WO2023108504A1 - 一种提高酵母菌株nadph和fadh 2供应的方法、工程菌及其应用 - Google Patents

一种提高酵母菌株nadph和fadh 2供应的方法、工程菌及其应用 Download PDF

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WO2023108504A1
WO2023108504A1 PCT/CN2021/138510 CN2021138510W WO2023108504A1 WO 2023108504 A1 WO2023108504 A1 WO 2023108504A1 CN 2021138510 W CN2021138510 W CN 2021138510W WO 2023108504 A1 WO2023108504 A1 WO 2023108504A1
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周雍进
张磊
陈瑞兵
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中国科学院大连化学物理研究所
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Definitions

  • the invention belongs to the application field of microbial genetic engineering and metabolic engineering, and in particular relates to a method for increasing the supply of yeast strains NADPH and FADH2, engineering bacteria and applications thereof.
  • cofactors there are more than 20 cofactors in cells that participate in different life metabolic processes. These cofactors have their own characteristics and are distributed in different organelles. The complex dynamic balance and biological functions make the precise regulation of cofactor engineering extremely challenging. For example, the synthesis of phenolic acid requires the participation of cofactors NADPH and FAD(H 2 ), but the importance of these cofactors in the synthesis process and the optimal regulation strategy under different conditions are still blank. Although it is sometimes possible to supply cofactors through in vitro feeding, cofactors are expensive and most cofactors cannot be directly absorbed across membranes, making in vitro feeding methods extremely costly and inefficient.
  • the present invention aims to optimize the supply of yeast endogenous cofactors NADPH and FAD (H 2 ), improve the synthesis efficiency of phenolic acid compounds, fill in the gaps in the field on the one hand, and explore the application of cofactor engineering as the target of cell factory engineering on the other hand potential and prospects.
  • a method for increasing the supply of NADPH and FADH2 in yeast strains is provided to strengthen the supply of intracellular NADPH and FAD(H 2 ), so as to solve the difficulty of insufficient supply of endogenous cofactors, thereby improving NADPH and FADH 2 dependence Synthesis efficiency of type natural products such as phenolic acids.
  • a method of increasing NADPH and FADH2 supply in a yeast strain comprising enhancing NADPH regeneration and increasing intracellular FADH2 supply;
  • Said enhanced NADPH regeneration includes:
  • Said increasing intracellular FADH supply comprises:
  • the RIB1 gene and the FLX1 gene were introduced into the yeast strain.
  • nucleotide sequence of the TKL1 gene is shown in SEQ ID NO: 2;
  • the nucleotide sequence of the TAL1 gene is shown in SEQ ID NO: 3;
  • the nucleotide sequence of the FLX1 gene is shown in SEQ ID NO: 4;
  • the nucleotide sequence of the RIB1 gene is shown in SEQ ID NO: 5;
  • the nucleotide sequence of the LmXFPK gene is shown in SEQ ID NO: 6;
  • the nucleotide sequence of the CkPTA gene is shown in SEQ ID NO: 7.
  • said enhancing NADPH regeneration comprises:
  • said increasing intracellular FADH 2 supply comprises:
  • a DNA fragment containing T CYC1 -FLX1-P tHXT7 -P TDH3 -RIB1-T TDH2 was integrated at the XI2 site of the yeast strain.
  • the yeast strain is selected from any one of Saccharomyces cerevisiae.
  • the yeast strain is selected from any of the engineering strains of phenolic acid-producing yeast;
  • the engineering strain of phenolic acid-producing yeast is obtained by introducing genes related to the phenolic acid synthesis pathway into the starting strain of Saccharomyces cerevisiae:
  • the phenolic acid synthesis pathway-related genes include at least one of Aro4 K229L , Aro7 G141S , EcAROL, ARO1, ARO2, ARO3, PHA2, MtPDH1, FjTAL, SbPAL1 H123F , AtCPR1, PtrC4H1, PtrC4H2, PtrC3H, PaHPAB, SeHPAC.
  • the phenolic acid synthesis pathway-related genes include Aro4 K229L having the nucleotide sequence shown in SEQ ID NO: 9, Aro7 G141S having the nucleotide sequence shown in SEQ ID NO: 10, Aro7 G141S having the nucleotide sequence shown in SEQ ID NO: EcAROL with the nucleotide sequence shown in ID NO: 11, ARO1 with the nucleotide sequence shown in SEQ ID NO: 12, ARO2 with the nucleotide sequence shown in SEQ ID NO: 13, and ARO2 with the nucleotide sequence shown in SEQ ID NO: ARO3 having the nucleotide sequence shown in 14, PHA2 having the nucleotide sequence shown in SEQ ID NO: 15, MtPDH1 having the nucleotide sequence shown in SEQ ID NO: 16, and having the nucleotide sequence shown in SEQ ID NO: 17 FjTAL with the nucleotide sequence shown, SbPAL1 H123F with the nucleotide sequence shown, S
  • the introduction site of ARO7 G141S , ARO4 K229L and EcAROL is ARO10;
  • the import site of FjTAL, SbPAL1 H123F and AtCPR1 is PDC5;
  • ARO1, ARO2, and ARO3 The import site of ARO1, ARO2, and ARO3 is XII1;
  • the import site of PahpaB and SehpaC is X2;
  • the import site of PHA2 and MtPDH1 is XI8.
  • a DNA fragment containing THIS3 -ARO7 G141S -P GAL10/1 -ARO4 K229L -T ENO2 -P GAL7 -EcAROL-T ADH1 is integrated into the ARO10 site of the starting strain;
  • a DNA fragment containing T CPS1 -PHA2-P GAL10/1 -MtPDH1-Sc OPT1 -T HIS5 was integrated into the XI8 site of the starting strain.
  • the GAL80 gene of the starting strain of S. cerevisiae was also knocked out.
  • the engineering bacterium obtained by the method described above is provided.
  • the engineering bacteria obtained by the above-mentioned method and the application of the above-mentioned engineering bacteria in the synthesis of NADPH and FADH 2 dependent natural products are provided.
  • the NADPH and FADH 2 dependent natural products are selected from one of phenolic acids.
  • a kind of preparation method of phenolic acid is provided, and described preparation method comprises the following steps:
  • the engineering bacteria are selected from the engineering bacteria obtained by the method described above.
  • the content of the glucose in the medium I is 10-20 g/L.
  • the fermentation I conditions include:
  • the temperature is 28 ⁇ 30°C;
  • the number of revolutions is 220 ⁇ 280rpm;
  • the time is 72 to 96 hours
  • the filling volume is 15-20/100mL.
  • the initial concentration of glucose in medium II is 15-20 g/L, and the glucose in medium II is supplemented to 15-20 g/L every 20-28 hours.
  • the batch fed-batch fermentation II conditions include:
  • the temperature is 28 ⁇ 30°C;
  • the number of revolutions is 800 ⁇ 1200rpm;
  • Dissolved oxygen is >40%
  • the pH is 5.6-6.0.
  • the above-mentioned method for increasing the supply of NADPH and FADH 2 to yeast strains is carried out using the CRISPR-Cas9 system.
  • the sgRNA expression vector pgRNA-GPP1 targeting the nucleotide sequence and the expression cassette GPP1up-T ENO2 -CkPTA-P GAL10/1 -LmXFPK-T ADH1 -GPP1dw were transferred into the yeast strain.
  • the sgRNA expression vector pgRNA-XI7 targeting the nucleotide sequence and the expression cassette XI7up-T PRM9 -P GAL10/1 -TAL1-T PYK1 -XI7dw are transferred into the yeast strain;
  • the sgRNA expression vector pgRNA-XI6 targeting the nucleotide sequence and the expression cassette XI6up-P GAL7 -TKL1-T ENO2 -XI6dw were transferred into the yeast strain.
  • the phenolic acid-producing yeast engineering strain is obtained by introducing genes related to phenolic acid synthesis pathway into the starting strain of Saccharomyces cerevisiae.
  • the sgRNA expression vector pgRNA-ARO10 targeting the nucleotide sequence and the expression cassette ARO10up-T HIS3 -ARO7 G141S -P GAL10/1 -ARO4 K229L -T ENO2 -P GAL7 -EcAROL-T ADH1 -ARO10dw were transferred to into the yeast starting strain; and/or
  • the sgRNA expression vector pgRNA-PDC5 targeting the nucleotide sequence and the expression cassette ScPDC5up-T CYC1 -FjTAL-Sc OPT1 -P GAL10/1 -SbPAL1 H123F -Sc OPT1 -T TDH2 -T FBA1 -AtCPR1-Sc OPT1 -P GAL7 -ScPDC5dw is transferred into the yeast starting strain; and/or
  • the sgRNA expression vector pgRNA-XI8 targeting the nucleotide sequence and the expression cassette XI8up-T CPS1 -PHA2-P GAL10/1 -MtPDH1-Sc OPT1 -T HIS5 -XI8dw were transferred into the yeast starting strain.
  • the present application provides an engineering strategy for the regeneration of the cofactor NADPH, which can significantly increase the accumulation of phenolic acids.
  • the sgRNA expression vector pgRNA-GAL80 having the targeted nucleotide sequence shown in SEQ ID NO: 8 was constructed; the sgRNA expression vector pgRNA-GAL80 was introduced into Saccharomyces cerevisiae, and the GAL80 gene was knocked out to obtain strain A0.
  • the genome editing of Saccharomyces cerevisiae in the present invention mainly relies on the self-constructed CRISPR/Cas9 system to complete.
  • Saccharomyces cerevisiae strain was made to overexpress LmXFPK and CkPTA genes through PGAL10/1 , and integrate them at the GPP1 gene locus, and at the same time complete the knockout of GPP1 and the expression of exogenous genes.
  • the specific steps of the above technical scheme are: constructing the sgRNA expression vector pgRNA-GPP1 targeting the nucleotide sequence, using fusion PCR to construct the expression cassette GPP1up-T ENO2 -CkPTA-P GAL10/1 -LmXFPK-T ADH1 -GPP1dw, and pgRNA -GPP1 together using the above method to transform Saccharomyces cerevisiae to complete the construction.
