WO2023108503A1 - 一种工程菌的构建方法及其应用 - Google Patents

一种工程菌的构建方法及其应用 Download PDF

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WO2023108503A1
WO2023108503A1 PCT/CN2021/138506 CN2021138506W WO2023108503A1 WO 2023108503 A1 WO2023108503 A1 WO 2023108503A1 CN 2021138506 W CN2021138506 W CN 2021138506W WO 2023108503 A1 WO2023108503 A1 WO 2023108503A1
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gene
host strain
construction method
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introducing
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周雍进
曹选
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中国科学院大连化学物理研究所
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    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention belongs to the field of microbial metabolism engineering and industrial biotechnology, and relates to a construction method and application of engineering bacteria.
  • Sclareol also known as hard tail alcohol, as a semi-helican-type diterpenoid tertiary alcohol, was originally extracted from Salvia sclarea, a lip-shaped spice plant of the genus Salvia, and thus got its name.
  • Sclareol is the most common starting material for the synthesis of ambrox, a substitute for ambergris.
  • Ambergris a waxy substance secreted by sperm whales, has been a fixture of high-end perfumes since ancient times for its musky and sweet earthy scent.
  • sclareol has strong antibacterial activity and plays a role in fungal growth regulation and plant growth inhibition, and has a strong effect on human leukemia cells (Dimas et al., Leuk Res, 1999, 23:217-234), tumor cells strain (Mahaira et al., Eur J Pharmacol, 2011,666:173-182), colon cancer cells and xenografts (Dimas et al., Apoptosis, 2007,12:685-694; Mahaira et al., Eur J Pharmacol, 2011, 666:173-182) has cytotoxicity.
  • LPP lysardene diol pyrophosphate
  • SsLPPS lysardene diol pyrophosphate
  • SsTPS sclareol synthase gene SsTPS in Saccharomyces cerevisiae
  • GGPP truncated hydroxy-3-methylpentanediol Acyl-CoA reductase gene tHMG1 and fusion expression GGPP synthase gene and farnesene synthase gene BTS1-ERG20 make sclareol production reach 9mg/L (Yang Wei et al., Biological Engineering Journal, 2013 (08): 152-159).
  • Ignea et al expressed the 8-hydroxypropyl diphosphate (8OH-CPP) synthase gene CcCLS from Cistus cerevisiae and the sclareol synthase gene SsTPS from Claryus cerevisiae in Saccharomyces cerevisiae chassis cell AM205 and The site-directed mutation of ERG20 to ERG20 F96C increased the production of sclareol to 403 mg/L (Ignea et al., Metabolic Engineering, 2015:65-75). In the same year, Trikka et al.
  • the invention constructs a yeast cell factory from global metabolism to realize efficient synthesis of sclareol.
  • the three modules were optimized separately, and the synergistic cooperation between the modules was realized, so that the production of sclareol in Saccharomyces cerevisiae reached 1.1g/L in shake flask, and 11.4g/L in batch fed-batch fermentation.
  • the technical problem to be solved by the present invention overcomes the low activity of terpene biosynthetic enzymes and mismatched metabolism in yeast engineering bacteria, and increases the biological activity of diterpenoids such as sclareol from terpene synthase optimization and global metabolic module optimization. Synthetic efficiency, providing a yeast genetically engineered strain producing sclareol and its application.
  • a method for constructing an engineering bacterium is provided.
  • the construction method optimizes the sclareol synthase of the host strain, which is beneficial to increase the yield of sclareol.
  • a construction method of engineering bacterium comprises the following steps:
  • the DNA fragment I includes in sequence from upstream to bottom: a promoter, a fusion gene, and a terminator;
  • the fusion gene includes: TPS gene, connecting peptide I gene, LPPS gene from upstream to bottom;
  • the host strain is selected from any one of Saccharomyces cerevisiae.
  • the fusion gene includes from upstream to bottom:
  • MBP gene connecting peptide II gene, TPS gene, connecting peptide I gene, LPPS gene;
  • Described fusion gene comprises sequentially from upstream to bottom:
  • TPS gene gene encoding connecting peptide I
  • LPPS gene gene encoding connecting peptide II
  • MBP gene gene encoding connecting peptide II
  • nucleotide sequence of the LPPS gene is shown in SEQ ID NO: 1;
  • the nucleotide sequence of the TPS gene is shown in SEQ ID NO: 2;
  • the nucleotide sequence of the MBP gene is shown in SEQ ID NO: 3;
  • the nucleotide sequence of the gene encoding the connecting peptide I is shown in SEQ ID NO: 6;
  • the nucleotide sequence of the gene encoding the connecting peptide II is shown in SEQ ID NO: 7;
  • the promoter is selected from any one of P eTDH3 and P GAL7 ;
  • the terminator is selected from any one of T PYX212 and T ADH1 .
  • introducing the DNA fragment I into the host strain is to integrate the DNA fragment I into the XI-3 gene locus of the host.
  • the construction method also includes optimizing the synthetic pathway of MVA, including at least one of (a) to (h):
  • the (a) to (h) are in no particular order.
  • the (a) to (h) are in no particular order.
  • the construction method also includes optimizing the central metabolic pathway, including at least one of (A) to (N):
  • the (A) to (N) are not in any particular order.
  • the construction method also includes optimizing the central metabolic pathway, including at least one of (A) to (G):
  • the (A) to (G) are not in any particular order.
  • the construction method also includes target regulation, including:
  • the construction method also includes at least one of the following:
  • At least one of ANB1 gene, DAN1 gene, HUG1 gene, PYK1 gene, LAC1 gene, CYB5 gene, YCL021W-A gene, YCL074W gene, OYE3 gene, OYE2 gene, HSP31 gene, BLI1 gene is introduced into the host bacterial strain ;
  • the construction method is constructed by using CRISPR/Cas9 technology.
  • the construction method also includes cell fusion to form a diploid.
  • an engineered bacterium constructed according to any one of the construction methods described above.
  • the engineering bacteria constructed according to any one of the above-mentioned construction methods, and the application of at least one of the above-mentioned engineering bacteria in the preparation of sclareol.
  • the application provides a Saccharomyces cerevisiae genetically engineered strain producing sclareol, which optimizes the sclareol synthase, expresses two enzymes that catalyze the synthesis of sclareol in tandem, and expresses them in N Maltose-binding protein MBP is fused at the end.
  • the central metabolic module, the isoprene biosynthesis pathway module and the regulatory module are optimized and constructed in yeast;
  • isoprene biosynthetic pathway rate-limiting enzyme genes including truncated hydroxy-3-methylglutaryl-CoA reductase gene tHMG1, mutant HMG2 K6R , from Silicibacter pomeroyi NADH-dependent SpHMGR, acetoacetyl-CoA thiolase gene ERG10, endogenous GGPP synthase and GGPP synthase fusion protein gene BTS1-PaGGPPS from Phomopsis amygdali and mutated the FPP synthase gene in the genome to ERG20 F96C and replaced ERG9 with PHXT1 . Dynamically regulate the expression of ERG9;
  • enhanced central metabolic pathways increase the supply of the precursor acetyl-CoA and the cofactor NADPH, including expression of the ATP-dependent citrate lyase gene MmACL from Mus musculus, NADP + -dependent malate synthesis from Rhodospuridium toruloides Enzyme gene RtME and citrate synthase 1 gene RtCIT1, malate dehydrogenase gene 'MDH3 to remove peroxisome signal peptide, citrate transporter gene CTP1, pyruvate carboxylase 1 gene PYC1, pyruvate transporter 1 and 3 genes MPC1 and MPC3, ATP-dependent citrate lyase a and b genes AnACLa and AnACLb from Aspergillus nidulans, NADP-specific isocitrate dehydrogenase 2 gene IDP2, citrate, alpha-ketoglutarate transporter Protein gene YHM2, glucose phosphate dehydrogenase isoform 1
  • knockout of regulatory factor genes including ROX1, DOS2, VBA5, YER134c, YNR063w, YGR259c;
  • a diploid strain is constructed, and the constructed ⁇ strain is matched to a type a strain, and then the two are fused to obtain a diploid strain.
  • the application provides a genetic engineering bacterium of Saccharomyces cerevisiae
  • the construction method of the genetic engineering bacterium of Saccharomyces cerevisiae comprises: combining the TPS gene shown in SEQ ID NO: 2 with the LPPS gene shown in SEQ ID NO: 1 Gene fusion, the connecting peptide is GGGS, to obtain fusion gene 1; the N-terminal of fusion gene 1 is fused with the gene encoding maltose binding protein shown in SEQ ID NO: 3 to obtain fusion gene 2; fusion gene 2 together with plasmid pgRNA-XI-3 Transform into the starting strain to obtain the Saccharomyces cerevisiae genetically engineered strain producing sclareol.
  • the target genomic site of the plasmid pgRNA-XI-3 is XI-3.
  • the application provides a fusion gene
  • the sequence of the fusion gene contains the LPPS gene sequence shown in SEQ ID NO: 1, the TPS gene sequence shown in SEQ ID NO: 2;
  • sequence of the fusion gene contains any one of the following fragments:
  • Fusion gene 1 The TPS gene shown in SEQ ID NO: 2 is fused with the LPPS gene shown in SEQ ID NO: 1 through the connecting peptide GGGS, and the fusion gene 1 is obtained according to the TPS gene-connecting peptide GGGGS-LPPS gene arrangement;
  • Fusion gene 2 the LPPS gene shown in SEQ ID NO: 1 is fused with the TPS gene shown in SEQ ID NO: 2 through the linker peptide GGGS to obtain fusion gene 2;
  • Fusion gene 3 The LPPS gene shown in SEQ ID NO: 1 and the terminator T PYK1 are fused to grow fragment 1, and the promoter P eTDH3 , the TPS gene shown in SEQ ID NO: 2 and the terminator T PRM9 are fused to grow fragment 2 , fusing long fragment 1 and long fragment 2 to obtain fusion gene 3;
  • nucleotide sequence of the connecting peptide GGGS is shown in SEQ ID NO: 6;
  • the fusion gene also contains the gene sequence encoding maltose binding protein shown in SEQ ID NO: 3;
  • sequence of the fusion gene contains the following fragments:
  • the TPS gene shown in SEQ ID NO: 2 is fused with the LPPS gene shown in SEQ ID NO: 1, and the connecting peptide is GGGS to obtain fusion gene 1; the N-terminal of fusion gene 1 is combined with maltose shown in SEQ ID NO: 3
  • the protein coding gene MBP is fused, and the connecting peptide is Gly6;
  • nucleotide sequence of the Gly6 is shown in SEQ ID NO: 7.
  • Saccharomyces cerevisiae genetically engineered strain is obtained by replacing the XI-3 gene in the starting strain with a fragment containing a fusion gene.
