杆菌K12MG1655的基因组为模板，PCR扩增panBC基因。扩增引物上设计引入强启动子Ptrc，引物两端同时设计BamHI和SphI限制性核酸内切酶位点。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。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-Ptrc-panBC。
以大肠杆菌K12MG1655的基因组为模板，PCR扩增panE基因。扩增引物上设计引入强启动子PJ23119，引物两端同时设计SphI和BsaBI限制性核酸内切酶位点。PCR扩增得到的PJ23119-panE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-Ptrc-panBC质粒。凝胶电泳回收双酶切的PJ23119-panE和pACYC184-Ptrc-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-Ptrc-panBC-PJ23119-panE，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。
大肠杆菌K12MG1655的ilvG基因突变失活，本发明引入了大肠杆菌BL21的具有活性的ilvG基因，提高了VB5的前体合成供应。本发明在大肠杆菌K12MG1655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。构建了工程菌E.coli MG1655 avtA:ilvG+M，提高了VB5前体乙酰乳酸的合成途径，弱化了缬氨酸竞争途径。
在大肠杆菌K12 MG1655的avtA基因上整合了上述筛选到的3个活性较高的panD基因，分别源于枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)和谷氨酸棒杆菌(Corynebacterium glutamicum)。这3个基因使用相同的强启动子PPL和相同的BCD2分别调控转录和翻译起始。
将上述构建的重组质粒pACYC184-Ptrc-panBC-PJ23119-panE转化到工程菌E.coli MG1655 avtA:panDBs-ilvG+M，工程菌E.coli MG1655 avtA:panDBl-ilvG+M和工程菌E.coli MG1655 avtA:panDCg-ilvG+M中，得到发酵法生产VB5的工程菌。通过摇瓶发酵比较工程菌的VB5产量，验证最优的PanD。
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。
通过催化生产β-丙氨酸和发酵生产VB5两步验证，从16种序列差异较大的不同来源的候选酶中筛选到了活性最高的L-天冬氨酸α-脱羧酶，在不添加β-丙氨酸时可高效发酵生产VB5，解除了生物合成瓶颈，降低了生产成本。
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。
下面结合实施例，进一步阐述本发明：
实施例1检测方法
采用HPLC法定量测定发酵液β-丙氨酸的积累量，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。采用邻苯二甲醛(OPA)在线柱前衍生法测定β-丙氨酸浓度，所用色谱柱为Agilent AdvanceBio AAA C18，4.6ⅹ100mm，2.7μm，柱温为40℃，检测波长为338nm，流动相流速为1mL/min。流动相A为10mM Na2HPO4和10mM Na2B4O7，调节pH至8.2，流动相B为乙腈：甲醇：水＝45：45：10。以从sigma公司购买的β-丙氨酸为标准品，测定该色谱条件下丙氨酸的浓度与光吸收值的标准曲线。
采用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过表达L-天冬氨酸α-脱羧酶的载体与全细胞催化产β-丙氨酸工程菌的构建
在基因合成公司合成了16个L-天冬氨酸α-脱羧酶基因panD，分别来源于解淀粉芽孢杆菌Bacillus amyloliquefaciens(如SEQ ID No.1所示)、枯草芽孢杆菌Bacillus subtilis(如SEQ ID No.2所示)、地衣芽孢杆菌Bacillus licheniformis(如SEQ ID No.3所示)、暗棕绿菌Chlorobium phaeobacteroides(如SEQ ID No.4所示)、有效棒杆菌Corynebacterium  efficiens(如SEQ ID No.5所示)、谷氨酸棒杆菌Corynebacterium glutamicum(如SEQ ID No.6所示)、海洋棒杆菌Corynebacterium marinum(如SEQ ID No.7所示)、大肠杆菌Escherichia coli(如SEQ ID No.8所示)、嗜盐古细菌甲烷暖球菌属Haloquadratum walsbyi(如SEQ ID No.9所示)、海底火山口宏基因组Hydrothermal vent metagenome(如SEQ ID No.10所示)、甲烷暖球菌Methanocaldococcus jannaschii(如SEQ ID No.11所示)、趋磁细菌Magnetospirillum magneticum(如SEQ ID No.12所示)、绿僵菌Metarhizium robertsii(如SEQ ID No.13所示)、矿山排水宏基因组Mine drainage metagenome(如SEQ ID No.14所示)、波罗的海红小梨形菌Rhodopirellula baltica(如SEQ ID No.15所示)和海栖热袍菌Thermotoga maritima(如SEQ ID No.16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。