  • sgRNA expression vector pgRNA-XI7 targeting the nucleotide sequence and the expression cassette XI7up-T PRM9 -P GAL10/1 -TAL1-T PYK1 -XI7dw
  • sgRNA expression vector pgRNA-XI6 targeting the nucleotide sequence
  • Transform yeast with the expression cassette XI6up-P GAL7 -TKL1-T ENO2 -XI6dw, respectively, and construct strain A1.
  • strains A0 and A1 phenolic acid synthesis pathways were expressed respectively, and strains RB103 and RB197 were obtained.
  • phenylpyruvate decarboxylase gene ARO10 and pyruvate decarboxylase gene PDC5 strengthening shikimate pathway gene 3-deoxy-D-arabino-heptulose-7-phosphate synthase Aro4 K229L (with Nucleotide sequence as shown in SEQ ID NO: 9), chorismate mutase Aro7 G141S (has the nucleotide sequence shown in SEQ ID NO: 10) and Escherichia coli derived shikimate kinase EcAROL (has as shown in The nucleotide sequence shown in SEQ ID NO: 11); strengthen endogenous shikimate pathway gene ARO1 (has the nucleotide sequence shown in SEQ ID NO: 12), ARO2 (has the nucleotide sequence shown in SEQ ID NO: 13
  • the specific steps of the above technical scheme are: through the above construction and transformation methods, use the sgRNA expression vector pgRNA-ARO10 targeting the nucleotide sequence and the expression cassette ARO10up-T HIS3 -ARO7 G141S -P GAL10/1 -ARO4 K229L -T ENO2 -P GAL7 -EcAROL-T ADH1 -ARO10dw; using the nucleotide sequence-targeted sgRNA expression vector pgRNA-PDC5 and the expression cassette ScPDC5up-T CYC1 -FjTAL-Sc OPT1 -P GAL10/1 -SbPAL1 H123F -Sc OPT1 - T TDH2 -T FBA1 -AtCPR1-Sc OPT1 -P GAL7 -ScPDC5dw; using the nucleotide sequence-targeting sgRNA expression vector pgRNA-XII4 and expression
  • RB103 and RB197 yeast strains were inoculated in the basal salt medium with 20g/L glucose as the substrate for phenolic acid fermentation.
  • the initial inoculation OD600 was 0.1. Fermentation conditions: liquid volume 20/100mL, 30°C, 220rpm, the fermentation time is 72 ⁇ 96h.
  • the fermentation products are extracted and monitored.
  • the yields of phenolic acid monomer caffeic acid in RB103 and RB197 were 286 and 385 mg/L, respectively, a significant increase of 35%.
  • the specific steps of the above technical scheme are: take 50 ⁇ L fermentation sample, add 450 ⁇ L sterile water, then add 500 ⁇ L ethanol and fully vortex and mix well; centrifuge at 13000 g for 5 minutes, take 500 ⁇ L supernatant and pass through a 0.22 ⁇ m water phase microporous membrane to obtain phenol acid sample.
  • Shimadzu HPLC was used for detection, the chromatographic column was 3 ⁇ 100mm 2.7um Poroshell 120 EC-C18 (Agilent), the flow rate was 0.8mL/min, mobile phase A was H2O+0.05%HCOOH, mobile phase B was ACN+0.05%HCOOH,
  • the detection wavelength of the ultraviolet detector is 280nm or 330nm.
  • the specific mobile phase gradient is 95%-A (0min), 90%-A (3min), 88%-A (4-5min), 85%-A (6-7min), 40%-A (10min), 95 %-A (12-14min).
  • an engineering strategy of the cytoplasmic compartmentalized localization and supply of the cofactor FADH 2 in the present application can significantly increase the accumulation of phenolic acids.
  • the method of the above technical solution is to overexpress endogenous FLX1 and RIB1 genes in Saccharomyces cerevisiae, transport mitochondrial FAD (H 2 ) to localize in the cytoplasm, increase the concentration and supply of FADH 2 in the cytoplasm, thereby increasing the production of phenolic acid.
  • the specific steps of the above technical solution are: constructing the sgRNA expression vector pgRNA-XI2 targeting the nucleotide sequence, using fusion PCR to construct the expression cassette XI2up-T CYC1 -FLX1-P tHXT7 -P TDH3 -RIB1-T TDH2 -XI2dw, and pgRNA-XI2 was used together to transform Saccharomyces cerevisiae strain RB197 using the above method to complete the construction and obtain strain RB209.
  • inoculate RB209 yeast strain for phenolic acid fermentation the initial inoculation OD600 is 0.1, fermentation conditions: liquid volume 20/100mL, 30°C, 220rpm, The fermentation time is 96h.
  • the fermentation products are extracted and monitored.
  • the results demonstrate that an engineering strategy of cytoplasmic compartmentalization and supply of the cofactor FADH 2 can significantly increase the accumulation of phenolic acids.
  • the output of phenolic acid monomer caffeic acid in RB209 was 518mg/L, which was significantly increased by 35% compared with strain RB197.
  • the present application optimized the conditions of batch fed-batch fermentation, and the accumulation of phenolic acid monomer caffeic acid in the cofactor engineering strain RB209 reached 5.8 g/L.
  • the specific steps of the above-mentioned technical solution are as follows: in a 1L fermenter, the basic salt medium with 20g/L glucose as the substrate starts to ferment, the initial inoculation volume is 0.25L, and the OD600 is 0.2. Dissolved oxygen is controlled to be 40% by the stirring paddle speed (800-1200rpm), and the fermentation temperature is 30°C. The pH was maintained at 5.6 using 4M KOH and 2M HCl. In the fed-batch phase, 200 g/L glucose solution was added, and the feeding rate was coupled to growth and increased exponentially (set ⁇ to 0.05 h -1 ) to maintain a constant biomass-to-glucose consumption rate.
  • This application provides a cofactor engineering strategy for enhancing the supply of NADPH and FADH 2 in yeast cells, and its application in the synthesis of phenolic acid compounds, which mainly includes the following steps:
  • Saccharomyces cerevisiae (CEN.PK113-11C) expressing Cas9 protein as the starting strain, the GAL80 gene was knocked out to obtain recombinant yeast A0;
  • the strain was fermented and cultured, and the yield of phenolic acid and its improvement were verified by extraction.
  • the GAL80 gene has the nucleotide sequence shown in SEQ ID NO: 1
  • the TKL gene has the nucleotide sequence shown in SEQ ID NO: 2
  • the TAL gene has the nucleotide sequence shown in SEQ ID NO: 3
  • the FLX1 gene has the nucleotide sequence shown in SEQ ID NO: 4
  • the RIB1 gene has the nucleotide sequence shown in SEQ ID NO: 5
  • the LmXFPK gene after codon optimization has the nucleotide sequence shown in SEQ ID NO: 6.
  • the nucleotide sequence shown, the CkPTA gene after codon optimization has the nucleotide sequence shown in SEQ ID NO:7.
  • a method for improving the synthesis of phenolic acid in yeast by NADPH and FADH 2 cofactor engineering described above uses the CRISPR-Cas9 gene editing technology to knock out or knock in the expression of the gene.
  • the expressed gene for enhanced cofactor supply may come from the coding gene of isozyme of other species or its codon-optimized gene.
  • This application provides the construction and application of a yeast strain that supplies NADPH and FADH 2 , and is used to improve the synthesis efficiency of phenolic acid in yeast, which belongs to the field of microbial technology. Since the synthesis of natural products such as phenolic acids requires the participation of cofactors NADPH and FAD(H 2 ), the inherent cofactor levels of yeast cannot fully meet the high-efficiency synthesis of phenolic acids, and the addition of exogenous cofactors is expensive. Therefore, the present invention establishes a strategy to enhance the supply of intracellular cofactors NADPH and FADH 2 and to increase the efficiency of phenolic acid biosynthesis.
  • the cofactor engineering strategy increased the yield of caffeic acid by 81%, and the batch fed-batch fermentation reached the highest yield so far of 5.8g/L.
  • This strategy has the characteristics of high conversion efficiency, low production cost, and broad prospects for industrial application. It has been confirmed that the enhancement of NADPH and FADH 2 in Saccharomyces cerevisiae can significantly improve the synthesis efficiency of NADPH and FADH 2 -dependent natural products such as phenolic acids, and has important application value. .
  • the method for improving the supply of NADPH and FADH 2 in yeast strains provided by the present application can effectively solve the problem of insufficient supply of endogenous cofactors by enhancing NADPH regeneration and increasing intracellular FADH 2 supply, thereby improving NADPH and FADH 2 -dependent Synthesis efficiency of natural products such as phenolic acids.
  • the method for increasing the supply of NADPH and FADH 2 in yeast strains provided by the present application is beneficial to the improvement of the supply of NADPH and FADH 2 by optimizing the codons of genes according to host preference.
  • the present invention provides a cofactor NADPH and FAD(H 2 ) engineering strategy to solve the difficulty of insufficient supply of endogenous cofactors during the synthesis of phenolic acids, thereby improving the NADPH/FAD(H 2 )-dependent natural Product synthesis efficiency.
  • the cofactor regulation method of the present invention involves regulation of the pentose phosphate shunt, FAD(H 2 ) synthesis and transport, which can strengthen the supply and self-circulation of endogenous cofactors NADPH and FAD(H 2 ), and avoid the need for exogenous addition of expensive cofactors cost consumption.
  • the present invention constructs the caffeic acid pathway of the phenolic acid compound in the cofactor engineering bacteria, and the yield reaches 5.8 g/L, which proves the application potential of the cofactor engineering strategy in the industrialization of cell factories.
  • Figure 1 shows a schematic diagram of the pgRNA plasmid construction process in the Saccharomyces cerevisiae CRISPR/Cas9 system.