  • the application provides a genetic engineering bacterium of Saccharomyces cerevisiae
  • the construction method of the genetic engineering bacterium of Saccharomyces cerevisiae comprises: combining the TPS gene shown in SEQ ID NO: 2 with the LPPS gene shown in SEQ ID NO: 1 Gene fusion, the connecting peptide is GGGS, to obtain fusion gene 1; the N-terminal of fusion gene 1 is fused with the gene encoding maltose binding protein shown in SEQ ID NO: 3 to obtain fusion gene 2; fusion gene 2 together with plasmid pgRNA-XI-3 Transferred into the starting strain, seamlessly knocked out the GAL80 gene, and obtained the Saccharomyces cerevisiae genetically engineered strain producing sclareol.
  • the present application provides a genetically engineered strain of Saccharomyces cerevisiae obtained by enhancing the expression of the rate-limiting enzyme gene of the isoprene biosynthetic pathway in the starting strain, including the truncated hydroxy-3-methylglutaryl coenzyme A reductase gene tHMG1, mutant HMG2 K6R derived from Silicibacter pomeroyi NADH-dependent SpHMGR, acetoacetyl-CoA thiolase gene ERG10, HMG2 K6R , endogenous GGPP synthase fusion protein gene BTS1 with GGPP synthase from Phomopsis amygdali -PaGGPPS and mutate at least one of the FPP synthetase gene in the genome to ERG20 F96C and use PHXT1 to replace the ERG9 promoter to dynamically regulate the expression of ERG9.
  • tHMG1 mutant HMG2 K6R from Silicibacter pomeroyi NADH-dependent SpHMGR
  • acetoacetyl-CoA thiolase gene ERG10 HMG2 K6R
  • BTS1-PaGGPPS from Phomopsis amygdali (the connecting peptide is GGGS) were mutated to ERG20 F96C and the ERG9 promoter was replaced by PHXT1 to dynamically regulate the expression of ERG9.
  • the starting strain is Saccharomyces cerevisiae genetically engineered strain CXM01*.
  • nucleotide sequence of the SpHMGR is shown in SEQ ID NO: 5.
  • nucleotide sequence of the PaGGPPS is shown in SEQ ID NO: 4.
  • the application provides a genetically engineered strain of Saccharomyces cerevisiae, which is obtained by strengthening the central metabolic pathway in the starting strain, including increasing the supply of precursor acetyl-CoA and cofactor NADPH, including expressing the protein from Mus musculus ATP-dependent citrate lyase gene MmACL, NADP + -dependent malate synthase gene RtME and citrate synthase 1 gene RtCIT1 from Rhodospuridium toruloides, malate dehydrogenase gene 'MDH3 with peroxisomal signal peptide removed , citrate transporter gene CTP1, pyruvate carboxylase 1 gene PYC1, pyruvate transporter 1 and 3 genes MPC1 and MPC3, ATP-dependent citrate lyase a and b genes AnACLa and AnACLb from Aspergillus nidulans, NADP specific Isocitrate dehydrogenase 2 gene IDP2,
  • acetyl-CoA and cofactor NADPH including expression of the ATP-dependent citrate lyase gene MmACL from Mus musculus, the NADP + -dependent malate synthase gene RtME from Rhodospuridium toruloides and Citrate synthase 1 gene RtCIT1, malate dehydrogenase gene 'MDH3 with peroxisome signal peptide removed, citrate transporter gene CTP1, pyruvate carboxylase 1 gene PYC1, pyruvate transporter 1 and 3 genes MPC1 and MPC3, ATP-dependent citrate lyase a and b genes AnACLa and AnACLb from Aspergillus nidulans, NADP-specific isocitrate dehydrogenase 2 gene IDP2, citrate, ⁇ -ketoglutarate transporter gene YHM2, Glucose phosphate dehydrogenase isoform 1 gene G
  • the present application provides a genetic engineering strain of Saccharomyces cerevisiae, in which the regulatory factor gene is knocked out in the starting strain, and the regulatory factor gene includes at least one of ROX1, DOS2, VBA5, YER134c, YNR063w, and YGR259c;
  • the regulatory factor genes include ROX1, DOS2, VBA5, YER134c, YNR063w, YGR259c;
  • the starting strain is Saccharomyces cerevisiae genetically engineered strain SCX38.
  • the present application provides a diploid strain of Saccharomyces cerevisiae, wherein the sclareol-producing Saccharomyces cerevisiae genetically engineered strain SCX42 is converted from MATa to MAT ⁇ to obtain SCX42- ⁇ , and SCX42 is transformed from defective ura3- , his3 - into ura3 - , met3 to get SCX42*, and then co-cultivate SCX42* and SCX42- ⁇ to get a diploid strain.
  • the present application provides a genetically engineered strain of Saccharomyces cerevisiae, wherein the genetically engineered strain of Saccharomyces cerevisiae is overexpressed in the starting strain: ANB1, DAN1, HUG1, PYK1, LAC1, CYB5, YCL021W-A, YCL074W, OYE3 , at least one of OYE2, HSP31, BLI1;
  • At least one of SPS4, YOR314W, SUL1, CDA2, ERV15, ZRT1, DSE1, ANB1 is obtained;
  • the starting strain is selected from any one of CXM01*, CXM17, CXM18, SCX38 and SCX42;
  • the CXM18 is obtained by knocking out transcription factors ROX1, DOS2, VBA5, YER134c, YNR063w, YGR259c from CXM17;
  • Saccharomyces cerevisiae genetically engineered bacteria are overexpressed in the starting strain: ANB1, DAN1, HUG1, PYK1, LAC1, CYB5, YCL021W-A, YCL074W, OYE3, OYE2, HSP31, BLI1, and knockout: SPS4, YOR314W, SUL1, CDA2, ERV15, ZRT1, DSE1, ANB1 were obtained.
  • the Saccharomyces cerevisiae engineered bacteria described in any one of the above uses the basic component medium, adds 20-25g/L glucose, the initial pH is 5-6, and shakes at room temperature ; transfer to a container containing a fermentation medium, shake and ferment at room temperature for 96-120 hours, and the fermentation liquid contains sclareol.
  • the construction method of the engineering bacteria provided in this application optimizes the sclareol synthase of the host strain, which is beneficial to increase the yield of sclareol.
  • the construction method of the engineering bacteria provided by the application improves the production of sclareol by strengthening the MVA pathway of Saccharomyces cerevisiae, strengthening central metabolism, and regulating transcription factors.
  • Figure 1 shows a schematic diagram of the metabolic engineering strategy to promote the synthesis of sclareol in Saccharomyces cerevisiae.
  • Figure 2 shows the production of sclareol when optimizing sclareol synthase.
  • Fig. 3 shows the conditions of strengthening the MVA pathway of Saccharomyces cerevisiae, strengthening the central metabolism, and regulating transcription factors to increase the production of sclareol.
  • Figure 4 shows the production of sclareol by target modification screened out by transcriptome sequencing, wherein A is the production of sclareol by overexpressing the target gene; B is the production of sclareol by knocking out the target gene.
  • Fig. 5 shows the production situation of sclareol in shake flask batch fed-batch fermentation, wherein A is the feeding time; B is the growth situation; C is the production situation of sclareol.
  • Fig. 6 shows the production situation of sclareol in fermenter batch fed-batch fermentation; A is the production situation of sclareol; B is the growth situation.
  • Fig. 7 shows the gas chromatogram of sclareol synthesized by Saccharomyces cerevisiae.
  • the transformation was carried out based on the CEN.PK113-11C strain (MATa MAL2-8c SUC2 his3 ⁇ 1 ura3-52) of Euroscarf (Oberursel, Germany).
  • the genetic modification of this application is completed with the help of the CRISPR/Cas9 system (refer to the literature Mans et al., FEMS Yeast Res. 2015 Mar; 15(2):fov004.)
  • the present application provides a method for constructing a sclareol-producing Saccharomyces cerevisiae strain, which can significantly increase the production of sclareol.
  • the construction method includes: optimization of sclareol synthase, strengthening of mevalonate pathway (MVA), optimization of central metabolic pathway, global regulation and diploid optimization.
  • MVA mevalonate pathway
  • plasmids pYX312-L+T, pYX312-LT and pYX312-TL were respectively constructed.
  • the pYX312 plasmid was constructed by our laboratory, and the P TPI promoter on the pYX212 plasmid was replaced with the enhanced P eTDH3 promoter, and the P eTDH3 promoter reference (Blazeck et al. Biotechnol Bioeng, 2012, 109 (11): 2884-2895 .) Amplified fusion gains.
  • LPPS gene and TPS gene were obtained by whole gene synthesis after codon optimization.
  • P eTDH3 was amplified using pYX312 as a template.
  • the terminators T PYK1 and T PRM9 were amplified using the CEN.PK113-11C genome (wild-type Saccharomyces cerevisiae) as a template.
  • Use the OE-PCR method to fuse the LPPS gene and T PYK1 (for the fusion fragment construction process, refer to Zhou et al. J Am Chem Soc 2012,134:3234-3241) into the growth fragment 1, and use OE-PCR to P eTDH3 , TPS and T PRM9 Methods fusion growth fragment 2; in order to construct pYX312-LT, LPPS gene and TPS gene were fused into LT gene by OE-PCR method.
  • TPS gene and LPPS gene were fused into TL gene by OE-PCR method.
  • the plasmids verified by colony PCR and sequenced correctly were named pYX312-L+T, pYX312-LT and pYX312-TL, respectively.
  • the plasmids PYX312-L+T, PYX312-LT and PYX312-TL were transformed into engineering bacteria XC07, genotype (MATa; MAL2-8c; SUC2; his3 ⁇ 1; ura3-52; XI-5:: PTEF1 -Cas9 -T CYC1 ; XI-3::P GAL7 -MBP-TL-T ADH1 ; gal80 ⁇ ; X4:: P TDH3 - SpHMGR - T PRM9 ; HXT1 ), sclareol was extracted by fermentation and tested and concluded that the fusion of TPS-LPPS is most beneficial to the synthesis of sclareol ( Figure 2).
  • the maltose binding protein encoding gene MBP is fused.
  • the specific implementation method is as follows: the MBP gene is fused with the N-terminus and the C-terminus of the TL gene respectively to obtain fusion genes MBP-TL and TL-MBP.
  • the donor DNA was obtained by fusing TL, fusion genes MBP-TL and TL-MBP with the upstream and downstream homology arms of promoter PGAL7 , terminator T ADH1 and XI-3 respectively.
  • the fusion genes MBP-TL, TL-MBP and TL were integrated into the engineering bacteria SCX22 (genotype: MATa; MAL2-8c; SUC2; ura3 ⁇ ; hfd1 ⁇ ; gal80 ⁇ ; gal1 ⁇ ; gal7 ⁇ ; his3 ⁇ : :(P TPI -MmACL-T FBA1 )+(P TDH3 -RtME-T CYC1 )+(P tHXT7 -'MDH3-T TDH2 )+(P PGK1 -CTP1-T HIS3 );
  • X1-5 ::P TEF1 - Cas9-T CYC1 ;
  • pyc1 :: PTEF1 -PYC1;
  • X1-4 (P TPI1 -MPC1)+(P PGK1 -MPC3-T DIT1 );
  • X1-2 (P GAL1-
  • the specific steps for the construction of the CRISPR/Cas9 system are as follows: the complete homologous recombination fragment including the upstream and downstream sequences of the integration site, the promoter P TEF1 , the terminator T CYC1 Cas9 gene and the screening resistance KanMX gene were obtained by the fusion PCR method. 500ng of the source recombinant fragment was transformed into the starting strain CENPK113-11C of Saccharomyces cerevisiae to obtain a Saccharomyces cerevisiae strain with Cas9 protein.