本发明将上述16种BCD2-panD序列连接到质粒上，构建16个重组质粒pET28a-BCD2-panD。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.17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16个正确的质粒pET28a-BCD2-panDBa、pET28a-BCD2-panDBs、pET28a-BCD2-panDBl、pET28a-BCD2-panDCp、pET28a-BCD2-panDCe、pET28a-BCD2-panDCg、pET28a-BCD2-panDCm、pET28a-BCD2-panDEc、pET28a-BCD2-panDHw、pET28a-BCD2-panDHv、pET28a-BCD2-panDMj、pET28a-BCD2-panDMm、pET28a-BCD2-panDMr、pET28a-BCD2-panDMd、pET28a-BCD2-panDRb、pET28a-BCD2-panDTm。在这16个重组载体中，16个不同来源的panD基因使用相同的启动子(T7)调控转录起始，使用相同的BCD2序列调控翻译起始。
将上述提取的16个表达不同来源panD基因的pET28a-BCD2-panD质粒转化到E.coli BL21(DE3)的感受态细胞，在含有50mg/L卡那霉素的LB平板上筛选，获得16株工程菌E.coli BL21/pET28a-BCD2-panDBa、E.coli BL21/pET28a-BCD2-panDBs、E.coli BL21/pET28a-BCD2-panDBl、E.coli BL21/pET28a-BCD2-panDCp、E.coli BL21/pET28a-BCD2-panDCe、E.coli BL21/pET28a-BCD2-panDCg、E.coli BL21/pET28a-BCD2-panDCm、E.coli BL21/pET28a-BCD2-panDEc、E.coli BL21/pET28a-BCD2-panDHw、E.coli BL21/pET28a-BCD2-panDHv、E.coli BL21/pET28a-BCD2-panDMj、E.coli BL21/pET28a-BCD2-panDMm、E.coli BL21/pET28a-BCD2-panDMr、E.coli BL21/pET28a-BCD2-panDMd、E.coli BL21/pET28a-BCD2-panDRb、E.coli BL21/pET28a-BCD2-panDTm，用于β-丙氨酸全细胞催化。
实施例3全细胞催化筛选最优的panD基因
刮取上述16个工程菌E.coli BL21/pET28a-BCD2-panD菌苔，接入含有3mL LB(含50mg/L卡那霉素)培养基的50mL无菌透气盖试管中，置于37℃摇床中220rpm培养12h，得到种子液，OD600为4-5；培养所得的种子液按2％的接种量接入含有30mL LB(含50mg/L卡那霉素)培养基的500mL挡板摇瓶中，置于37℃摇床中220rpm培养2h，加入0.3mM诱导剂IPTG，继续按照相同的条件诱导4h。诱导后的三角瓶中加入30mL天冬氨酸溶液(使用氢氧化钠调节pH至6.0),置于37℃摇床中220rpm培养30分钟。离心后取上清液检测β-丙氨酸的产量，每个工程菌设置三个平行实验，取平均值。过表达不同来源panD基因的工程菌的β-丙氨酸产量如表1所示，过表达来源于枯草芽孢杆菌B.subtilis(SEQ ID No.2所示)、地衣芽孢杆菌B.licheniformis(如SEQ ID No.3所示)、谷氨酸棒杆菌C.glutamicum和趋磁细菌M.magneticum的panD基因的工程菌的β-丙氨酸的产量比其它工程菌高十倍左右，其中来源于地衣芽孢杆菌的L-天冬氨酸α-脱羧酶的催化效率最高。
表1


进一步使用5L发酵罐验证工程菌E.coli BL21/pET28a-BCD2-panDBl的催化性能。刮取工程菌E.coli BL21/pET28a-BCD2-panDBl菌苔接入含有50mL的LB(含50mg/L卡那霉素(5～200mg/L均可)培养基的500mL三角瓶中，在37℃ 220rpm摇床中培养4h，得到种子液，OD600为4-5；培养所得的种子液按2％的接种量接入含有2L无机盐培养基的5L发酵罐中，培养温度为37℃，DO控制在30％以上，罐压控制在0.02～0.10MPa。反馈补加氨水控制pH维持在6.9，通过流加补料液使培养液中葡萄糖的浓度维持在5g/L以下。当培养液中菌体OD600达到30时加入0.1mM诱导剂IPTG，4h后菌体培养液OD600达到80左右。补加10g/L的L-天冬氨酸作为底物，催化过程中不调控pH，当pH不再上升时再加入10g/L的L-天冬氨酸。间歇补加25次固体天冬氨酸，催化36h，β-丙氨酸的产量达到140.5g/L。