  • Figure 2 shows a schematic diagram of the NADPH cofactor engineering strategy.
  • Figure 3 shows a schematic diagram of the metabolic engineering strategy to realize the synthesis of phenolic acid compounds by Saccharomyces cerevisiae.
  • Figure 4 shows a schematic diagram of the FAD(H 2 ) cofactor engineering strategy.
  • Figure 5 shows the yield of the phenolic acid compound caffeic acid synthesized by cofactor engineered yeast.
  • Fig. 6 shows the relative content of the cofactor FAD(H 2 ) in the cofactor-engineered yeast.
  • Figure 7 shows the yield of the phenolic acid compound caffeic acid produced by cofactor-engineered yeast strains in fed-batch fermentation.
  • Example 1 NADPH cofactor engineering enhances the construction of recombinant yeast for phenolic acid synthesis
  • the laboratory independently constructed the recombinant Saccharomyces cerevisiae CEN.PK113-11C-CAS (genotype MATa, SUC2, MAL2-8c, his3 ⁇ , ura3 ⁇ , XI5::(P TEF1 -CAS9-T CYC1 )) integrating the CAS9 gene, and Use this as the host strain to construct the cofactor engineering yeast strain.
  • CEN.PK113-11C-CAS the construction method of CEN.PK113-11C-CAS is as follows: First, directly chemically synthesize CAS9 and the resistance gene KanMX expression cassette T AgTEF -KanMX-P AgTEF -P TEF1 -CAS9-T CYC1 , and combine the expression cassette with XI5 by fusion PCR
  • the donor DNA fragment XI5up-T AgTEF -KanMX-P AgTEF -P TEF1 -CAS9-T CYC1 -XI5dw was obtained by fusion of upstream and downstream 500bp, transformed into Saccharomyces cerevisiae CEN.PK113-11C, and integrated into the XI5 site by homologous recombination Geneticin G418 was used to screen positive strains, and the strain CEN.PK113-11C-CAS+KanMX was obtained.
  • pgRNA-KanMX was transformed with the donor DNA fragment XI5up- PTEF1 obtained by fusion PCR.
  • chemical transformation method about 500ng of pgRNA-KanMX and donor DNA were simultaneously transformed into the yeast strain integrated with the Cas9 protein, and cultured statically at 30°C for 2 days on the SD plate with histidine added; the transformant was added with After cultured in the liquid SD medium of histidine, it was verified correctly by PCR, and the plasmid was lost by spreading on the solid medium containing 5-fluoroorotic acid. After the plasmid was lost, the strain CEN.PK113-11C-CAS was obtained, and the strain was preserved for future use. .
  • sgRNA-1 uses primers p1 (SEQ ID NO: 26; GCGGTTAGTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATC) and pX1 (AAACTTCTCCGCAGTGAAAGATAAATGATC( M20 ) GTTTTAGAGCTAGAAATAG, wherein M20 is an alternative 20bp targeting sequence)
  • sgRNA-2 uses primer p 2 (SEQ ID NO: 27; GATAACACTGCGG
  • the replacement sequences of M20 and N20 in pX1 and pX2 and the corresponding sequence names after replacement are shown in Table 1.
  • the sgRNA expression vectors involved in the present invention include pgRNA-GAL80 (targeting gene GAL80, SEQ ID NO: 8), pgRNA -GPP1 (targeting gene GPP1), pgRNA-XI7 (targeting site XI7), pgRNA-XI6 (targeting site XI6), pgRNA-ARO10 (targeting gene ARO10), pgRNA-PDC5 (targeting gene PDC5) , pgRNA-XII4 (targeting site XII4), pgRNA-XII1 (targeting site XII1), pgRNA-X2 (targeting site X2), pgRNA-XI8 (targeting site XI8), pgRNA-XI2 (targeting site to site XI2).
  • the donor DNA respectively amplify about 500 bp downstream of the target site of the pgRNA vector as homology arms, and then assemble each fragment to be inserted by conventional fusion PCR to obtain a complete donor DNA molecule, and The pgRNAs of the corresponding sites are transformed into yeast together for gene editing and strain transformation.
  • the gene information involved is shown in Table 4.
  • pgRNA-XI2 SEQ ID NO: 48; GCATGATAAATCGGTAGAAT SEQ ID NO: 49; GGTTCTAGAAGTGCCCTTTG pgRNA-KanMX SEQ ID NO: 50; GGTTCTAGAAGTGCCCTTTG SEQ ID NO: 51; ATTCTACCGATTTATCATGC
  • NADPH regeneration in Saccharomyces cerevisiae is mainly accomplished through the oxidation step of the pentose phosphate pathway (PPP). Because the exogenous phenolic acid pathway was engineered to express using the promoters PGAL7 and PGAL10/1 , phenolic acids would only be synthesized under glucose-restricted conditions.
  • PPP pentose phosphate pathway
  • the phenolic acid pathway was constructed on the basis of strains A0 and A1 (as shown in Figure 3). Sequentially transform pgRNA-ARO10 and ARO10up-T HIS3 -ARO7 G141S -P GAL10/1 -ARO4 K229L -T ENO2 -P GAL7 -EcAROL-T ADH1 -ARO10dw; pgRNA-PDC5 and ScPDC5up-T CYC1 -FjTAL-Sc OPT1 - P GAL10/1 -SbPAL1 H123F -Sc OPT1 -T TDH2 -T FBA1 -AtCPR1-Sc OPT1 -P GAL7 -ScPDC5dw; pgRNA-XII4 and XII4up-T PRM9t -PtrC4H2-Sc OPT1 -P GAL10/1 -PtrC4H1-Sc
  • strain RB197 pgRNA-XI2 and XI2up-T CYC1 -FLX1-P tHXT7 -P TDH3 -RIB1-T TDH2 -XI2dw were further transformed to express RIB1 gene enhancement
  • the synthesis of FADH 2 the expression of FLX1 gene transfers FADH 2 from the mitochondria to the cytoplasm, and finally achieves the purpose of increasing the FADH 2 in the cytoplasm.
  • the obtained FAD(H 2 ) cofactor engineering strain is RB209, which is used for subsequent fermentation and synthesis analysis.
  • YPD medium 20g/L glucose, 20g/L peptone, 10g/L yeast powder;
  • SD medium 20g/L glucose, 6.7g/L YNB, add histidine 0.02g/L when used;
  • Fermentation medium (basic component medium): (NH 4 ) 2 SO 4 2.5g/L, KH 2 PO 4 14.4g/L, MgSO 4 7H 2 O 0.5g/L, add about 900mL ddH 2 O, adjust The pH is 5.6, the volume is adjusted to 950mL, and sterilized at 115°C for 30min. After sterilization, add 1mL vitamin solution (recipe in Table 3) and 2mL trace metal solution (recipe in Table 2), and add histidine and uracil (40mg/L) as needed. Glucose was added to the fermentation medium to 20g/L for phenolic acid fermentation.
  • the liquid volume is 20mL/100mL Erlenmeyer flask, and the fermentation is carried out under the conditions of 30°C and 220rpm for 72-96h. Point sampling or endpoint sampling for biomass (expressed as absorbance at 600nm) and phenolic acid production analysis.
  • the fermentation experiment of phenolic acid was carried out under the condition of basal medium containing 20g/L glucose, the initial inoculation OD600 was 0.1, the fermentation conditions: liquid volume 20/100mL, 30°C, 220rpm, fermentation time 96h. Then extract the phenolic acid compound, take 50 ⁇ L fermentation sample, add 450 ⁇ L sterile water, then add 500 ⁇ L ethanol, vortex and mix well; centrifuge at 13,000 g for 5 minutes, take 500 ⁇ L supernatant and pass through a 0.22 ⁇ m aqueous microporous membrane to obtain the phenolic acid sample .
  • the sample was detected by Shimadzu HPLC, the chromatographic column was 3 ⁇ 100mm 2.7um Poroshell 120 EC-C18 (Agilent), the flow rate was 0.8mL/min, the mobile phase A was H2O+0.05%HCOOH, and the mobile phase B was ACN+0.05%HCOOH ,
  • the detection wavelength of the ultraviolet detector is 280nm or 330nm.
  • the specific mobile phase gradient is 95%-A (0min), 90%-A (3min), 88%-A (4-5min), 85%-A (6-7min), 40%-A (10min), 95 %-A (12-14min).
  • RB209 was subjected to fed-batch fermentation.
  • start fermentation with basal salt medium with 20g/L glucose as the substrate, the initial inoculum volume is 0.25L, and the OD600 is 0.2.
  • Dissolved oxygen is controlled to be 40% by the stirring paddle speed (800-1200rpm), and the fermentation temperature is 30°C.
  • the pH was maintained at 5.6 using 4M KOH and 2M HCl.
  • 500 g/L glucose solution was added every 24 hours to bring the glucose concentration in the culture medium to 20 g/L.
  • Sc OPT1 indicates that the gene is codon-optimized according to S. cerevisiae preference.