  • an sgRNA expression vector targeting chromosomal integration site XI-3 constructs an sgRNA expression vector targeting chromosomal integration site XI-3; then, transform the sgRNA expression vector and donor DNA (500ng each) into SCX22 by chemical transformation, and apply to a screening plate at 30°C After static culture for 3 days, the transformants were cultured in liquid SD medium, verified by colony PCR, and spread on a plate containing 5-fluoroorotic acid for plasmid loss.
  • the strains after plasmid loss were named engineering bacteria SCX23, SCX24 and SCX25.
  • sclareol was extracted by fermentation and tested to confirm that the MBP-TL fusion mode (SCX23) is most conducive to the synthesis of sclareol ( FIG. 2 ).
  • MBP-TL fusion gene was integrated into the starting strain CENPK113-11C and the GAL80 gene was knocked out to obtain the engineering strain CXM01*.
  • the mevalonate-related rate-limiting genes were constructed with different promoters and terminators, and integrated into different sites in the genome (site selection reference Mikkelsen et al., Metab.Eng.2012, 14:104-111), to obtain the MVA pathway optimized engineering strain CXM17. Its main genes and loci (not limited to) are shown in Table 1.
  • sgRNA expression vectors and gene expression cassettes targeting chromosomal integration sites Gene reading frame, promoter and terminator and obtain complete donor DNA fragment by fusion PCR method (fusion fragment construction process refers to J Am Chem Soc 2012,134:3234-3241); Subsequently, sgRNA expression vector and gene expression cassette ( Each 500ng) was transformed into Saccharomyces cerevisiae by chemical transformation method, spread on the screening plate and cultured at 30°C for 3 days, after the transformant was cultured in liquid SD medium, the correctness was verified by colony PCR, and spread on a plate containing 5- Fluororotic acid plates were used for plasmid loss, and the strains after plasmid loss were preserved for future use.
  • the central metabolic pathway was strengthened, and with the help of the CRISPR/Cas9 system, the expression cassettes related to the central metabolic rate-limiting genes were constructed using different promoters and terminators (Table 2), and integrated into different sites in the genome to obtain the MVA pathway and central pathway.
  • the specific implementation method takes the ROX1 gene as an example: first, construct an sgRNA expression vector targeting the ORF box of the ROX1 gene; then, respectively amplify the upstream and downstream sequences of 300 bp of the ROX1 gene and obtain the complete donor DNA fragment by fusion PCR method; Subsequently, the gRNA expression vector and the gene expression cassette (500ng each) were transformed into Saccharomyces cerevisiae by a chemical transformation method, spread onto a screening plate and cultured at 30°C for 3 days, and the transformants were cultured in liquid SD medium, and passed Colony PCR verification is correct, spread on a plate containing 5-fluoroorotic acid for plasmid loss, and the strain after plasmid loss is preserved for future use. The other five transcription factor target knockouts followed the above process to achieve seamless knockout.
  • a diploid strain capable of efficiently biosynthesizing sclareol is constructed.
  • the construction method includes: the type of engineering bacteria is converted from MATa to MAT ⁇ , and MATa and MAT ⁇ realize diploid mating.
  • the engineering bacteria SCX42 is converted from MATa to MAT ⁇ , and with the help of the CRISPR/Cas9 system, firstly, an sgRNA expression vector targeting the MATa region is constructed; then, the genome matching region is amplified using the MAT ⁇ genome as a template; Subsequently, the gRNA expression vector and donor DNA (500ng each) were transformed into Saccharomyces cerevisiae by chemical transformation method, spread on the screening plate and cultured at 30°C for 3 days, and the transformants were cultured in liquid SD medium, and passed After PCR amplification of the colony, the transformant was sent to sequencing and showed that the type was ⁇ , and it was spread on a plate containing 5-fluoroorotic acid for plasmid loss, and the strain after plasmid loss was named SCX42- ⁇ .
  • the SCX42 is transformed from defective ura3 - , his3 - to ura3 - , met3 - , the specific implementation is: construct an sgRNA expression vector targeting the MET3 gene region, and then amplify 300 bp upstream and downstream of the MET3 gene and the ORF of the HIS3 gene box, and obtain donor DNA by fusion PCR.
  • the gRNA expression vector and the gene expression cassette (500ng each) were transformed into Saccharomyces cerevisiae by a chemical transformation method, spread onto a screening plate and cultured at 30°C for 3 days, and the transformants were cultured in liquid SD medium, and passed The colony PCR was verified to be correct, and the plasmid was spread on a plate containing 5-fluoroorotic acid for plasmid loss, and the strain after the plasmid loss was named SCX42*.
  • the engineering bacteria SCX42- ⁇ and SCX42* were separately cultured in YPD medium, and then 0.5 mL of bacterial liquid was mixed and inoculated in 20 mL of YPD medium for 48 hours. The fermentation broth was taken and centrifuged, washed twice with water, diluted appropriately and spread on SD-URA3 solid medium. The single colony grown was diploid.
  • transcriptome sequencing analysis is carried out on engineering bacteria to obtain gene targets that can further increase the production of sclareol.
  • the method includes: carrying out transcriptome sequencing on engineering bacteria CXM01*, CXM17, CXM18 (obtained by knocking out transcription factors ROX1, DOS2, VBA5, YER134c, YNR063w, YGR259c), SCX38 and SCX42, and analyzing the obtained
  • the modification target that is beneficial to the synthesis of sclareol, and the target that can further improve the synthesis of sclareol can be found through overexpression or knockout.
  • the bacteria with glucose residue 10 g/L in the fermentation broth and the bacteria in the ethanol utilization period were respectively subjected to transcriptome sequencing, and by comparing the above five Overexpression and knockout transformation targets were obtained by differential gene expression of the strains.
  • overexpression or knockout analysis was performed on the above-mentioned targets.
  • the specific embodiment is: with the help of CRISPR/Cas9 system, the gene overexpression target sites are respectively fused with the promoter PGAL7 and the terminator TENO2 , and integrated into the XI-6 site of the engineering bacteria.
  • seamless gene knockout is implemented.
  • the sclareol production of engineering bacteria was extracted and tested through fermentation.
  • the overexpression of target genes DAN1, HUG1, LAC1, OYE3 and OYE2 and the knockout of target genes SUL1, CDA2, ERV15, ZRT1, DSE1 and ANB1 can all promote the synthesis of sclareol.
  • the highest yield is 1.1g/L.
  • the chromosomal integration site sequence XI-3, XI-1, XII-2, XII-3, XI-2 reference (Mikkelsen et al., Metab.Eng.2012, 14:104-111 )get.
  • Table 3 for the targeting sequence of the chromosomal integration site.
  • Table 4 for the medium formula.
  • Plasmids pYX312-L+T, pYX312-LT and pYX312-TL were constructed respectively.
  • the pYX312 plasmid was constructed by our laboratory and obtained by replacing the P TPI promoter on the pYX212 plasmid (purchased from Addgene) with the enhanced P eTDH3 promoter, wherein the P eTDH3 promoter reference (Blazeck et al. Biotechnol Bioeng, 2012, 109 (11 ):2884-2895.) Amplified fusion was obtained.
  • the LPPS gene and the TPS gene were codon-optimized and obtained by whole gene synthesis.
  • the optimized nucleotide sequence of the LPPS gene is shown in SEQ ID NO: 1, and the optimized nucleotide sequence of the TPS gene As shown in SEQ ID NO:2.
  • P eTDH3 was amplified using PYX312 as a template.
  • the terminators TPYK1 and TPRM9 were amplified using the CEN.PK113-11C genome as a template.
  • the LPPS gene and T PYK1 were fused to grow fragment 1 by OE-PCR method (for the fusion fragment construction process, refer to J Am Chem Soc 2012,134:3234-3241), and the P eTDH3 , TPS and T PRM9 were fused to grow fragment by OE-PCR method two.
  • pYX312-LT the LPPS gene and the TPS gene were fused into the LT gene by OE-PCR method (the connecting peptide is GGGS, and the nucleotide sequence encoding the gene is shown in SEQ ID NO: 6); in order to construct pYX312-TL, The TPS gene and LPPS gene were fused into TL gene by OE-PCR method (connecting peptide is GGGS). Connect pYX312 (EcoRI/HindIII) with long fragments 1 and 2, LT fusion gene and TL fusion gene respectively by seamless cloning method, and transform Escherichia coli competent. The plasmids verified by colony PCR and sequenced correctly were named pYX312-L+T, pYX312-LT and pYX312-TL, respectively.
  • the plasmids pYX312-L+T, pYX312-LT and pYX312-TL were transformed into engineering bacteria XC07, which was transformed on the basis of CEN.PK-113-11C, and the genotype was (MATa; MAL2-8c; SUC2; his3 ⁇ 1; ura3-52; XI-5:: PTEF1 -Cas9-T CYC1 ; XI-3::P GAL7 -MBP-TL-T ADH1 ; gal80 ⁇ ; X4:: PTDH3- SpHMGR-T PRM9; X2:: P TPI1 -BTS1-ERG20-T FBA1 ; P ERG9 :: P HXT1 ), sclareol was extracted by fermentation and detected and confirmed that the fusion mode of TPS ⁇ LPPS is most conducive to the synthesis of sclareol (Figure 2), fermentation conditions: activation The resulting strains were cultured in culture
  • the maltose binding protein coding gene MBP is fused.
  • the specific implementation method is as follows: the MBP gene is fused with the 5' end and the 3' end of the TL gene respectively (the connecting peptide is Gly6, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 7), and the fusion gene MBP- TL and TL-MBP.
  • the donor DNA was obtained by fusing TL, fusion genes MBP-TL and TL-MBP with the upstream and downstream homology arms of PGAL7 and terminator T ADH1 and XI-3 sites, respectively. Referring to the CRISPR/Cas9 system (Mans R, et al.
  • the fusion gene was integrated into the genome of the engineering strain SCX22, which is in the CEN.PK-113 Transformation on the basis of -11C, genotype : MATa ; MAL2-8c; SUC2; ura3 ⁇ ; hfd1 ⁇ ; gal80 ⁇ ; +(P tHXT7 -'MDH3-T TDH2 )+(P PGK1 -CTP1-T HIS3 ); X1-5::P TEF1 -Cas9-T CYC1 ; pyc1::P TEF1 -PYC1 ; X1-4::(P TPI1 -MPC1)+(P PGK1 -MPC3-T DIT1 ); X1-2::(P GAL1- AnACLa-T CYC1 )+(P GAL10- AnACLb-T ADH1 ); gal10 ⁇ ::(
  • Example 2 The effect of MVA pathway supply on the synthesis of sclareol by Saccharomyces cerevisiae
  • CEN.PK113-11C as the starting bacterium, integrate the MBP-TL fusion gene at the XI-3 site (the integration method is the same as in Example 1), knock out the GAL80 gene, and obtain the engineering bacterium CXM01*.