其中无机盐培养基成分和补料液成分如下：无机盐培养基：2g/L(NH4)2HPO4,4g/L KH2PO4,0.85g/L Citric acid(柠檬酸),0.7g/L MgSO4·7H2O，10mg/L FeSO4·7H2O,2.25mg/L ZnSO4·7H2O,0.2mg/L CuSO4·5H2O,0.5mg/L MnSO4·5H2O,0.23mg/L NaB4O7·10H2O,2.0mg/L CaCl2·2H2O,0.1mg/L NH4Mo7O24，0.15mg/L CoCl2·6H2O，余量为水。补料液包含700g/L葡萄糖和20g/L MgSO4·7H2O，余量为水。
实施例4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K12MG1655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.18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.24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.25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K12MG1655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.19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.26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.27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-Cas9System,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.28所示)；
P6：GCTTGGAAAG GACTAGTATT ATACCTAGG(如SEQ ID No.29所示)；
P7:CG GACTGGAAGA AGATCTG(如SEQ ID No.30所示)；
P8:TTTCTTAGAC GTCGGAATTG AGACTCATGC ACAGCACGA(如SEQ ID No.31所示)；
P9:TCGTGCTGT GCATGAGTCTCAATTCCGACGTCTAAGAAAC(如SEQ ID No.32所示)；
P10:GATCTCCTTT TTAAGTGAAC TTGGGGTCAG TGCGTCCTGC TGAT(如SEQ ID No.33所示)；
P11:ATCAGCAGGACGCACTGACCCCAAGTTCACTTAAAAAGGAGA TC(如SEQ ID No.34所示)；
P12:TGCCGTTCAT ATTGGTGATG CAAAAAACCC CTCAAGACC(如SEQ ID No.35所示)；
P13:GGTCTTGAGGGGTTTTTTGCATC ACCAATATGAACGGCA(如SEQ ID No.36所示)；
P14:GCTGATAGAG CTGCTTGGT(如SEQ ID No.37所示)；
P15:GGAGCTACTC ACACTGCTTG(如SEQ ID No.38所示)；
P16:CGCATACATT GATGCGTATG(如SEQ ID No.39所示)；
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分别以pET28a-BCD2-panDBs、pET28a-BCD2-panDBl、E.coli BL21/pET28a-BCD2-panDCg为模板扩增获得BCD2-panDBs-Ter、BCD2-panDBl-Ter、BCD2-panDCg-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DonorBs(如SEQ ID No.20所示)、DonorBl(如SEQ ID No.21所示)和DonorCg(如SEQ ID No.22所示)，作为基因编辑的模板。其中SEQ ID No.20、21和22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。SEQ ID No.20的560nt-943nt为panDBs序列，944nt-995nt为终止子序列，996-1261nt为avtA基因下游游序列。SEQ ID No.21的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。SEQ ID No.22的560nt-970nt为panDCg序列，971nt-1022nt为终止子序列，1023-1288nt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:panDBs/pCas，E.coli MG1655 avtA:panDBl/pCas，E.coli MG1655 avtA:panDCg/pCas，仍按照上述方法制备感受态备用。