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Abstract

一种提高酵母菌株NADPH和FADH 2供应的方法、工程菌及其应用。该方法包括增强NADPH再生和提高胞内FADH 2供给；所述增强NADPH再生包括：在酵母菌株中导入LmXFPK基因、CkPTA基因、TAL1基因和TKL1基因；所述提高胞内FADH 2供给包括：在酵母菌株中导入RIB1基因和FLX1基因；可以解决酚酸等合成过程中内源辅因子供应不足的困难，从而提高酚酸等NADPH/FADH 2依赖型天然产物的合成效率。

Description

一种提高酵母菌株NADPH和FADH 2供应的方法、工程菌及其应用 技术领域
本发明属于微生物基因工程与代谢工程应用领域，具体涉及一种提高酵母菌株NADPH和FADH2供应的方法、工程菌及其应用。
技术背景
构建微生物工厂合成天然产物过程中，催化元件优化，前体供应增强和代谢重塑被认为是最重要的代谢工程策略。而当复杂的代谢工程策略将细胞的固有代谢流定向到目标化合物时，原有的辅因子代谢可能不再适用于新的细胞状态，辅因子的不足或缺乏往往容易被忽视。由于辅因子不足或缺失，来自其它物种的催化酶在酵母中可能出现活性低下甚至失活，给微生物异源高效合成小分子化合物带来挑战。因此，目前急需对参与小分子合成的重要辅因子进行研究，开发高效的辅因子工程策略进一步提高合成效率。
细胞中存在超过20种的辅因子参与不同的生命代谢过程，这些辅因子各具特色，分布于不同细胞器，且复杂的动态平衡和生物学功能使得辅因子工程的精确调控极具挑战。例如酚酸合成过程需要辅因子NADPH和FAD(H 2)的参与，这些辅因子在其合成过程中的重要性以及不同条件下的最适调控策略还是一片空白。虽然有时可以通过体外饲喂辅因子进行供应，但是辅因子价格昂贵，且多数辅因子无法直接跨膜吸收，使得体外饲喂的方法成本极高且效率低下。因此，本发明旨在优化酵母内源辅因子NADPH和FAD(H 2)供应，提高酚酸化合物的合成效率，一方面填补领域空白，另一方面探索辅因子工程作为细胞工厂工程靶点的应用潜力与前景。
发明内容
根据本申请的一个方面，提供一种提高酵母菌株NADPH和FADH2供应的方法强化胞内NADPH和FAD(H 2)的供应，以解决内源辅因子供应不足的困难，从而提高NADPH和FADH 2依赖型天然产物(例如酚酸)的合成效率。
一种提高酵母菌株NADPH和FADH 2供应的方法，所述方法包括增强NADPH再生和提高胞内FADH 2供给；
所述增强NADPH再生包括：
在酵母菌株中导入LmXFPK基因、CkPTA基因、TAL1基因和TKL1基因；
所述提高胞内FADH 2供给包括：
在酵母菌株中导入RIB1基因和FLX1基因。
可选地，TKL1基因的核苷酸序列如SEQ ID NO：2所示；
TAL1基因的核苷酸序列如SEQ ID NO：3所示；
FLX1基因的核苷酸序列如SEQ ID NO：4所示；
RIB1基因的核苷酸序列如SEQ ID NO：5所示；
LmXFPK基因的核苷酸序列如SEQ ID NO：6所示；
CkPTA基因的核苷酸序列如SEQ ID NO：7所示。
可选地，所述增强NADPH再生包括：
将含有T ENO2-CkPTA-P GAL10/1-LmXFPK-T ADH1的DNA片段整合在酵母菌株的GPP1位点；
将含有T PRM9-P GAL10/1-TAL1-T PYK1的DNA片段整合在酵母菌株的XI7位点；
将含有P GAL7-TKL1-T ENO2的DNA片段整合在酵母菌株的XI6位点；
优选地，所述提高胞内FADH 2供给包括：
将含有T CYC1-FLX1-P tHXT7-P TDH3-RIB1-T TDH2的DNA片段整合在酵母菌株的XI2位点。
可选地，所述酵母菌株选自酿酒酵母中的任一种。
可选地，所述酵母菌株选自产酚酸酵母工程菌株中的任一种；
可选地，所述产酚酸酵母工程菌株通过在酿酒酵母出发菌株中导入酚酸合成途径相关基因得到：
所述酚酸合成途径相关基因包括Aro4 K229L、Aro7 G141S、EcAROL、ARO1、ARO2、ARO3、PHA2、MtPDH1、FjTAL、SbPAL1 H123F、AtCPR1、PtrC4H1、PtrC4H2、PtrC3H、PaHPAB、SeHPAC中的至少一种。
可选地，所述酚酸合成途径相关基因包括具有如SEQ ID NO：9所示核苷酸序列的Aro4 K229L、具有如SEQ ID NO：10所示核苷酸序列的Aro7 G141S、具有如SEQ ID NO：11所示核苷酸序列的EcAROL、具有如SEQ ID NO：12所示的核苷酸序列的ARO1、具有SEQ ID NO：13所示核苷酸序列的ARO2、具有如SEQ ID NO：14所示核苷酸序列的ARO3、具有如SEQ ID NO：15所示核苷酸序列的PHA2、具有如SEQ ID NO：16所示核苷酸序列的MtPDH1、具有如SEQ ID NO：17所示核苷酸序列的FjTAL，具有如SEQ ID NO：18所示核苷酸序列的SbPAL1 H123F、具有如SEQ ID NO：19所示核苷酸序列的AtCPR1、具有如SEQ ID NO：20所示核苷酸序列的PtrC4H1、具有如SEQ ID NO：21所示核苷酸序列的PtrC4H2、具有如SEQ ID NO：22所示核苷酸序列的PtrC3H、具有如SEQ ID NO：23所示核苷酸序列的PaHPAB、具有如SEQ ID NO：24所示核苷酸序列的SeHPAC中的至少一种。
可选地，ARO7 G141S、ARO4 K229L、EcAROL的导入位点为ARO10；
FjTAL、SbPAL1 H123F、AtCPR1的导入位点为PDC5；
PtrC4H2、PtrC4H1、PtrC3H的导入位点为XII4；
ARO1、ARO2、ARO3的导入位点为XII1；
PahpaB、SehpaC的导入位点为X2；
PHA2、MtPDH1的导入位点为XI8。
可选地，将含有T HIS3-ARO7 G141S-P GAL10/1-ARO4 K229L-T ENO2-P GAL7-EcAROL-T ADH1的DNA片段整合到出发菌株的ARO10位点；
将含有T CYC1-FjTAL-Sc OPT1-P GAL10/1-SbPAL1 H123F-Sc OPT1-T TDH2-T FBA1-AtCPR1-Sc OPT1-P GAL7的DNA片段整合到出发菌株的PDC5位点；
将含有T PRM9t-PtrC4H2-Sc OPT1-P GAL10/1-PtrC4H1-Sc OPT1-T PYK1-P GAL7-PtrC3H-Sc OPT1-T DIT1的DNA片段整合到出发菌株的XII4位点；、
将含有P GAL7-ARO1-T ENO2-T CPS1-ARO2-P GAL10/1-ARO3-T HIS5的DNA片段整合到出发菌株的XII1位点；
将含有T PRM9-PahpaB-Sc OPT1-P GAL10/1-SehpaC-Sc OPT1-T HIS3的DNA片段整合到出发菌株的X2位点；
将含有T CPS1-PHA2-P GAL10/1-MtPDH1-Sc OPT1-T HIS5的DNA片段整合到出发菌株的XI8位点。
可选地，还敲除酿酒酵母出发菌株的GAL80基因。
根据本申请的另一个方面，提供上述所述的方法得到的工程菌。
根据本申请的另一个方面，提供上述所述的方法得到的工程菌、上述所述的工程菌在合成NADPH和FADH 2依赖型天然产物中的应用。
可选地，所述NADPH和FADH 2依赖型天然产物选自酚酸中的一种。
根据本申请的另一个方面，提供一种酚酸的制备方法，所述制备方法包括以下步骤：
将工程菌接种于含葡萄糖的培养基I中，发酵I，得到所述酚酸；
或；
将工程菌接种于含葡萄糖的培养基II中进行批式补料发酵II，得到所述酚酸；
所述工程菌选自上述所述方法得到的工程菌。
可选地，所述葡萄糖在所述培养基I中的含量为10～20g/L。
可选地，所述发酵I条件包括：
温度为28～30℃；
转数为220～280rpm；
时间为72～96小时；
装液量15～20/100mL。
可选地，所述批式补料发酵II为培养基II中葡萄糖初始浓度为15～20g/L，每隔20～28小时将培养基II中葡萄糖补充至15～20g/L。
可选地，所述批式补料发酵II条件包括：
温度为28～30℃；
转数为800～1200rpm；
溶氧为>40％；
pH为5.6～6.0。
作为一种实施方案，上述提高酵母菌株NADPH和FADH 2供应的方法利用CRISPR-Cas9系统进行。
可选地，将靶向核苷酸序列的sgRNA表达载体pgRNA-XI2、表达盒XI2up-T CYC1-FLX1-P tHXT7-P TDH3-RIB1-T TDH2-XI2dw转入酵母菌株中
将靶向核苷酸序列的sgRNA表达载体pgRNA-GPP1、表达盒GPP1up-T ENO2-CkPTA-P GAL10/1-LmXFPK-T ADH1-GPP1dw转入酵母菌株中。
可选地，将靶向核苷酸序列的sgRNA表达载体pgRNA-XI7、表达盒XI7up-T PRM9-P GAL10/1-TAL1-T PYK1-XI7dw转入酵母菌株中；
将靶向核苷酸序列的sgRNA表达载体pgRNA-XI6和表达盒XI6up-P GAL7-TKL1-T ENO2-XI6dw转入酵母菌株中。
所述产酚酸酵母工程菌株通过在酿酒酵母出发菌株中导入酚酸合成途径相关基因得到。可选地，将靶向核苷酸序列的sgRNA表达载体pgRNA-ARO10和表达盒ARO10up-T HIS3-ARO7 G141S-P GAL10/1-ARO4 K229L-T ENO2-P GAL7-EcAROL-T ADH1-ARO10dw转入酵母出发菌株中；和/或
将靶向核苷酸序列的sgRNA表达载体pgRNA-PDC5和表达盒ScPDC5up-T CYC1-FjTAL-Sc OPT1-P GAL10/1-SbPAL1 H123F-Sc OPT1-T TDH2-T FBA1-AtCPR1-Sc OPT1-P GAL7-ScPDC5dw转入酵母出发菌株中；和/或