  • Overexpression truncated tHMG1 gene in engineering bacterium CXM01* at FAA4 site (donor DNA is FAA4up-FAA4-T AHD1 -kanMX-P THD3 -tHMG1-FAA4dw); Overexpression SpHMGR gene (nucleotide sequence such as SEQ ID NO: 5) at the POX1 site (the donor DNA is POX1up-POX1-T TDH2 -kanMX-P tHXT7 -SpHMGR-POX1dw).
  • To mutate the ERG20 gene on the genome to ERG20 F96C is to mutate the base TTC to TGT.
  • HMG2 K6R and ERG10 genes into site XII-2 (donor DNA is XII-2up-T ENO2 -HMG2 K6R -P GAL10-1 -ERG10-T PYK1 -XII-2dw); integrate at site XII-3 Two copies of HMG2 K6R (donor DNA is XII-3up-T PDC1 -HMG2 K6R -P GAL10-1 -HMG2 K6R -T ENO2 -XII-3dw); ERG9 gene promoter was replaced by PHXT1 ; BTS1 and The PaGGPPS (nucleotide sequence shown in SEQ ID NO: 4) gene is connected and integrated into the XI-2 site through protein fusion expression (donor DNA is XI-2up-P GAL7 -BTS1-PaGGPPS-T TDH2 -XI -2dw), to obtain engineering bacteria CXM17.
  • MVA pathway-related gene expression cassette construction and genome integration take the integration of tHMG1 to FAA4 locus as an example.
  • FAA4up-FAA4, TAHD1 , kanMX, P THD3 , tHMG1, and T FAA4 were amplified respectively, and the complete sequence was obtained by fusion PCR.
  • Donor DNA fragment FAA4up-FAA4-T AHD1 -kanMX-P THD3 -tHMG1-T FAA4 Subsequently, 500ng of the donor DNA fragment was transformed into Saccharomyces cerevisiae by chemical transformation method, spread onto the screening plate and cultured statically at 30°C On day 3, after the transformants were cultured in liquid YPD+G418 medium, the correctness was verified by colony PCR, and the pgRNA-kanMX and donor T AHD1 -P THD3 were transformed to lose the KanMX selection resistance gene. The verified correct transformants were spread on plates containing 5-fluoroorotic acid for plasmid loss, and the strains after plasmid loss were preserved for future use.
  • HMG2 K6R gene was amplified using primers (F: ATGTCACTTCCCTTAAGAACGATAGTACATTTG; R: TAATAATGCTGAGGTTTTACAGGGGGG) using CEN.PK113-11C as a template.
  • the engineering bacteria fermentation process used 15mL Delft-D fermentation medium/100mL shake flask, and the initial pH was 5.6.
  • Initial OD 600 0.1, fermented for 96 hours at 30°C and 220 rpm, and the biomass and sclareol production were measured.
  • CXM17 was obtained by strengthening the MVA pathway, and the yield of sclareol per OD 600 reached 13.4 mg/L/OD 600 .
  • the ATP-dependent citrate lyase MmACL gene, malate synthase RtME gene, malate dehydrogenase 'MDH3 gene with signal peptide removed and citrate transporter gene CTP1 were integrated into the HIS3 locus (the donor DNA was HIS3up -HIS3-P TPI1 -MmACL-T FBA1 -P TDH3 -RtME-T CYC1 -P tHXT7 -'MDH3-T TDH2 -P PGK1 -CTP1-T ADH1 -P TEF1 -'tesA-T HIS3 );
  • the enzyme gene promoter P PYC1 was replaced by the promoter P TEF1 ; overexpression of the pyruvate transporter coding genes MPC1 and MPC3 at the XI-4 site (the donor DNA was XI-4up-P tHXT7 -MPC3-T DIT1 -T MPC
  • the specific transformation methods of the engineering bacteria in this example can be obtained by referring to the literature (Yu et al., Cell, 2018:174:1549-1558e1514.).
  • Sclareol was extracted with n-hexane, and sclareolide was used as internal standard, and detected by GC-MS. As shown in Figure 3, the production of sclareol was significantly increased to 26.2 mg/L/OD 600 .
  • Example 4 The effect of target regulation on the synthesis of sclareol by Saccharomyces cerevisiae
  • the engineering strain SCX38 knocked out the genes ROX1, DOS2, VBA5, YER134c, YNR063w, and YGR259c to obtain the engineering strain SCX42.
  • the specific implementation method takes the ROX1 gene as an example: first, construct an sgRNA expression vector targeting the ORF box of the ROX1 gene; then, respectively amplify the 300 bp sequences upstream and downstream of the ROX1 gene and obtain a complete donor DNA fragment by fusion PCR;
  • the gRNA expression vector and gene expression cassette (500ng each) were transformed into Saccharomyces cerevisiae by chemical transformation method, spread on the screening plate and cultured at 30°C for 3 days, and the transformant was cultured in liquid SD medium, and verified by colony PCR Correct, spread on a plate containing 5-fluoroorotic acid for plasmid loss, and the strain after plasmid loss is preserved for future use.
  • the other five transcription factor target knockouts followed the above process to achieve seamless knockout
  • the engineered bacteria SCX42 was converted from MATa to MAT ⁇ .
  • the sgRNA expression vector targeting the MATa region was constructed; then, the MAT ⁇ genome was used as a template to amplify the genome matching region; then, the gRNA-HMRa was expressed
  • the vector and donor DNA 500ng each
  • the strain after plasmid loss was named SCX42- ⁇ .
  • the transformation method of mating type conversion can be obtained by referring to the patent (Tianjin University. A method for converting yeast mating type: CN201610102424.8[P].2016-04-13.).
  • the specific implementation is: construct an sgRNA expression vector targeting the MET3 gene region, then amplify 300 bp upstream and downstream of the MET3 gene and the ORF frame of the HIS3 gene, and Donor DNA was obtained by fusion PCR.
  • the gRNA expression vector and the gene expression cassette (500ng each) were transformed into Saccharomyces cerevisiae by a chemical transformation method, spread onto a screening plate and cultured at 30°C for 3 days, and the transformants were cultured in liquid SD medium, and passed The colony PCR was verified to be correct, and the plasmid was spread on a plate containing 5-fluoroorotic acid for plasmid loss, and the strain after the plasmid loss was named SCX42*.
  • the engineering bacteria SCX42- ⁇ and SCX42* were separately cultured in YPD medium, and then 0.5 mL of bacterial liquid was mixed and inoculated in 20 mL of YPD medium for 48 hours. The fermentation broth was taken and centrifuged, washed twice with water, diluted appropriately and spread on SD-URA3 solid medium. The single colony grown was diploid.
  • the engineering bacteria CXM01*, CXM17, CXM18 was obtained by knocking out the transcription factors ROX1, DOS2, VBA5, YER134c, YNR063w, YGR259c from the engineering bacteria CXM17), SCX38 and SCX42 in the basic component medium ((Delft-D added glucose concentration 20g/L) fermentation culture, and respectively at the time point when the residual glucose concentration was 10g/L and when the glucose was exhausted and the ethanol was used, the bacterial cells were taken for transcriptome sequencing, and the transformation targets that might be beneficial to the synthesis of sclareol were analyzed and obtained.
  • the basic component medium ((Delft-D added glucose concentration 20g/L) fermentation culture
  • Overexpression targets ANB1, DAN1, HUG1, PYK1, LAC1, CYB5, YCL021W-A, YCL074W, OYE3, OYE2, HSP31, BLI1; Knockout targets: SPS4, YOR314W, SUL1, CDA2, ERV15, ZRT1, DSE1 , ANB1.
  • the modified targets can be found in the Saccharomyces Genome Database (https://www.yeastgenome.org/).
  • the gene overexpression target sites were fused with the promoter P GAL7 and the terminator T ENO2 , respectively, and integrated into the XI-6 site of the engineered bacteria.
  • ORF seamless knockout is implemented.
  • Shake flask batch test fed-batch fermentation adopts 50mL fermentation broth/250mL Erlenmeyer flask culture system, and selects engineering bacteria SCX42 and diploid.
  • 3xDelft-A medium add 500g/L glucose in Delft-A medium
  • 3xDelft-D medium add 500g/L glucose in Delft-D medium
  • the feeding time point that is, the time point corresponding to the sugar feeding time point in the figure
  • Figure 5A is shown in Figure 5A below, in which diploids are fed at the time points indicated by black dots and gray dots, while other engineering bacteria are only fed at the time points indicated by black dots. Feed at the indicated time points.
  • the growth of the engineered strain SCX42 was slightly better than that of Delft-A medium when supplemented with Delft-D, and the content of sclareol was significantly better than that of Delft-A (2.2g/L), reaching 4.5g /L; under the same condition of supplementing Delft-A, SCX42 grew faster than the diploid, but the sclareol content of the diploid was slightly higher than that of the engineering strain SCX42, which was 2.4g/L.