工程菌E.coli MG1655 avtA:panDBs/pCas，E.coli MG1655 avtA:panDBl/pCas和E.coli MG1655 avtA:panDCg/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:panDBs，E.coli MG1655 avtA:panDBl和E.coli  MG1655 avtA:panDCg。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K12MG1655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K12MG1655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.40所示)；
P18:CGTAGTTACA GACTAGTATT ATACCTAGG(如SEQ ID No.41所示)；
P19:GGCAGAAAAT CAGCCAGTTC(如SEQ ID No.42所示)；
P20:TCCACACATT ATACGAGCCG GATGATTAAT TGTCAAGAAC TCTGTAGCAA GGAAGG(如SEQ ID No.43所示)；
P21:TTGA CAATTAATCATCCGGCTCGTATAATGTGTGGACAAGATT CAGGACGGGG AAC(如SEQ ID No.44所示)；
P22:CGAAAAAAGA CGCTCTAAAA GCGTCTCTTT TCTGGTATATTCCTTTTGCG CTCAG(如SEQ ID No.45所示)；
P23:CAGAAAAGAGACGCTTTTAGAGCGTCTTTTTTCGTTTTGGAGC TACTC ACACTGCTTG(如SEQ ID No.46所示)；
P24:GCCAATATGC AGATGCTCATGAGCATCTGCATATTGG C(如SEQ ID No.47所示)；
P25:CACGTTCGGA TATGAACTG(如SEQ ID No.48所示)；
P26:CGTCAAGCTT CAGCAACTC(如SEQ ID No.49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TTGA CAATTAATCATCCGGCTCGTATAATGTGTGGA，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.23所示)，作为基因编辑的模板。SEQ ID No.23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，分别电转化至E.coli MG1655 avtA:panDBs/pCas，E.coli MG1655 avtA:panDBl/pCas，E.coli MG1655 avtA:panDCg/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:panDBs-ilvG+M，E.coli MG1655 avtA:panDBl-ilvG+M和E.coli MG1655 avtA:panDCg-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。
将上述构建的载体pACYC184-panBCE转化至上述工程菌E.coli MG1655 avtA:panDBs-ilvG+M，E.coli MG1655 avtA:panDBl-ilvG+M和E.coli MG1655 avtA:panDCg-ilvG+M中，分别获得工程菌E.coli MG1655 avtA:panDBs-ilvG+M/pACYC184-panBCE，E.coli MG1655 avtA:panDBl-ilvG+M/pACYC184-panBCE和E.coli MG1655 avtA:panDCg-ilvG+M/pACYC184-panBCE，用于发酵生产VB5。
其中：


















实施例5VB5工程菌的发酵试验
取试验菌株E.coli MG1655 avtA:panDBs-ilvG+M/pACYC184-panBCE，E.coli MG1655 avtA:panDBl-ilvG+M/pACYC184-panBCE和E.coli MG1655 avtA:panDCg-ilvG+M/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，用氨水调节反应体系的pH值使其维持在6.8-7.0。培养过程中，每隔4h取样一次，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(表2)。
表2
本发明通过β-丙氨酸全细胞催化和VB5发酵两个方面，验证了本发明筛选到的来源于地衣芽孢杆菌的PanD活性最高，可显著提高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L-天冬氨酸α-脱羧酶基因panD的表达在生产维生素B5中的应用；
    所述L-天冬氨酸α-脱羧酶来源于地衣芽孢杆菌(Bacillus licheniformis)。
  2. 如权利要求1所述的应用，其特征在于，所述L-天冬氨酸α-脱羧酶基因panD具有：
    (I)、如SEQ ID No.3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avtA基因中插入强启动子和/或强RBS，其中强启动子为PPL，强RBS为BCD2；和/或
    (2)、表达了来源于大肠杆菌BL21的ilvGM基因；和/或