将靶向核苷酸序列的sgRNA表达载体pgRNA-XII4和表达盒XII4up-T PRM9t-PtrC4H2-Sc OPT1-P GAL10/1-PtrC4H1-Sc OPT1-T PYK1-P GAL7-PtrC3H-Sc OPT1-T DIT1-XII4dw转入酵母出发菌株中；和/或将用靶向核苷酸序列的sgRNA表达载体pgRNA-XII1和表达盒XII1up-P GAL7-ARO1-T ENO2-T CPS1-ARO2-P GAL10/1-ARO3-T HIS5-XII1dw；使用靶向核苷酸序列的sgRNA表达载体pgRNA-X2和表达盒X2up-T PRM9-PahpaB-Sc OPT1-P GAL10/1-SehpaC-Sc OPT1-T HIS3-X2dw转入酵母出发菌株中；和/或
将靶向核苷酸序列的sgRNA表达载体pgRNA-XI8和表达盒XI8up-T CPS1-PHA2-P GAL10/1-MtPDH1-Sc OPT1-T HIS5-XI8dw转入酵母出发菌株中。
作为一种实施方案，本申请一种辅因子NADPH再生的工程策略，能够显著增加酚酸的积累量。
构建具有SEQ ID NO：8所示的靶向核苷酸序列的sgRNA表达载体pgRNA-GAL80；将所述sgRNA表达载体pgRNA-GAL80导入酿酒酵母，敲除GAL80基因，获得菌株A0。
上述技术方案的具体步骤是：以无缝敲除基因GAL80为例，本发明对酿酒酵母的基因组编辑主要依托自主构建的CRISPR/Cas9系统完成。首先，构建序列如SEQ ID NO：8所示的靶向基因GAL80的表达载体pgRNA-GAL80，其中下划线标记的2个20bp核苷酸序列为靶向GAL80的sgRNA；然后构建修复基因组断裂并敲除GAL80的供体DNA分子，分别扩增GAL80基因上下游非编码区域各500bp序列(GAL80up和GAL80dw)，通过融合PCR方法获得上下游融合DNA片段GAL80up-GAL80dw，即供体DNA片段；使用化学转化法将约500ng的pgRNA80和供体DNA同时转化进入整合有Cas9蛋白的酵母菌株，在加有组氨酸的SD平板上于30℃静置培养2天；转化子经加有组氨酸的液体SD培养基培养后，通过PCR验证正确，涂布于含有5-氟乳清酸的固体培养基进行质粒丢失，质粒丢失后的菌株保存备用。下文中的其它基因组编辑工作均遵循相似流程。
进一步，使所述酿酒酵母菌株通过P GAL10/1过表达LmXFPK和CkPTA基因，整合在GPP1基因位点，同时完成GPP1的敲除和外源基因表达。
上述技术方案的具体步骤是：构建靶向核苷酸序列的sgRNA表达载体pgRNA-GPP1，使用融合PCR构建表达盒GPP1up-T ENO2-CkPTA-P GAL10/1-LmXFPK-T ADH1-GPP1dw，和pgRNA-GPP1一起使用上述方法转化酿酒酵母，完成构建。
进一步，使用靶向核苷酸序列的sgRNA表达载体pgRNA-XI7和表达盒XI7up-T PRM9-P GAL10/1-TAL1-T PYK1-XI7dw，以及靶向核苷酸序列的sgRNA表达载体pgRNA-XI6和表达盒XI6up-P GAL7-TKL1-T ENO2-XI6dw分别转化酵母，构建菌株A1。
进一步，在菌株A0和A1基础上，分别表达酚酸合成途径，获得菌株RB103和RB197。包括敲除内源苯丙酮酸脱羧酶基因ARO10和丙酮酸脱羧化酶基因PDC5，强化莽草酸途径基因3-脱氧-D-阿拉伯糖型-庚酮糖酸-7-磷酸合成酶Aro4 K229L(具有如SEQ ID NO：9所示的核苷酸序列)，分支酸变位酶Aro7 G141S(具有如SEQ ID NO：10所示的核苷酸序列)和大肠杆菌来源的莽草酸激酶EcAROL(具有如SEQ ID NO：11所示的核苷酸序列)；强化内源莽草酸途径基因ARO1(具有如SEQ ID NO：12所示的核苷酸序列)，ARO2(具有如SEQ ID NO：13所示的核苷酸序列)和ARO3(具有如SEQ ID NO：14所示的核苷酸序列)；表达芳香氨基酸途径基因PHA2(具有如SEQ ID NO：15所示的核苷酸序列)和密码子优化的Medicago truncatula来源基因MtPDH1(具有如SEQ ID NO：16所示的核苷酸序列)；然后表达植物来源和细菌来源的咖啡酸合成途径，包括酪氨酸氨解酶基因FjTAL(具有如SEQ ID NO：17所示的核苷酸序列)，高粱苯丙氨酸氨解酶基因SbPAL1 H123F(具有如SEQ ID NO：18所示的核苷酸序列)，拟南芥细胞色素P450还原酶基因AtCPR1(具有如SEQ ID NO：19所 示的核苷酸序列)，杨树肉桂酸羟化酶基因PtrC4H1(具有如SEQ ID NO：20所示的核苷酸序列)和PtrC4H2(具有如SEQ ID NO：21所示的核苷酸序列)，杨树香豆酸-3-羟化酶基因PtrC3H(具有如SEQ ID NO：22所示的核苷酸序列)，绿脓假单胞菌4-羟基苯乙酸3-加氧酶基因PaHPAB(具有如SEQ ID NO：23所示的核苷酸序列)和肠道沙门氏菌黄素氧化还原酶基因SeHPAC(具有如SEQ ID NO：24所示的核苷酸序列)，实现了酚酸的合成。
上述技术方案的具体步骤是：通过上述使用构建和转化方法，使用靶向核苷酸序列的sgRNA表达载体pgRNA-ARO10和表达盒ARO10up-T HIS3-ARO7 G141S-P GAL10/1-ARO4 K229L-T ENO2-P GAL7-EcAROL-T ADH1-ARO10dw；使用靶向核苷酸序列的sgRNA表达载体pgRNA-PDC5和表达盒ScPDC5up-T CYC1-FjTAL-Sc OPT1-P GAL10/1-SbPAL1 H123F-Sc OPT1-T TDH2-T FBA1-AtCPR1-Sc OPT1-P GAL7-ScPDC5dw；使用靶向核苷酸序列的sgRNA表达载体pgRNA-XII4和表达盒XII4up-T PRM9t-PtrC4H2-Sc OPT1-P GAL10/1-PtrC4H1-Sc OPT1-T PYK1-P GAL7-PtrC3H-Sc OPT1-T DIT1-XII4dw；使用靶向核苷酸序列的sgRNA表达载体pgRNA-XII1和表达盒XII1up-P GAL7-ARO1-T ENO2-T CPS1-ARO2-P GAL10/1-ARO3-T HIS5-XII1dw；使用靶向核苷酸序列的sgRNA表达载体pgRNA-X2和表达盒X2up-T PRM9-PahpaB-Sc OPT1-P GAL10/1-SehpaC-Sc OPT1-T HIS3-X2dw；使用靶向核苷酸序列的sgRNA表达载体pgRNA-XI8和表达盒XI8up-T CPS1-PHA2-P GAL10/1-MtPDH1-Sc OPT1-T HIS5-XI8dw；依次完成转化构建，分别在A0和A1中构建获得生产酚酸的菌株RB103和RB197。
进一步地，在以20g/L葡萄糖为底物的基础盐培养基中，接种RB103和RB197酵母菌株，进行酚酸发酵，初始接种OD600为0.1，发酵条件：装液量20/100mL，30℃，220rpm，发酵时间为72～96h。
进一步地，对发酵产物进行提取和监测。结果证明NADPH再生能够显著增加酚酸的积累量。RB103和RB197中酚酸单体咖啡酸的产量分别为286和385mg/L，显著提高35％。
以上技术方案的具体步骤是：取50μL发酵样品，加入450μL无菌水，再加入500μL乙醇后充分涡旋混匀；13000g离心5min，取500μL上清过0.22μm水相微孔滤膜后获得酚酸样品。检测使用岛津HPLC，色谱柱为3×100mm 2.7um Poroshell 120 EC-C18(Agilent)，流速为0.8mL/min，流动相A为H2O+0.05％HCOOH，流动相B为ACN+0.05％HCOOH，紫外检测器检测波长为280nm或330nm。具体流动相梯度为95％-A(0min)，90％-A(3min)，88％-A(4-5min)，85％-A(6-7min)，40％-A(10min)，95％-A(12-14min)。
作为一种实施方案，本申请一种辅因子FADH 2胞浆区室化定位和供应的工程策略，能够显著增加酚酸的积累量。
上述技术方案的方法是在酿酒酵母中过表达内源的FLX1和RIB1基因，转运线粒体FAD(H 2)在胞浆定位，增加胞浆中FADH 2浓度和供应，从而提高酚酸产量。
上述技术方案的具体步骤是：构建靶向核苷酸序列的sgRNA表达载体pgRNA-XI2，使用融合PCR构建表达盒XI2up-T CYC1-FLX1-P tHXT7-P TDH3-RIB1-T TDH2-XI2dw，和pgRNA-XI2一起使用上述方法转化酿酒酵母菌株RB197，完成构建，获得菌株RB209。
进一步地，在以20g/L葡萄糖为底物的基础盐培养基中，接种RB209酵母菌株，进行酚酸发酵，初始接种OD600为0.1，发酵条件：装液量20/100mL，30℃，220rpm，发酵时间为96h。
进一步地，对发酵产物进行提取和监测。结果证明辅因子FADH 2胞浆区室化定位和供应的工程策略能够显著增加酚酸的积累量。RB209中酚酸单体咖啡酸的产量分别为518mg/L，比菌株RB197显著提高35％。
作为一种实施方案，本申请通过批式补料发酵的条件优化，辅因子工程菌株RB209的酚酸单体咖啡酸积累量达到5.8g/L。
进一步地，上述技术方案的具体步骤是：在1L发酵罐中，以20g/L葡萄糖为底物的基础盐培养基开始发酵，起始接种体积为0.25L，OD600为0.2。通过搅拌桨速度(800-1200rpm)控制溶氧为40％，发酵温度为30℃。使用4M的KOH和2M的HCl控制pH维持在5.6。在补料批次阶段，补加200g/L葡萄糖溶液，补料速率与生长偶联，呈指数增加(将μ设置为0.05h -1)，以保持恒定的生物量比葡萄糖消耗速率。