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Abstract

提供了一种工程菌的构建方法、构建得到的工程菌及其在制备香紫苏醇中的应用，该构建方法包括以下步骤：在酿酒酵母宿主菌株中导入从上游到下依次包括启动子、融合基因、终止子的DNA片段I，融合基因从上游到下依次包括：TPS基因、连接肽I的编码基因、LPPS基因。该构建方法优化了宿主菌株的香紫苏醇合成酶，有利于提高香紫苏醇的产率。

Description

一种工程菌的构建方法及其应用 技术领域
本发明属于微生物代谢工程及工业生物技术领域，涉及一种工程菌的构建方法及其应用。
背景技术
香紫苏醇(sclareol)又称硬尾醇，作为一种半日花烷型二萜叔醇，最初在鼠尾草属唇形香料植物香紫苏(Salvia sclarea)中提取并由此得名。香紫苏醇是龙涎香替代品——龙涎醚合成最普遍的原料。龙涎香是抹香鲸分泌的一种蜡质物质，自古以来因其麝香和甜土气味被用作高端香水的固定成分。然而，抹香鲸濒临灭绝的现状造就了龙涎醚不可再生的宝贵性，因此利用香紫苏醇合成龙涎醚已经成为香水工业生产天然龙涎香替代品的主要方法。香紫苏醇在医药和农药上同样具有重要的生物活性。如香紫苏醇具有很强的抗菌活性以及在真菌生长调节和植物生长抑制方面起作用，并且对人类的白血病细胞(Dimas et al.,Leuk Res,1999,23:217-234)、肿瘤细胞株(Mahaira et al.,Eur J Pharmacol,2011,666:173-182)、结肠癌细胞及异种移植物(Dimas et al.,Apoptosis,2007,12:685-694；Mahaira et al.,Eur J Pharmacol,2011,666:173-182)具有细胞毒性。最近，有研究以香紫苏醇和香紫苏醇内酯为原料，将生物催化与自由基反应相结合，成功合成八种氧化型混源萜天然产物及一种类似物，说明香紫苏醇对混源萜类天然产物合成也至关重要(Li et al.,Nat.Chem.,2020,12:173-179)。
目前，香紫苏醇的生产大多以植物香紫苏花序及茎叶提油后的香紫苏浸膏为原料，根据AFNOR指令(NFT75-255)和工业水平，从1000kg干植物中获得的精油中含有20-260g香紫苏醇，即香紫苏醇得率为0.002％-0.026％。植物香紫苏的生长周期长，且受限于土地、环境及气候因素的影响，不能完全满足工业化生产的需求。微生物是一种非常理想的“细胞工厂”，具有生长周期短、可高密度培养、不受外界环境干扰等优势。2009年，瑞士日内瓦芬美意有限公司的Schalk克隆出植物香紫苏中香紫苏醇合成的两个关键基因，一是，焦磷酸赖百当烯二醇酯(labdenediol diphosphate,LPP)合酶(SsLpps，Ⅱ型萜类合酶)，催化二萜类化合物前体物质GGPP生成LPP；二是，香紫苏醇合酶(SsTps，Ⅰ型萜类合酶)，催化LPP生成香紫苏醇(US patent 9267155)。在大肠杆菌Escherichia coli中表达这两个酶，并优化异戊二烯(MEP)途径，批式补料发酵香紫苏醇产量达到1.5g/L(Schalk et al.,J.Am.Chem.Soc.,2012,134(46):18900-18903)。2013年，杨薇等人在酿酒酵母中表达焦磷酸赖百当烯二醇酯(LPP)合酶基因SsLPPS和香紫苏醇合酶基因SsTPS，并过表达截断型羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1及融合表达GGPP合成酶基因和法尼烯合成酶基因BTS1-ERG20，使得香紫苏醇产量达到9mg/L(杨薇等.,生物工程学报,2013(08):152-159)。2015年，Ignea等人在酿酒酵母底盘细胞AM205中通过表达来自岩蔷薇的8-羟丙基二磷酸(8OH-CPP)合成酶基因CcCLS和来自香紫苏的香紫苏醇合成酶基因SsTPS并对ERG20定点突变为ERG20 F96C，使得香紫苏醇产量提高至403mg/L(Ignea et al.,Metabolic Engineering,2015:65-75)。同一年，Trikka等人在酿酒酵母中利用类胡萝卜素显色筛选到6个敲除靶点ROX1、DOS2、VBA5、YER134c、YNR063w和YGR259c，在底盘细胞AM238中对其进行敲除后，香紫苏醇产量在摇瓶中达到750mg/L(Trikka et al.,Microb.Cell Fact.,2015,14(1):60-79)。目前所构建工程菌株产量还比较低，难以满足市场需求。
本发明从全局代谢构建酵母细胞工厂，实现香紫苏醇高效合成。首先优化香紫苏醇合成酶基因，提高其生物合成效率；并将细胞初级代谢和香紫苏醇生物合成途径整合并分成三个模块：1)初级代谢模块提供前体乙酰辅酶A和辅因子NADPH；2)甲羟戊酸生物合成模块；3)转录因子调控模块。分别优化三个模块，并实现模块间协同合作，使得酿酒酵母中香紫苏醇产量在摇瓶中达到1.1g/L，批式补料发酵达到11.4g/L。
发明内容
本发明要解决的技术问题克服目前酵母工程菌的中萜类生物合成酶的活性低和代谢不匹配等难题，从萜类合成酶优化和全局代谢模块优化增加香紫苏醇等二萜的生物合成效率，提供一种产香紫苏醇的酵母基因工程菌及其应用。
根据本申请的一个方面，提供一种工程菌的构建方法，所述构建方法优化了宿主菌株的香紫苏醇合成酶，有利于提高香紫苏醇的产率。
一种工程菌的构建方法，所述构建方法包括以下步骤：
在宿主菌株中导入DNA片段I，得到所述工程菌；
所述DNA片段I从上游到下依次包括：启动子、融合基因、终止子；
所述融合基因从上游到下依次包括：TPS基因、连接肽I基因、LPPS基因；
所述宿主菌株选自酿酒酵母中的任一种。
可选地，所述融合基因从上游到下依次包括：
MBP基因、连接肽II基因、TPS基因、连接肽I基因、LPPS基因；
或
所述融合基因从上游到下依次包括：
TPS基因、连接肽I的编码基因、LPPS基因、连接肽II的编码基因、MBP基因。
可选地，所述LPPS基因的核苷酸序列如SEQ ID NO：1所示；
所述TPS基因的核苷酸序列如SEQ ID NO：2所示；
所述MBP基因的核苷酸序列如SEQ ID NO：3所示；
所述连接肽I的编码基因的核苷酸序列如SEQ ID NO：6所示；
所述连接肽II的编码基因的核苷酸序列如SEQ ID NO：7所示；
所述启动子选自P eTDH3、P GAL7中的任意一种；
所述终止子选自T PYX212、T ADH1中的任意一种。
可选地，所述在宿主菌株中导入DNA片段I为将DNA片段I整合到宿主的XI-3基因位点。
可选地，所述构建方法还包括优化MVA的合成途径，包括(a)～(h)中的至少一种：
(a)敲除宿主菌株的GAL80的编码基因；
(b)将tHMG1基因导入宿主菌株；
(c)将SpHMGR基因导入宿主菌株；
(d)将宿主菌株基因组上ERG20基因突变为ERG20 F96C基因；
(e)将HMG2 K6R基因和ERG10基因导入宿主菌株；
(f)将含有2个拷贝HMG2 K6R基因的DNA片段导入宿主菌株；
(g)将宿主菌株中ERG9基因的启动子替换为P HXT1；
(h)将含有BTS1和PaGGPPS基因的DNA片段导入宿主菌株；
所述(a)～(h)不分先后顺序。
可选地，包括(a)～(h)中的至少一种：
(a)敲除宿主菌株的GAL80基因；
(b)将含有FAA4-T AHD1-P THD3-tHMG1的DNA片段整合到宿主菌株的FAA4基因位点；
(c)将含有POX1-T TDH2-P tHXT7-SpHMGR的DNA片段整合到宿主菌株的POX1基因位点；
(d)将宿主菌株基因组上ERG20基因突变为ERG20 F96C基因；
(e)将含有T ENO2-HMG2 K6R-P GAL10-1-ERG10-T PYK1的DNA片段整合到宿主菌株的XII-2基因位点；
(f)将含有T PDC1-HMG2 K6R-P GAL10-1-HMG2 K6R-T ENO2的DNA片段整合到宿主菌株的XII-3基因位点；
(g)将宿主菌株中ERG9基因的启动子替换为P HXT1；
(h)将含有P GAL7-BTS1-PaGGPPS-T TDH2的DNA片段整合到宿主菌株的XI-2基因位点；
所述(a)～(h)不分先后顺序。
可选地，所述构建方法还包括优化中心代谢途径，包括(A)～(N)中的至少一种：
(A)将MmACL基因导入宿主菌株；
(B)将RtME基因导入宿主菌株；
(C)将‘MDH3基因导入宿主菌株；
(D)将CTP1基因导入宿主菌株；
(E)将宿主菌株的PYC1基因启动子P PYC1替换为启动子P TEF1；
(F)将MPC1基因导入宿主菌株；
(G)将MPC3基因导入宿主菌株；
(H)将AnACLa基因、AnACLb基因导入宿主菌株；
(I)将RtCIT1基因导入宿主菌株；
(J)将IDP2基因导入宿主菌株；
(K)将YHM2基因导入宿主菌株；
(L)将宿主菌株的PGI1基因的启动子替换为启动子P COX9；
(M)将GND1基因、TKL1基因、TAL1基因、ZWF1基因导入宿主菌株；
(N)将宿主菌株的IDH2基因的启动子替换为启动子P GSY1；
所述(A)～(N)不分先后顺序。
可选地，所述构建方法还包括优化中心代谢途径，包括(A)～(G)中的至少一种：
(A)将含有HIS3-P TPI1-MmACL-T FBA1-P TDH3-RtME-T CYC1-P tHXT7-’MDH3-T TDH2-P PGK1-CTP1-T ADH1的DNA片段整合到宿主菌株的HIS3基因位点；
(B)将宿主菌株的PYC1基因的启动子P PYC1替换为启动子P TEF1；
(C)将含有P tHXT7-MPC3-T DIT1-T MPC1-MPC1-P TPI1的DNA片段整合到宿主菌株的XI-4位点；
(D)将含有T CYC1-AnACLa-P GAL1-P GAL10-AnACLb-T ADH1的DNA片段整合到宿主菌株的X2位点；
(E)将含有P TPI1-RtCIT1-T FBA1-T CYC1-IDP2-P THD3-P TEF1-YHM2的DNA片段整合到宿主菌株的GAL1、GAL7、GAL10位点；
(F)将含有P COX9-T CYC1-GND1-P THD3-P tHXT7-TKL1-T TDH2-T ADH1-TAL1-P PGK1-P TEF1-ZWF1-T ZWF1的DNA片段整合到宿主菌株的PGI1位点；
(G)将宿主菌株的IDH2基因的启动子替换为启动子P GSY1；
所述(A)～(G)不分先后顺序。
可选地，所述构建方法还包括靶点调控，包括：
敲除宿主菌株的ROX1基因、DOS2基因、VBA5基因、YER134c基因、YNR063w基因、YGR259c基因中的至少一种。
可选地，所述构建方法还包括以下的至少一种：
(I)在宿主菌株中导入ANB1基因、DAN1基因、HUG1基因、PYK1基因、LAC1基因、CYB5基因、YCL021W-A基因、YCL074W基因、OYE3基因、OYE2基因、HSP31基因、BLI1基因中的至少一种；
和/或
(II)敲除宿主菌株的SPS4基因、YOR314W基因、SUL1基因、CDA2基因、ERV15基因、ZRT1基因、DSE1基因、ANB1基因中的至少一种。
可选地，所述构建方法通过采用CRISPR/Cas9技术进行构建。
可选地，所述构建方法还包括细胞融合形成双倍体。
根据本申请的另一个方面，提供根据上述任一项所述的构建方法构建得到的工程菌。
根据本申请的另一个方面，提供根据上述任一项所述的构建方法构建得到的工程菌、上述所述的工程菌中的至少一种在制备香紫苏醇中的应用。
作为一种实施方案，本申请提供一种产香紫苏醇的酿酒酵母基因工程菌，优化了香紫苏醇合成酶，将催化香紫苏醇合成的两个酶进行串联表达，并且在N端融合了麦芽糖结合蛋白MBP。
可选地，在酵母中将中心代谢模块、异戊二烯生物合成途径模块和调控模块进行优化后构建而成；