    (3)、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L-天冬氨酸α-脱羧酶基因panD；
    所述L-天冬氨酸α-脱羧酶来源于地衣芽孢杆菌(Bacillus licheniformis)。
  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；和/或
    (ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
    (iii)、增加了拷贝数的panB、panC和/或panE基因。
  6. 宿主，其特征在于，表达了来源于地衣芽孢杆菌(Bacillus licheniformis)的L-天冬氨酸α-脱羧酶基因panD。
  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；和/或
    (ii)、来源于大肠杆菌BL21的ilvGM基因；和/或
    (iii)、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，无需添加β-丙氨酸，发酵，收集发酵液，离心取上清液，获得维生素B5。
PCT/CN2023/076309 2022-03-07 2023-02-16 天冬氨酸脱羧酶在发酵生产维生素b5中的应用 WO2023169168A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1420931A (zh) * 1999-09-21 2003-05-28 Basf公司 制备泛化合物的方法和微生物
CN1639349A (zh) * 2002-07-03 2005-07-13 巴斯福股份公司 用于提高泛酸产量的微生物和方法
CN1788089A (zh) * 2002-07-03 2006-06-14 巴斯福股份公司 用于提高泛酸产量的微生物和方法
CN115595328A (zh) * 2022-03-07 2023-01-13 中国科学院微生物研究所（Cn） 天冬氨酸脱羧酶在发酵生产维生素b5中的应用
CN115595314A (zh) * 2022-03-07 2023-01-13 中国科学院微生物研究所（Cn） 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

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1420931A (zh) * 1999-09-21 2003-05-28 Basf公司 制备泛化合物的方法和微生物
CN1639349A (zh) * 2002-07-03 2005-07-13 巴斯福股份公司 用于提高泛酸产量的微生物和方法
CN1788089A (zh) * 2002-07-03 2006-06-14 巴斯福股份公司 用于提高泛酸产量的微生物和方法
CN115595328A (zh) * 2022-03-07 2023-01-13 中国科学院微生物研究所（Cn） 天冬氨酸脱羧酶在发酵生产维生素b5中的应用
CN115595314A (zh) * 2022-03-07 2023-01-13 中国科学院微生物研究所（Cn） 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

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 16 November 2015 (2015-11-16), ANONYMOUS : "aspartate 1-decarboxylase PanD [Bacillus paralicheniformis ATCC 9945a]", XP093090475, retrieved from NCBI Database accession no. AGN36811.1 *
DATABASE Protein 30 May 2019 (2019-05-30), ANONYMOUS : "MULTISPECIES: aspartate 1-decarboxylase [Bacillus]", XP093090473, retrieved from NCBI Database accession no. WP_003182918.1 *
JIANG, Y.CHEN, B.DUAN, C. L.SUN, B. BYANG, J. JYANG, S.: "Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System", APPL ENVIRON MICROB, vol. 81, 2015, pages 2506 - 2514, XP055444637, DOI: 10.1128/AEM.04023-14
NATURE METHODS, vol. 10, no. 4, 2013, pages 354 - 360
SAHM, H. ET AL., APPL ENVIRON MICROB, vol. 65, 1999, pages 1973 - 1979
ZHANG, B. ET AL., FOOD CHEMISTRY, vol. 294, 2019, pages 267 - 275

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