本申请提供了一种强化酵母胞内NADPH和FADH 2供应的辅因子工程策略，及其在酚酸化合物合成的应用，主要包括以下步骤：
以表达Cas9蛋白的酿酒酵母(CEN.PK113-11C)为出发菌株，敲除GAL80基因，得到重组酵母菌A0；
改造磷酸戊糖支路，敲除GPP1基因，通过启动子P GAL10/1或P GAL7表达密码子优化后的来源Leuconostoc mesenteroides的LmXFPK基因和来源Clostridium kluyveri的CkPTA基因，以及酿酒酵母来源基因TKL和TAL，实现增强NADPH再生供应酚酸合成的目的，得到重组酵母菌A1；
进一步，改造细胞质和线粒体的FADH 2合成与穿梭途径，通过启动子P GAL10/1或P GAL7组合表达酿酒酵母来源基因FLX1和RIB1，得到重组酵母菌A2，实现增强FADH 2合成以及胞浆定位，支持酚酸在胞浆中高效合成的目的；
进一步，构建以NADPH和FADH 2为核心关键辅因子的酚酸合成途径，显著提高酚酸化合物咖啡酸的产量。
菌株发酵培养，提取验证酚酸产量及其提高情况。
GAL80基因具有如SEQ ID NO：1所示的核苷酸序列，TKL基因具有如SEQ ID NO：2所示的核苷酸序列，TAL基因具有如SEQ ID NO：3所示的核苷酸序列，FLX1基因具有如SEQ ID NO：4所示的核苷酸序列，RIB1基因具有如SEQ ID NO：5所示的核苷酸序列，密码子优化后的LmXFPK基因具有如SEQ ID NO：6所示的核苷酸序列，密码子优化后的CkPTA基因具有如SEQ ID NO：7所示的核苷酸序列。
上述所述的一种NADPH和FADH 2辅因子工程提高酵母中酚酸合成的方法，运用CRISPR-Cas9基因编辑技术，对基因进行敲除或敲入表达。
所述增强辅因子供应所表达的基因可以来自于其他物种同工酶的编码基因或其密码子优化基因。
上述任一工程酵母菌合成酚酸等NADPH/FADH 2依赖型天然产物的应用。
本申请提供了一种供应NADPH和FADH 2酵母菌株的构建及应用，并用于提高酵母中酚酸合成效率，属于微生物技术领域。由于酚酸等天然产物合成需要辅因子NADPH和FAD(H 2)的参与，酵母固有辅因子水平无法充分满足酚酸的高效合成，而外源添加辅因子价格昂贵。因此，本发明建立了一种强化供应胞内辅因子NADPH和FADH 2的策略，并用于提高酚酸生物合成效率。该辅因子工程策略使得咖啡酸产量提高了81％，批式补料发酵达到迄今最高产量5.8g/L。这一策略具有转化效率高、生产成本低、工业化应用前景广等特点，证实了酿酒酵母NADPH和FADH 2强化能显著提高酚酸等NADPH及FADH 2依赖型天然产物的合成效率，具有重要应用价值。
本申请所提供的提高酵母菌株NADPH和FADH 2供应的方法，通过增强NADPH再生和提高胞内FADH 2供给，可以有效解决以解决内源辅因子供应不足的困难，从而提高NADPH和FADH 2依赖型天然产物(例如酚酸)的合成效率。
本申请所提供的提高酵母菌株NADPH和FADH 2供应的方法，通过将基因按照宿主偏好性进行密码子优化，有利于NADPH和 FADH 2供应的提高。
本申请能产生的有益效果包括：
本发明提供了一种辅因子NADPH和FAD(H 2)工程策略，以解决酚酸等合成过程中内源辅因子供应不足的困难，从而提高酚酸等NADPH/FAD(H 2)依赖型天然产物的合成效率。
本发明的辅因子调控方法涉及调控磷酸戊糖支路，FAD(H 2)合成和转运，可以强化内源辅因子NADPH和FAD(H 2)供应和自循环，避免外源添加昂贵辅因子的成本消耗。
本发明在辅因子工程菌中，构建酚酸化合物咖啡酸途径，产量达到5.8g/L，证明了该辅因子工程策略在细胞工厂工业化的应用潜力。
附图说明
图1示出了酿酒酵母CRISPR/Cas9系统中pgRNA质粒构建过程示意图。
图2示出了NADPH辅因子工程策略示意图。
图3示出了代谢工程改造策略实现酿酒酵母合成酚酸化合物的示意图。
图4示出了FAD(H 2)辅因子工程策略示意图。
图5示出了辅因子工程酵母菌合成酚酸化合物咖啡酸的产量。
图6示出了辅因子工程酵母菌中辅因子FAD(H 2)的相对含量。
图7示出了辅因子工程酵母菌批式补料发酵生产酚酸化合物咖啡酸的产量。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学公司购买。
本申请实施例基于Euroscarf(Oberursel,Germany)公司的CEN.PK113-11C菌株(MATa MAL2-8c SUC2 his3Δ1 ura3-52)进行改造。
实施例1 NADPH辅因子工程增强酚酸合成的重组酵母构建
酚酸的生产过程极度消耗NADPH，当以葡萄糖为底物时，尽管细胞能够依靠磷酸戊糖途径提供NADPH，仍然可能供给不足。因此，我们增强磷酸戊糖途径下游非氧化步骤，为酚酸合成所需的高水平NADPH供给提供条件。
(1)基因编辑CRISPR/Cas9系统构建
实验室前期自主构建了整合CAS9基因的重组酿酒酵母CEN.PK113-11C-CAS(基因型MATa,SUC2,MAL2-8c,his3Δ,ura3Δ,XI5::(P TEF1-CAS9-T CYC1)),并以此为宿主菌株进行辅因子工程酵母菌株的构建。其中，CEN.PK113-11C-CAS构建方法如下：首先，直接化学合成CAS9和抗性基因KanMX表达盒T AgTEF-KanMX-P AgTEF-P TEF1-CAS9-T CYC1，将表达盒通过融合PCR与XI5上下游500bp融合获得供体DNA片段XI5up-T AgTEF-KanMX-P AgTEF-P TEF1-CAS9-T CYC1-XI5dw，转化酿酒酵母CEN.PK113-11C，以同源重组方式整合到XI5位点，通过遗传霉素G418进行阳性菌株筛选，获得菌株CEN.PK113-11C-CAS+KanMX。为了从基因组中去除KanMX抗性基因标签，使用pgRNA-KanMX与融合PCR获得的供体DNA片段XI5up-P TEF1进行转化。使用化学转化法将约500ng的pgRNA-KanMX和供体DNA同时转化进入整合有Cas9蛋白的酵母菌株，在加有组氨酸的SD平板上于30℃静置培养2天；转化子经加有组氨酸的液体SD培养基培养后，通过PCR验证正确，涂布于含有5-氟乳清酸的固体培养基进行质粒丢失，质粒丢失后获得菌株CEN.PK113-11C-CAS，菌株保存备用。进一步转化pgRNA-GAL80(靶向基因GAL80,SEQ ID NO：8)和供体DNA片段GAL80up-GAL80dw完成GAL80敲除，获得菌株A0(MATa,SUC2,MAL2-8c,his3Δ,ura3Δ,gal80Δ,XI5::(P TEF1-CAS9-T CYC1))，可实现P GAL7和P GAL10/1表达的途径基因在限糖条件下高效表达。
以上sgRNA表达载体构建过程如图1所示，本发明中使用的所有sgRNA表达载体除20bp靶向序列不同以外，其余部分完全相同。简单来说，首先使用引物6005(SEQ ID NO：25；GATCATTTATCTTTCACTGCGGAGAAG)从pgRNA-GAL80(靶向基因GAL80,SEQ ID NO：8)中扩增载体骨架S1；接着，分别扩增sgRNA-1和sgRNA-2，其中sgRNA-1使用引物p1(SEQ ID NO：26；GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATC)和pX1(AAACTTCTCCGCAGTGAAAGATAAATGATC(M 20)GTTTTAGAGCTAGAAATAG，其中M 20为可替换20bp靶向序列)，sgRNA-2使用引物p2(SEQ ID NO：27；GATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGC)和pX2(AAACTTCTCCGCAGTGAAAGATAAATGATC(N 20)GTTTTAGAGCTAGAAATAG，其中N(N 20)为可替换的另外20bp靶向序列)；最后，sgRNA片段和载体骨架片段使用Gibson Assembly的方法进行克隆连接，获得的重组载体测序后进行应用。pX1和pX2中M 20和N 20的替换序列及替换后对应的序列名称详见表1，本发明中涉及的sgRNA表达载体包括pgRNA-GAL80(靶向基因GAL80,SEQ ID NO：8)，pgRNA-GPP1(靶向基因GPP1)，pgRNA-XI7(靶向位点XI7)，pgRNA-XI6(靶向位点XI6)，pgRNA-ARO10(靶向基因ARO10)，pgRNA-PDC5(靶向基因PDC5)，pgRNA-XII4(靶向位点XII4)，pgRNA-XII1(靶向位点XII1)，pgRNA-X2(靶向位点X2)，pgRNA-XI8(靶向位点XI8)，pgRNA-XI2(靶向位点XI2)。对于供体DNA，分别扩增pgRNA载体靶向位点的下游各约500bp序列作为同源臂，之后通过常规融合PCR的方式将需要插入的各个片段进行组装，获得完整的供体DNA分子，与对应位点的pgRNA一起转化酵母，用于基因编辑和菌株改造。涉及到的基因信息见表4。
表1
序列名称 M 20 N 20
pgRNA-GAL80 SEQ ID NO：28；TTCCAAGGCACATTGTTAAA SEQ ID NO：29；CACAATTTGTAATGCAAGGT
pgRNA-GPP1 SEQ ID NO：30；GTCTGGAGCGAACTTGGCAA SEQ ID NO：31；GAATACGTTAACAAGCTAGA
pgRNA-XI7 SEQ ID NO：32；GTCAGTAACAGTGATTGCTG SEQ ID NO：33；TATGTAGGTTCCGATTAAAG