可选地，强化表达了异戊二烯生物合成途径限速酶基因，包括截断型羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1，突变型 HMG2 K6R，来源于Silicibacter pomeroyi NADH依赖型SpHMGR，乙酰乙酰辅酶A硫解酶基因ERG10，内源GGPP合成酶与来自Phomopsis amygdali的GGPP合成酶融合蛋白基因BTS1-PaGGPPS并突变了基因组中FPP合成酶基因为ERG20 F96C及采用P HXT1替换ERG9启动子动态调控ERG9表达；
可选地，强化了中心代谢途径增加了前体乙酰辅酶A和辅因子NADPH的供给，包括表达来自Mus musculus的ATP依赖型柠檬酸裂解酶基因MmACL，来自Rhodospuridium toruloides的NADP +依赖型苹果酸合成酶基因RtME和柠檬酸合成酶1基因RtCIT1，去除过氧化物酶体信号肽的苹果酸脱氢酶基因’MDH3，柠檬酸转运蛋白基因CTP1，丙酮酸羧化酶1基因PYC1，丙酮酸转运蛋白1和3基因MPC1和MPC3，来自Aspergillus nidulans的ATP依赖型柠檬酸裂解酶a和b基因AnACLa和AnACLb，NADP特异性异柠檬酸脱氢酶2基因IDP2，柠檬酸、α-酮戊二酸转运蛋白基因YHM2，磷酸葡萄糖脱氢酶异构体1基因GND1，转酮酶基因TKL1，转醛缩酶基因TAL1，6-磷酸葡萄糖脱氢酶基因ZWF1，线粒体NAD +依赖异柠檬酸脱氢酶基因IDH2并采用P GSY1替换PGI1启动子弱化PGI1表达；
可选地，敲除了调控因子基因，包括ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c；
可选地，构建了二倍体菌株，将构建的α菌株配型转换为a型菌株，然后两者融合获得二倍体菌株。
作为一种实施方案，本申请提供一种酿酒酵母基因工程菌，所述酿酒酵母基因工程菌的构建方法包括：将SEQ ID NO：2所示的TPS基因与SEQ ID NO：1所示的LPPS基因融合，连接肽为GGGS，获得融合基因1；融合基因1的N端与SEQ ID NO：3所示的麦芽糖结合蛋白编码基因融合，获得融合基因2；融合基因2连同质粒pgRNA-XI-3转入出发菌株中，获得产香紫苏醇的酿酒酵母基因工程菌。
可选地，所述质粒pgRNA-XI-3的靶向基因组位点是XI-3。
作为一种实施方案，本申请提供一种融合基因，所述融合基因的序列中含有SEQ ID NO：1所示的LPPS基因序列、SEQ ID NO：2所示的TPS基因序列；
可选地，所述融合基因的序列含有以下片段中的任一种：
融合基因1：SEQ ID NO：2所示的TPS基因通过连接肽GGGS与SEQ ID NO：1所示的LPPS基因融合，按照TPS基因-连接肽GGGGS-LPPS基因排列，获得融合基因1；
融合基因2：SEQ ID NO：1所示的LPPS基因通过连接肽GGGS与SEQ ID NO：2所示的TPS基因融合，获得融合基因2；
融合基因3：将SEQ ID NO：1所示的LPPS基因和终止子T PYK1融合成长片段一，将启动子P eTDH3、SEQ ID NO：2所示的TPS基因和终止子T PRM9融合成长片段二，将长片段一和长片段二融合，获得融合基因3；
可选地，所述连接肽GGGS的核苷酸序列如SEQ ID NO：6所示；
可选地，所述融合基因还含有SEQ ID NO：3所示的麦芽糖结合蛋白编码基因序列；
可选地，所述融合基因的序列含有以下片段：
SEQ ID NO：2所示的TPS基因与SEQ ID NO：1所示的LPPS基因融合，连接肽为GGGS，获得融合基因1；融合基因1的N端与SEQ ID NO：3所示的麦芽糖结合蛋白编码基因MBP融合，连接肽为Gly6；
可选地，所述Gly6的核苷酸序列如SEQ ID NO：7所示。
可选地，所述酿酒酵母基因工程菌通过用含有融合基因的片段替换出发菌株中的XI-3基因得到。
作为一种实施方案，本申请提供一种酿酒酵母基因工程菌，所述酿酒酵母基因工程菌的构建方法包括：将SEQ ID NO：2所示的TPS基因与SEQ ID NO：1所示的LPPS基因融合，连接肽为GGGS，获得融合基因1；融合基因1的N端与SEQ ID NO：3所示的麦芽糖结合蛋白编码基因融合，获得融合基因2；融合基因2连同质粒pgRNA-XI-3转入出发菌株，无缝敲除GAL80基因，获得产香紫苏醇的酿酒酵母基因工程菌。
作为一种实施方案，本申请提供一种酿酒酵母基因工程菌，通过在出发菌株强化表达了异戊二烯生物合成途径限速酶基因得到，包括截断型羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1，突变型HMG2 K6R，来源于Silicibacter pomeroyi NADH依赖型SpHMGR，乙酰乙酰辅酶A硫解酶基因ERG10，HMG2 K6R，内源GGPP合成酶与来自Phomopsis amygdali的GGPP合成酶融合蛋白基因BTS1-PaGGPPS并突变了基因组中FPP合成酶基因为ERG20 F96C及采用P HXT1替换ERG9启动子动态调控ERG9表达中的至少一种。
可选地，包括截断型羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1，突变型HMG2 K6R，来源于Silicibacter pomeroyi NADH依赖型SpHMGR，乙酰乙酰辅酶A硫解酶基因ERG10，HMG2 K6R，内源GGPP合成酶与来自Phomopsis amygdali的GGPP合成酶融合蛋白基因BTS1-PaGGPPS(连接肽为GGGS)并突变了基因组中FPP合成酶基因为ERG20 F96C及采用P HXT1替换ERG9启动子动态调控ERG9表达。
可选地，所述出发菌株为酿酒酵母基因工程菌CXM01*。
可选地，所述SpHMGR的核苷酸序列如SEQ ID NO：5所示。
可选地，所述PaGGPPS的核苷酸序列如SEQ ID NO：4所示。
作为一种实施方案，本申请提供一种酿酒酵母基因工程菌，通过在出发菌株中强化了中心代谢途径得到，包括增加了前体乙酰辅酶A和辅因子NADPH的供给，包括表达来自Mus musculus的ATP依赖型柠檬酸裂解酶基因MmACL，来自Rhodospuridium toruloides的NADP +依赖型苹果酸合成酶基因RtME和柠檬酸合成酶1基因RtCIT1，去除过氧化物酶体信号肽的苹果酸脱氢酶基因’MDH3，柠檬酸转运蛋白基因CTP1，丙酮酸羧化酶1基因PYC1，丙酮酸转运蛋白1和3基因MPC1和MPC3，来自Aspergillus nidulans的ATP依赖型柠檬酸裂解酶a和b基因AnACLa和AnACLb，NADP特异性异柠檬酸脱氢酶2基因IDP2，柠檬酸、α-酮戊二酸转运蛋白基因YHM2，磷酸葡萄糖脱氢酶异构体1基因GND1，转酮酶基因TKL1，转醛缩酶基因TAL1，6-磷酸葡萄糖脱氢酶基因ZWF1，线粒体NAD+依赖异柠檬酸脱氢酶基因IDH2并采用P GSY1替换PGI1启动子弱化PGI1表达中的至少一种。
可选地，包括增加了前体乙酰辅酶A和辅因子NADPH的供给，包括表达来自Mus musculus的ATP依赖型柠檬酸裂解酶基因MmACL，来自Rhodospuridium toruloides的NADP +依赖型苹果酸合成酶基因RtME和柠檬酸合成酶1基因RtCIT1，去除过氧化物酶体信号肽的苹果酸脱氢酶基因’MDH3，柠檬酸转运蛋白基因CTP1，丙酮酸羧化酶1基因PYC1，丙酮酸转运蛋白1和3基因MPC1和MPC3，来自Aspergillus nidulans的ATP依赖型柠檬酸裂解酶a和b基因AnACLa和AnACLb，NADP特异性异柠檬酸脱氢酶2基因IDP2，柠檬酸、α-酮戊二酸转运蛋白基因YHM2，磷酸葡萄糖脱氢酶异构体1基因GND1，转酮酶基因TKL1，转醛缩酶基因TAL1，6-磷酸葡萄糖脱氢酶基因ZWF1，线粒体NAD+依赖异柠檬酸脱氢酶基因IDH2并采用P GSY1替换PGI1启动子弱化PGI1表达。
作为一种实施方案，本申请提供一种酿酒酵母基因工程菌，在出发菌株中敲除了调控因子基因，所述调控因子基因包括ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c中的至少一种；
可选地，所述调控因子基因包括ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c；
可选地，所述出发菌株为酿酒酵母基因工程菌SCX38。
作为一种实施方案，本申请提供一种酿酒酵母二倍体菌株，将产香紫苏醇的酿酒酵母基因工程菌SCX42由MATa转换为MATα得到SCX42-α，将SCX42由缺陷型ura3 -、his3 -转变为ura3 -、met3得到SCX42*，然后将SCX42*与SCX42-α共培养获得二倍体菌株。
作为一种实施方案，本申请提供一种酿酒酵母基因工程菌，所述酿酒酵母基因工程菌通过在出发菌株过表达：ANB1，DAN1，HUG1，PYK1，LAC1，CYB5，YCL021W-A，YCL074W，OYE3，OYE2，HSP31，BLI1中的至少一种；
或敲除：SPS4，YOR314W，SUL1，CDA2，ERV15，ZRT1，DSE1，ANB1中的至少一种得到；
可选地，所述出发菌株选自CXM01*、CXM17、CXM18、SCX38和SCX42中的任一种；
可选地，所述CXM18由CXM17敲除转录因子ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c获得；
可选地，所述酿酒酵母基因工程菌通过在出发菌株过表达：ANB1，DAN1，HUG1，PYK1，LAC1，CYB5，YCL021W-A，YCL074W，OYE3，OYE2，HSP31，BLI1，和敲除：SPS4，YOR314W，SUL1，CDA2，ERV15，ZRT1，DSE1，ANB1得到。
作为一种实施方案，提供上述任一项所述的酿酒酵母基因工程菌或上述任一项所述的酿酒酵母二倍体菌株在合成香紫苏醇中的应用；
可选地，所述香紫苏醇的制备方法，将上述任一项所述的酿酒酵母工程菌采用基础成分培养基，添加20-25g/L葡萄糖，初始pH为5-6，室温震荡培养；转接于含有发酵培养基的容器中，室温震荡发酵培养96-120h，发酵液中即包含了香紫苏醇。
本申请可产生的有益效果包括：
(1)本申请所提供的工程菌的构建方法，优化了宿主菌株的香紫苏醇合成酶，有利于提高香紫苏醇的产率。
(2)本申请所提供的工程菌的构建方法，通过强化酿酒酵母MVA途径、强化中心代谢、转录因子调控提高香紫苏醇产量。
附图说明
图1示出了代谢工程改造策略促进酿酒酵母香紫苏醇合成示意图。
图2示出了优化香紫苏醇合成酶时的香紫苏醇产量情况。
图3示出了强化酿酒酵母MVA途径、强化中心代谢、转录因子调控提高香紫苏醇产量情况。
图4示出了转录组测序筛出的靶点改造香紫苏醇产量情况，其中A为过表达靶点基因香紫苏醇产量情况；B为敲除靶点基因香紫苏醇产量情况。
图5示出了摇瓶批式补料发酵香紫苏醇的产量情况，其中A为补料时间；B为生长情况；C为香紫苏醇产量情况。
图6示出了发酵罐批式补料发酵香紫苏醇的产量情况；A为香紫苏醇产量情况；B为生长情况。
图7示出了酿酒酵母合成香紫苏醇的气相色谱图。
具体实施方式
下述实施例中所使用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材料、试剂等如无特殊说明，均可从商业途径得到。
本申请实施例中基于Euroscarf(Oberursel,Germany)公司的CEN.PK113-11C菌株(MATa MAL2-8c SUC2 his3Δ1 ura3-52)进行改造。本申请的基因改造借助CRISPR/Cas9系统完成(可参考文献Mans et al.,FEMS Yeast Res.2015 Mar；15(2):fov004.)