pgRNA-XI6 SEQ ID NO：34；CGCTACAATTCGGTAAGTTT SEQ ID NO：35；TAGTAAATACAACTATTGGA
pgRNA-ARO10 SEQ ID NO：36；GAAAACTGATTTCGTATCGA SEQ ID NO：37；AATATACGAACGAAACAATG
pgRNA-PDC5 SEQ ID NO：38；GATAAGCTTTATGAAGTCAA SEQ ID NO：39；ATTTGCCTTGCAAAAATTGT
pgRNA-XII4 SEQ ID NO：40；TAATGGGGAATTGTACAAAT SEQ ID NO：41；GTTACCCGCGCTATTTCACA
pgRNA-XII1 SEQ ID NO：42；CCACTTCTCATGACATATAT SEQ ID NO：43；CAATACTAATTAGTCTTTGC
pgRNA-X2 SEQ ID NO：44；CGGGTCTAGGCCTGCATAAT SEQ ID NO：45；GCTCGTTTCTTTTTTCAGTG
pgRNA-XI8 SEQ ID NO：46；GATGAAATAGCCTCAGTTAC SEQ ID NO：47；TTGTCGTGTTACTGATAGTA
pgRNA-XI2 SEQ ID NO：48；GCATGATAAATCGGTAGAAT SEQ ID NO：49；GGTTCTAGAAGTGCCCTTTG
pgRNA-KanMX SEQ ID NO：50；GGTTCTAGAAGTGCCCTTTG SEQ ID NO：51；ATTCTACCGATTTATCATGC
(2)NADPH辅因子工程酵母菌株构建
酿酒酵母中NADPH再生主要通过磷酸戊糖途径(PPP)的氧化步骤完成。因为设计使用启动子P GAL7和P GAL10/1表达外源酚酸途径，因此只有在限糖条件下才会合成酚酸。
在限糖条件下磷酸戊糖途径的氧化步骤通量已经较大，真正限制NADPH再生的限速步骤是下游非氧化步骤。因此，我们开创地设计增强下游非氧化步骤增强NADPH再生，从而为酚酸合成提供充足的辅因子，NADPH再生的设计路线如图2所示。
利用上述构建的基因编辑菌株A0，通过转化pgRNA-GPP1和GPP1up-T ENO2-CkPTA-P GAL10/1-LmXFPK-T ADH1-GPP1dw敲除GPP1的同时表达CkPTA和LmXFPK，进一步转化pgRNA-XI7和XI7up-T PRM9-P GAL10/1-TAL1-T PYK1-XI7dw表达TAL1，然后转化pgRNA-XI6和XI6up-P GAL7-TKL1-T ENO2-XI6dw表达TKL1，获得辅因子NADPH工程酵母菌株A1。
(3)酚酸合成途径的构建
在菌株A0和A1基础上构建酚酸途径(如图3)。分别依次转化pgRNA-ARO10和ARO10up-T HIS3-ARO7 G141S-P GAL10/1-ARO4 K229L-T ENO2-P GAL7-EcAROL-T ADH1-ARO10dw；pgRNA-PDC5和ScPDC5up-T CYC1-FjTAL-Sc OPT1-P GAL10/1-SbPAL1 H123F-Sc OPT1-T TDH2-T FBA1-AtCPR1-Sc OPT1-P GAL7-ScPDC5dw；pgRNA-XII4和XII4up-T PRM9t-PtrC4H2-Sc OPT1-P GAL10/1-PtrC4H1-Sc OPT1-T PYK1-P GAL7-PtrC3H3-Sc OPT1-T DIT1-XII4dw；pgRNA-XII1和XII1up-P GAL7-ARO1-T ENO2-T CPS1-ARO2-P GAL10/1-ARO3-T HIS5-XII1dw；pgRNA-X2和X2up-T PRM9-PahpaB-Sc OPT1-P GAL10/1-SehpaC-Sc OPT1-T HIS3-X2dw；pgRNA-XI8和XI8up-T CPS1-PHA2-P GAL10/1-MtPDH1-Sc OPT1-T HIS5-XI8dw，完成酚酸途径的构建和增强，获得菌株RB103和RB197，供后续发酵和合成分析使用。
实施例2 FAD(H 2)辅因子工程增强酚酸合成的重组酵母构建
酚酸的生产过程极度消耗NADH和FADH 2，当以葡萄糖为底物时，胞浆内NADH的水平远远高于FADH 2，相比之下，FADH 2可能供给不足。因此，我们通过增强FADH 2的合成，并将线粒体的FADH 2外排到胞浆，提高胞内FADH 2供给(图4)。
具体是使用上述CRISPR-Cas9基因敲除和插入技术，在菌株RB197中，进一步转化pgRNA-XI2和XI2up-T CYC1-FLX1-P tHXT7-P TDH3-RIB1-T TDH2-XI2dw，从而表达RIB1基因增强FADH 2的合成，表达FLX1基因从线粒体转运FADH 2到胞浆，最终达到提高胞浆FADH 2的目的。获得的FAD(H 2)辅因子工程菌株为RB209，供后续发酵和合成分析使用。
实施例3重组酿酒酵母发酵合成酚酸
(1)培养基
YPD培养基：20g/L葡萄糖，20g/L蛋白胨，10g/L酵母粉；
SD培养基：20g/L葡萄糖，6.7g/L YNB，使用时添加组氨酸0.02g/L；
发酵培养基(基础成分培养基)：(NH 4) 2SO 4 2.5g/L，KH 2PO 4 14.4g/L，MgSO 4·7H 2O 0.5g/L，加入约900mL ddH 2O，调节pH为5.6，定容至950mL，115℃灭菌30min。灭菌后，补加1mL维生素溶液(配方见表3)和2mL微量金属溶液(配方见表2)，根据需要添加组氨酸和尿嘧啶(40mg/L)。向发酵培养基中添加葡糖糖到20g/L，用于酚酸发酵。
表2微量金属溶液配置方法(1L溶液)：
编号 试剂 重量[g]
1 FeSO 4·7H 2O 3.0
2 ZnSO 4·7H 2O 4.5
3 CaCl 2·2H 2O 4.5
4 MnCl 2·4H 2O 1.03
5 CoCl 2·6H 2O 0.3
6 CuSO 4·5H 2O 0.3
7 Na 2MoO 4·2H 2O 0.4
8 H 3BO 3 1.0
9 KI 0.1
10 Na 2EDTA·2H 20 19.0
表3维生素溶液配置方法(1L溶液)：
编号 名称 重量[g]
1 D-生物素 0.05
2 D-泛酸钙 1.0
3 硫胺 1.0
4 吡哆醇 1.0
5 烟酸 1.0
6 对氨基苯甲酸 0.2
7 肌醇 25.0
(2)实验流程及条件
将RB103，RB197和RB209菌株活化，分别挑取单菌落于3/15mL YPD培养基中，30℃，220rpm震荡培养16h；接种，将种子液用发酵培养基清洗2次，按初始OD600＝0.1接种于发酵培养基，装液量为20mL/100mL锥形瓶，30℃，220rpm条件下进行发酵72～96h。定点取样或者终点取样，用于生物量(以600nm处的吸光值表示)和酚酸产量分析。
(3)以葡萄糖为底物的酚酸合成
在含20g/L葡萄糖的基础培养基条件下进行酚酸的发酵实验，初始接种OD600为0.1，发酵条件：装液量20/100mL，30℃，220rpm， 发酵时间为96h。然后提酚酸化合物，取50μL发酵样品，加入450μL无菌水，再加入500μL乙醇后充分涡旋混匀；13000g离心5min，取500μL上清过0.22μm水相微孔滤膜后获得酚酸样品。样品检测使用岛津HPLC，色谱柱为3×100mm 2.7um Poroshell 120 EC-C18(Agilent)，流速为0.8mL/min，流动相A为H2O+0.05％HCOOH，流动相B为ACN+0.05％HCOOH，紫外检测器检测波长为280nm或330nm。具体流动相梯度为95％-A(0min)，90％-A(3min)，88％-A(4-5min)，85％-A(6-7min)，40％-A(10min)，95％-A(12-14min)。发酵96h结束，三种菌株RB103，RB197和RB209的酚酸产量存在显著差异，结果如图5所示。通过磷酸戊糖途径非氧化步骤的改造增强辅因子NADPH供应，使得酚酸单体咖啡酸在RB197中的产量达到385mg/L，相对于RB103的286mg/L显著提高35％。进一步通过辅因子FAD(H 2)工程，RB209中咖啡酸的产量达到518mg/L，相对于RB197显著提高35％。检测发现RB209中的辅因子FAD(H2)水平比对照菌株RB197提高34％(图6)。
为了验证辅因子工程合成酚酸的潜力，对RB209进行批式补料发酵。在1L发酵罐中，以20g/L葡萄糖为底物的基础盐培养基开始发酵，起始接种体积为0.25L，OD600为0.2。通过搅拌桨速度(800-1200rpm)控制溶氧为40％，发酵温度为30℃。使用4M的KOH和2M的HCl控制pH维持在5.6。在补料批次阶段，每24小时补加500g/L葡萄糖溶液至培养基中的葡萄糖浓度到20g/L。最终发酵86h达到迄今为止最高的酚酸单体咖啡酸产量5.8g/L(图7)。这一结果证实了酿酒酵母NADPH和FADH 2辅因子工程在酚酸合成中的潜力和重要应用价值。
表4基因名称及序列编号
编号 基因名称 来源 序列编号
1 GAL80 Saccharomyces cerevisiae SEQ ID NO：1
2 TKL1 Saccharomyces cerevisiae SEQ ID NO：2
3 TAL1 Saccharomyces cerevisiae SEQ ID NO：3
4 FLX1 Saccharomyces cerevisiae SEQ ID NO：4
5 RIB1 Saccharomyces cerevisiae SEQ ID NO：5
6 LmXFPK-Sc OPT1 Leuconostoc mesenteroides SEQ ID NO：6
7 CkPTA-Sc OPT1 Clostridium kluyveri SEQ ID NO：7
8 pgRNA-GLA80 / SEQ ID NO：8
9 ARO4 K229L Saccharomyces cerevisiae SEQ ID NO：9
10 ARO7 G141S Saccharomyces cerevisiae SEQ ID NO：10
11 EcAROL Escherichia coli SEQ ID NO：11
12 ARO1 Saccharomyces cerevisiae SEQ ID NO：12
13 ARO2 Saccharomyces cerevisiae SEQ ID NO：13
14 ARO3 Saccharomyces cerevisiae SEQ ID NO：14
15 PHA2 Saccharomyces cerevisiae SEQ ID NO：15
16 MtPDH1-Sc OPT1 Medicago truncatula SEQ ID NO：16
17 FjTAL-Sc OPT1 Flavobacterium johnsoniae SEQ ID NO：17
18 SbPAL1 H123F-Sc OPT1 Sorghum bicolor SEQ ID NO：18
19 AtCPR1-Sc OPT1 Arabidopsis thaliana SEQ ID NO：19
20 PtrC4H1-Sc OPT1 Populus trichocarpa SEQ ID NO：20
21 PtrC4H2-Sc OPT1 Populus trichocarpa SEQ ID NO：21
22 PtrC3H-Sc OPT1 Populus trichocarpa SEQ ID NO：22
23 PaHPAB-Sc OPT1 Pseudomonas aeruginosa SEQ ID NO：23
24 SeHPAC-Sc OPT1 Sulfobacillus acidophilus SEQ ID NO：24
注：Sc OPT1表明将基因按照酿酒酵母偏好性进行密码子优化。
以上所述，仅是本申请的几个实施例，并非对本申请做任何形式的限制，虽然本申请以较佳实施例揭示如上，然而并非用以限制本申请，任何熟悉本专业的技术人员，在不脱离本申请技术方案的范围内，利用上述揭示的技术内容做出些许的变动或修饰均等同于等效实施案例，均属于技术方案范围内。

Claims (19)

  1. 一种提高酵母菌株NADPH和FADH 2供应的方法，其特征在于，所述方法包括增强NADPH再生和提高胞内FADH 2供给；
    所述增强NADPH再生包括：
    在酵母菌株中导入LmXFPK基因、CkPTA基因、TAL1基因和TKL1基因；
    所述提高胞内FADH 2供给包括：
    在酵母菌株中导入RIB1基因和FLX1基因。
  2. 根据权利要求1所述的方法，其特征在于，TKL1基因的核苷酸序列如SEQ ID NO：2所示；
    TAL1基因的核苷酸序列如SEQ ID NO：3所示；
    FLX1基因的核苷酸序列如SEQ ID NO：4所示；
    RIB1基因的核苷酸序列如SEQ ID NO：5所示；
    LmXFPK基因的核苷酸序列如SEQ ID NO：6所示；
    CkPTA基因的核苷酸序列如SEQ ID NO：7所示。
  3. 根据权利要求1所述的方法，其特征在于，所述增强NADPH再生包括：
    将含有T ENO2-CkPTA-P GAL10/1-LmXFPK-T ADH1的DNA片段整合在酵母菌株的GPP1位点；
    将含有T PRM9-P GAL10/1-TAL1-T PYK1的DNA片段整合在酵母菌株的XI7位点；
    将含有P GAL7-TKL1-T ENO2的DNA片段整合在酵母菌株的XI6位点。
  4. 根据权利要求1所述的方法，其特征在于，所述提高胞内FADH 2供给包括：
    将含有T CYC1-FLX1-P tHXT7-P TDH3-RIB1-T TDH2的DNA片段整合在酵母菌株的XI2位点。
  5. 根据权利要求1所述的方法，其特征在于，所述酵母菌株选自酿酒酵母中的任一种。
  6. 根据权利要求1所述的方法，其特征在于，所述酵母菌株选自产酚酸酵母工程菌株中的任一种。
  7. 根据权利要求6所述的方法，其特征在于，所述产酚酸酵母工程菌株通过在酿酒酵母出发菌株中导入酚酸合成途径相关基因得到：
    所述酚酸合成途径相关基因包括Aro4 K229L、Aro7 G141S、EcAROL、ARO1、ARO2、ARO3、PHA2、MtPDH1、FjTAL、SbPAL1 H123F、AtCPR1、PtrC4H1、PtrC4H2、PtrC3H、PaHPAB、SeHPAC中的至少一种。
  8. 根据权利要求7所述的方法，其特征在于，所述酚酸合成途径相关基因包括具有如SEQ ID NO：9所示核苷酸序列的Aro4 K229L、具有如SEQ ID NO：10所示核苷酸序列的Aro7 G141S、具有如SEQ ID NO：11所示核苷酸序列的EcAROL、具有如SEQ ID NO：12所示的核苷酸序列的ARO1、具有SEQ ID NO：13所示核苷酸序列的ARO2、具有如SEQ ID NO：14所示核苷酸序列的ARO3、具有如SEQ ID NO：15所示核苷酸序列的PHA2、具有如SEQ ID NO：16所示核苷酸序列的MtPDH1、具有如SEQ ID NO：17所示核苷酸序列的FjTAL，具有如SEQ ID NO：18所示核苷酸序列的SbPAL1 H123F、具有如SEQ ID NO：19所示核苷酸序列的AtCPR1、具有如SEQ ID NO：20所示核苷酸序列的PtrC4H1、具有如SEQ ID NO：21所示核苷酸序列的PtrC4H2、具有如SEQ ID NO：22所示核苷酸序列的PtrC3H、具有如SEQ ID NO：23所示核苷酸序列的PaHPAB、具有如SEQ ID NO：24所示核苷酸序列的SeHPAC中的至少一种。
  9. 根据权利要求7所述的方法，其特征在于，ARO7 G141S、ARO4 K229L、EcAROL的导入位点为ARO10；
    FjTAL、SbPAL1 H123F、AtCPR1的导入位点为PDC5；
    PtrC4H2、PtrC4H1、PtrC3H的导入位点为XII4；
    ARO1、ARO2、ARO3的导入位点为XII1；
    PahpaB、SehpaC的导入位点为X2；
    PHA2、MtPDH1的导入位点为XI8。
  10. 根据权利要求7所述的方法，其特征在于，将含有T HIS3-ARO7 G141S-P GAL10/1-ARO4 K229L-T ENO2-P GAL7-EcAROL-T ADH1的DNA片段整合到出发菌株的ARO10位点；
    将含有T CYC1-FjTAL-Sc OPT1-P GAL10/1-SbPAL1 H123F-Sc OPT1-T TDH2-T FBA1-AtCPR1-Sc OPT1-P GAL7的DNA片段整合到出发菌株的PDC5位点；
    将含有T PRM9t-PtrC4H2-Sc OPT1-P GAL10/1-PtrC4H1-Sc OPT1-T PYK1-P GAL7-PtrC3H-Sc OPT1-T DIT1的DNA片段整合到出发菌株的XII4位点；、
    将含有P GAL7-ARO1-T ENO2-T CPS1-ARO2-P GAL10/1-ARO3-T HIS5的DNA片段整合到出发菌株的XII1位点；
    将含有T PRM9-PahpaB-Sc OPT1-P GAL10/1-SehpaC-Sc OPT1-T HIS3的DNA片段整合到出发菌株的X2位点；
    将含有T CPS1-PHA2-P GAL10/1-MtPDH1-Sc OPT1-T HIS5的DNA片段整合到出发菌株的XI8位点。
  11. 根据权利要求7所述的方法，其特征在于，还敲除酿酒酵母出发菌株的GAL80基因。
  12. 权利要求1～11任一项所述的方法得到的工程菌。
  13. 权利要求1～11任一项所述的方法得到的工程菌、权利要求12所述的工程菌在合成NADPH和FADH 2依赖型天然产物中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述NADPH和FADH 2依赖型天然产物选自酚酸中的一种。
  15. 一种酚酸的制备方法，其特征在于，所述制备方法包括以下步骤：
    将工程菌接种于含葡萄糖的培养基I中，发酵I，得到所述酚酸；
    或；
    将工程菌接种于含葡萄糖的培养基II中进行批式补料发酵II，得到所述酚酸；
    所述工程菌选自权利要求6～11任一项所述方法得到的工程菌。
  16. 根据权利要求15所述的制备方法，其特征在于，所述葡萄糖在所述培养基I中的含量为10～20g/L。
  17. 根据权利要求15所述的制备方法，其特征在于，所述发酵I条件包括：
    温度为28～30℃；
    转数为220～280rpm；
    时间为72～96小时；
    装液量15～20/100mL。
  18. 根据权利要求15所述的制备方法，其特征在于，所述批式补料发酵II为培养基II中葡萄糖初始浓度为15～20g/L，每隔20～28小时将培养基II中葡萄糖补充至15～20g/L。
  19. 根据权利要求15所述的制备方法，其特征在于，所述批式补料发酵II条件包括：
    温度为28～30℃；
    转数为800～1200rpm；
    溶氧为>40％；
    pH为5.6～6.0。
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CN111778167A (zh) * 2019-04-04 2020-10-16 暨南大学 高产桦木酸的酿酒酵母工程菌及其构建方法与应用
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