本申请代谢工程改造策略促进酿酒酵母香紫苏醇合成示意图如图所示。
表1.本申请实施例中优化MVA合成途径涉及到的基因相关信息
Figure PCTCN2021138506-appb-000001
表2.本申请实施例强化中心代谢途径涉及到的基因相关信息
Figure PCTCN2021138506-appb-000002
Figure PCTCN2021138506-appb-000003
表3.本申请实施例涉及到的部分位点靶向序列
Figure PCTCN2021138506-appb-000004
表4.本申请实施例中培养基中的组分组成
Figure PCTCN2021138506-appb-000005
作为一种实施方案，本申请提供一种产香紫苏醇的酿酒酵母菌株的构建方法，能够显著增加香紫苏醇的产量。
所述构建方法包括：香紫苏醇合成酶的优化，甲羟戊酸途径(MVA)强化，中心代谢途径优化、全局调控以及二倍体优化。
在一个具体实施方案中，分别构建质粒pYX312-L+T，pYX312-LT和pYX312-TL。pYX312质粒由本实验室构建，将pYX212质粒上P TPI启动子替换为增强型P eTDH3启动子获得，其中P eTDH3启动子参考文献(Blazeck et al.Biotechnol Bioeng,2012,109(11):2884-2895.)扩增融合获得。为了构建pYX312-L+T，LPPS基因和TPS基因由密码子优化后全基因合成获得。P eTDH3以pYX312为模板扩 增获得。终止子T PYK1和T PRM9以CEN.PK113-11C基因组(野生型酿酒酵母)为模板扩增获得。将LPPS基因和T PYK1用OE-PCR方法(融合片段构建过程参考Zhou et al.J Am Chem Soc 2012,134:3234-3241)融合成长片段一，将P eTDH3、TPS和T PRM9用OE-PCR方法融合成长片段二；为了构建pYX312-LT，将LPPS基因和TPS基因用OE-PCR方法融合成LT基因。为了构建pYX312-TL，将TPS基因和LPPS基因用OE-PCR方法融合成TL基因。将pYX312(EcoRI/HindIII)分别与长片段一、二，LT融合基因，TL融合基因用无缝克隆方法连接，并转化大肠杆菌感受态。经菌落PCR验证和测序正确的质粒分别命名为pYX312-L+T，pYX312-LT和pYX312-TL。
进一步地，将质粒PYX312-L+T，PYX312-LT和PYX312-TL转入工程菌XC07，基因型(MATa；MAL2-8c；SUC2；his3Δ1；ura3-52；XI-5::P TEF1-Cas9-T CYC1；XI-3::P GAL7-MBP-TL-T ADH1；gal80Δ；X4::P TDH3-SpHMGR-T PRM9；X2::P TPI1-BTS1-ERG20-T FBA1；P ERG9::P HXT1)，经过发酵提取香紫苏醇并检测得出结论：TPS～LPPS融合方式最有利于香紫苏醇合成(图2)。
进一步地，在TL融合基因基础上，融合麦芽糖结合蛋白编码基因MBP。具体实施方式如下：将MBP基因分别与TL基因的N端和C端融合，获得融合基因MBP-TL和TL-MBP。将TL、融合基因MBP-TL和TL-MBP分别与启动子P GAL7和终止子T ADH1及XI-3位点的上下游同源臂融合获得供体DNA。
进一步地，借助CRISPR/Cas9系统，将融合基因MBP-TL、TL-MBP和TL整合到工程菌SCX22(基因型：MATa；MAL2-8c；SUC2；ura3Δ；hfd1Δ；gal80Δ；gal1Δ；gal7Δ；his3Δ::(P TPI-MmACL-T FBA1)+(P TDH3-RtME-T CYC1)+(P tHXT7-’MDH3-T TDH2)+(P PGK1-CTP1-T HIS3)；X1-5::P TEF1-Cas9-T CYC1；pyc1::P TEF1-PYC1；X1-4::(P TPI1-MPC1)+(P PGK1-MPC3-T DIT1)；X1-2::(P GAL1-AnACLa-T CYC1)+(P GAL10-AnACLb-T ADH1)；gal10Δ::(P TPI1-RtCIT1-T FBA1)+(P TDH3-IDP2-T CYC1)+(P TEF1-YHM2-T GAL1)；pgi1Δ::(P COX9-PGI1)+(P TDH3-GND1-T CYC1)+(P tHXT7-TKL1-T TDH2)+(P PGK1-TAL1-T ADH1)+(P TEF1-ZWF1)；idh2Δ::P GSY1-IDH2；ERG20::P TDH3-ERG20(F96C)；FAA4::FAA4+P TDH3-tHMG1-T FAA4；POX1::POX1+P tHXT7-SpHMGR-T POX1；’tesAΔ；Rtfas1Δ；Rtfas2Δ；P ACC1-ACC1)基因组中。CRISPR/Cas9系统构建的具体步骤是：通过融合PCR方法获得完整同源重组片段包含整合位点的上下游序列、启动子P TEF1、终止子T CYC1的Cas9基因及筛选抗性KanMX基因，将同源重组片段500ng转化入酿酒酵母出发菌株CENPK113-11C，获得带有Cas9蛋白的酿酒酵母菌株。
进一步地，构建靶向染色体整合位点XI-3的sgRNA表达载体；随后，将sgRNA表达载体和供体DNA(各500ng)，通过化学转化法转化到SCX22中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR验证正确，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株分别命名为工程菌SCX23、SCX24和SCX25。
进一步地，经过发酵提取香紫苏醇并检测确认MBP-TL融合方式(SCX23)最有利于香紫苏醇合成(图2)。
进一步地，将MBP-TL融合基因整合到出发菌株CENPK113-11C并敲除GAL80基因获得工程菌CXM01*。
进一步地，优化MVA合成途径。借助CRISPR/Cas9系统，将甲羟戊酸相关限速基因采用不同启动子和终止子构建表达盒，并整合到基因组不同位点(位点选择参考文献Mikkelsen et al.,Metab.Eng.2012,14:104-111)，获得MVA途径优化工程菌CXM17。其主要基因及位点(不限于)如表1所示。
进一步地，MVA途径相关基因表达盒构建和基因组整合的具体步骤是：首先，构建靶向染色体整合位点的sgRNA表达载体和基因表达盒；然后，分别扩增整合位点上下游各300bp序列、基因阅读框、启动子和终止子并通过融合PCR方法(融合片段构建过程参考J Am Chem Soc 2012,134:3234-3241)获得完整供体DNA片段；随后，将sgRNA表达载体和基因表达盒(各500ng)，通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR验证正确，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株保存备用。下文中酿酒酵母其它基因组编辑工作均遵循相似流程。以整合HMG2 K6R和ERG10基因到染色体XII-2位点为例：构建靶向染色体XII-2位点的sgRNA表达载体；与此同时，构建HMG2 K6R和ERG10基因表达盒，分别扩增基因XII-2位点上下游各300bp序列，基因HMG2 K6R，ERG10，双向启动子P GAL10-1，终止子T PYK1和终止子T ENO2，通过融合PCR方法获得完整供体DNA片段；随后经上述方法转化、验证，获得正确转化子。
进一步地，经过发酵提取并检测，工程菌CXM17香紫苏醇单位OD 600产量达到13.37mg/L/OD 600(图3)。
进一步地，强化中心代谢途径，借助CRISPR/Cas9系统，将中心代谢相关限速基因采用不同启动子和终止子构建表达盒(表2)，并整合到基因组不同位点上，获得MVA途径和中心代谢途径优化的工程菌SCX38。
进一步地，经过发酵提取并检测，工程菌SCX38香紫苏醇产量达到26.29mg/L/OD 600(图3)。
进一步地，敲除影响萜类合成的转录因子ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c，获得工程菌SCX42。
进一步地，具体实施方法以ROX1基因为例：首先，构建靶向ROX1基因ORF框的sgRNA表达载体；然后，分别扩增ROX1基因上下游各300bp序列并通过融合PCR方法获得完整供体DNA片段；随后，将gRNA表达载体和基因表达盒(各500ng)，通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR验证正确，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株保存备用。其它5个转录因子靶点敲除均遵循上述过程，实现无缝敲除。
进一步地，经过发酵提取并检测，工程菌SCX42香紫苏醇产量达到75.38mg/L/OD 600(图3)。
本发明的第二方面，构建一株能高效生物合成香紫苏醇的二倍体。
所述构建方法包括：工程菌配型由MATa转换为MATα，以及MATa和MATα实现二倍体交配。
在一个具体实施方案中，将工程菌SCX42由MATa转换为MATα，借助CRISPR/Cas9系统，首先，构建靶向MATa区域的sgRNA表达载体；然后，以MATα基因组为模板，扩增基因组配型区域；随后，将gRNA表达载体和供体DNA(各500ng)，通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR扩增后送测序显示配型为α型的转化子，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株命名为SCX42-α。
进一步地，将SCX42由缺陷型ura3 -、his3 -转变为ura3 -、met3 -，具体实施方案是：构建靶向MET3基因区域的sgRNA表达载体，然后扩增MET3基因上下游各300bp及HIS3基因ORF框，并通过融合PCR获得供体DNA。随后，将gRNA表达载体和基因表达盒(各500ng)，通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR验证正确，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株命名为SCX42*。
进一步地，将工程菌SCX42-α与SCX42*分别在YPD培养基中单独培养，然后各取0.5mL菌液混合接种于20mL YPD培养基，培养48h。取发酵液离心，水洗两遍后适当稀释并涂布于SD-URA3固体培养基，长出的单菌落即为二倍体。
本发明的第三方面，对工程菌实施转录组测序分析，获得能进一步提高香紫苏醇产量的基因靶点。
所述方法包括：对工程菌CXM01*、CXM17、CXM18(由工程菌CXM17敲除转录因子ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c获得)、SCX38和SCX42进行转录组测序，并分析获得可能有利于香紫苏醇合成的改造靶点，并通过过表达或者敲除方式找到能进一步提高香紫苏醇合成的靶点。
在一个具体实施方案中，将工程菌CXM01*、CXM17、CXM18、SCX38和SCX42发酵培养后分别取发酵液中葡萄糖残余10g/L和乙醇利用时期的菌体进行转录组测序，并通过对比上述五株菌基因表达差异获得过表达和敲除改造靶点。
进一步地，对上述靶点进行过表达或者敲除分析。具体实施方案是：借助CRISPR/Cas9系统，将基因过表达靶点分别与启动子P GAL7和终止子T ENO2融合，并整合到工程菌XI-6位点。对于基因敲除靶点，则实施基因无缝敲除。
进一步地，经过发酵提取并检测工程菌香紫苏醇产量。如图4所示，靶点基因DAN1，HUG1，LAC1，OYE3和OYE2过表达及靶点基因SUL1，CDA2，ERV15，ZRT1，DSE1和ANB1敲除对香紫苏醇合成都能起到促进作用。其中最高产量为1.1g/L。
本申请具体实施例中，染色体整合位点序列XI-3，XI-1，XII-2，XII-3，XI-2参考文献(Mikkelsen et al.,Metab.Eng.2012,14:104-111)获得。染色体整合位点靶向序列参考表3。培养基配方参考表4。
实施例1 香紫苏醇合成酶基因的优化
分别构建质粒pYX312-L+T，pYX312-LT和pYX312-TL。pYX312质粒由本实验室构建，将pYX212质粒(购于Addgene)上P TPI启动子替换为增强型P eTDH3启动子获得，其中P eTDH3启动子参考文献(Blazeck et al.Biotechnol Bioeng,2012,109(11):2884-2895.)扩增融合获得。为了构建pYX312-L+T，LPPS基因和TPS基因经密码子优化后全基因合成获得，LPPS基因优化后的核苷酸序列如SEQ ID NO：1所示，TPS基因优化后的核苷酸序列如SEQ ID NO：2所示。P eTDH3以PYX312为模板扩增获得。终止子T PYK1和T PRM9以CEN.PK113-11C基因组为模板扩增获得。将LPPS基因和T PYK1用OE-PCR方法(融合片段构建过程参考J Am Chem Soc 2012,134:3234-3241)融合成长片段一，将P eTDH3、TPS和T PRM9用OE-PCR方法融合成长片段二。为了构建pYX312-LT，将LPPS基因和TPS基因用OE-PCR方法融合成LT基因(连接肽为GGGS，编码基因的核苷酸序列如SEQ ID NO：6所示)；为了构建pYX312-TL，将TPS基因和LPPS基因用OE-PCR方法融合成TL基因(连接肽为GGGS)。将pYX312(EcoRI/HindIII)分别与长片段一、二，LT融合基因，TL融合基因用无缝克隆方法连接，并转化大肠杆菌感受态。经菌落PCR验证和测序正确的质粒分别命名为pYX312-L+T，pYX312-LT和pYX312-TL。
将质粒pYX312-L+T，pYX312-LT和pYX312-TL转入工程菌XC07，工程菌XC07为在CEN.PK-113-11C的基础上改造，基因型为(MATa；MAL2-8c；SUC2；his3Δ1；ura3-52；XI-5::P TEF1-Cas9-T CYC1；XI-3::P GAL7-MBP-TL-T ADH1；gal80Δ；X4::P TDH3-SpHMGR-T PRM9；X2::P TPI1-BTS1-ERG20-T FBA1；P ERG9::P HXT1)，经过发酵提取香紫苏醇并检测确认TPS～LPPS融合方式最有利于香紫苏醇合成(图2)，发酵条件：活化后的菌株于培养基中，30℃，220rpm震荡培养24h，转接于15mL Delft-D发酵培养基/100mL摇瓶，初始OD 600＝0.1，30℃，220rpm条件下发酵96h，测定生物量及香紫苏醇产量。
在TL融合基因基础上，融合麦芽糖结合蛋白编码基因MBP。具体实施方式如下：将MBP基因分别与TL基因的5’端和3’端融合(连接肽为Gly6，其编码基因的核苷酸序列如SEQ ID NO：7所示)，获得融合基因MBP-TL和TL-MBP。将TL、融合基因MBP-TL和TL-MBP分别与P GAL7和终止子T ADH1及XI-3位点的上下游同源臂融合获得供体DNA。参考CRISPR/Cas9系统(Mans R,et al.FEMS Yeast Res.2015 Mar；15(2):fov004.)，将融合基因整合到工程菌SCX22的基因组中，工程菌SCX22为在CEN.PK-113-11C的基础上改造，基因型：MATa；MAL2-8c；SUC2；ura3Δ；hfd1Δ；gal80Δ；gal1Δ；gal7Δ；his3Δ::(P TPI-MmACL-T FBA1)+(P TDH3-RtME-T CYC1)+(P tHXT7-’MDH3-T TDH2)+(P PGK1-CTP1-T HIS3)；X1-5::P TEF1-Cas9-T CYC1；pyc1::P TEF1-PYC1；X1-4::(P TPI1-MPC1)+(P PGK1-MPC3-T DIT1)；X1-2::(P GAL1-AnACLa-T CYC1)+(P GAL10-AnACLb-T ADH1)；gal10Δ::(P TPI1-RtCIT1-T FBA1)+(P TDH3-IDP2-T CYC1)+(P TEF1-YHM2-T GAL1)；pgi1Δ::(P COX9-PGI1)+(P TDH3-GND1-T CYC1)+(P tHXT7-TKL1-T TDH2)+(P PGK1-TAL1-T ADH1)+(P TEF1-ZWF1)；idh2Δ::P GSY1-IDH2；Erg20::P TDH3-Erg20(F96C)；FAA4::FAA4+P TDH3-tHMG1-T FAA4；POX1::POX1+P tHXT7-SpHMGR-T POX1；’tesAΔ；Rtfas1Δ；Rtfas2Δ；P ACC1-ACC1。整合方法如下：构建靶向染色体整合位点XI-3的sgRNA表达载体；随后，将sgRNA表达载体和供体DNA(XI-3up-P GAL7-MBP-TL-T ADH1-XI-3dw；XI-3up-P GAL7-TL-MBP-T ADH1-XI-3dw；XI-3up-P GAL7-TL-T ADH1-XI-3dw)各500ng，通过化学分别转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR验证正确，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株保存备用。质粒丢失后的菌株分别命名为工程菌SCX23、SCX24和SCX25。经过发酵提取香紫苏醇并检测确认MBP-TL融合方式(SCX23)最有利于香紫苏醇合成(图2)。
实施例2 MVA途径供给对酿酒酵母合成香紫苏醇的影响
以CEN.PK113-11C为出发菌，在XI-3位点整合MBP-TL融合基因(整合方法同实施例1)，敲除GAL80基因，获得工程菌CXM01*。在工程菌CXM01*中过表达截断型tHMG1基因于FAA4位点(供体DNA为FAA4up-FAA4-T AHD1-kanMX-P THD3-tHMG1-FAA4dw)；过表达SpHMGR基因(核苷酸序列如SEQ ID NO：5所示)于POX1位点(供体DNA为POX1up-POX1-T TDH2-kanMX-P tHXT7-SpHMGR-POX1dw)。将基因组上ERG20基因突变为ERG20 F96C即将碱基TTC突变为TGT。将HMG2 K6R和ERG10基因整合到XII-2位点(供体DNA为XII-2up-T ENO2-HMG2 K6R-P GAL10-1-ERG10-T PYK1-XII-2dw)；在XII-3位点整合两个拷贝的HMG2 K6R(供体DNA为XII-3up-T PDC1-HMG2 K6R-P GAL10-1-HMG2 K6R-T ENO2-XII-3dw)；将ERG9基因启动子替换为P HXT1；将BTS1和PaGGPPS(核苷酸序列如SEQ ID NO：4所示)基因通过蛋白融合表达方式连接并整合到XI-2位点(供体DNA为XI-2up-P GAL7-BTS1-PaGGPPS-T TDH2-XI-2dw)，获得工程菌CXM17。
MVA途径相关基因表达盒构建和基因组整合的具体步骤以整合tHMG1到FAA4位点为例，首先分别扩增FAA4up-FAA4、T AHD1、kanMX、P THD3、tHMG1、T FAA4并通过融合PCR方法获得完整供体DNA片段FAA4up-FAA4-T AHD1-kanMX-P THD3-tHMG1-T FAA4；随后，将供体DNA片段500ng通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体YPD+G418培养基培养后，通过菌落PCR验证正确的，继续转化pgRNA-kanMX和供体T AHD1-P THD3以丢失KanMX筛选抗性基因。经验证正确的转化子涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株保存备用。下文中酿酒酵母其它基因组编辑工作均遵循相似流程。其中HMG2 K6R基因采用引物(F：ATGTCACTTCCCTTAAGAACGATAGTACATTTG；R：TAATAATGCTGAGGTTTTACAGGGGGG)以CEN.PK113-11C为模板扩增获得。
工程菌发酵过程采用15mL Delft-D发酵培养基/100mL摇瓶，初始pH为5.6。初始OD 600＝0.1，30℃，220rpm条件下发酵96h，测定生物量及香紫苏醇产量。如图3所示，通过强化MVA途径获得CXM17，单位OD 600香紫苏醇产量达到13.4mg/L/OD 600。
实施例3 强化中心代谢提高香紫苏醇合成效率
在MVA优化菌株CXM17的基础上，优化酿酒酵母中心代谢途径以强化前体乙酰辅酶A和辅因子NADPH的供应，得到工程菌SCX38。详细过程如下所示。将ATP依赖的柠檬酸裂解酶MmACL基因、苹果酸合成酶RtME基因、去除信号肽的苹果酸脱氢酶’MDH3基因和柠檬酸转运蛋白编码基因CTP1整合到HIS3基因位点(供体DNA为HIS3up-HIS3-P TPI1-MmACL-T FBA1-P TDH3-RtME-T CYC1-P tHXT7-’MDH3-T TDH2-P PGK1-CTP1-T ADH1-P TEF1-‘tesA-T HIS3)；将丙酮酸羧化酶基因启动子P PYC1替换为启动子P TEF1；过表达丙酮酸转运蛋白编码基因MPC1和MPC3于XI-4位点(供体DNA为XI-4up-P tHXT7-MPC3-T DIT1-T MPC1-MPC1-P TPI1-XI-4dw)；将AnACL基因(AnACLa和AnACLb)整合到X2位点(供体DNA为X2up-T CYC1-AnACLa-P GAL1-P GAL10-AnACLb-T ADH1-X2dw)；将柠檬酸合成酶基因RtCIT1、异柠檬酸脱氢酶基因IDP2和柠檬酸、草酰乙酸和α-酮戊二酸转运蛋白编码基因YHM2基因整合到GAL1、GAL7和GAL10位点(供体DNA为T GAL7-P TPI1-RtCIT1-T FBA1-T CYC1-IDP2-P THD3-P TEF1-YHM2-T GAL1)，即整合该DNA片段的同时敲除GAL1、GAL7、GAL10三个基因；将6磷酸葡萄糖异构酶PGI1基因启动子替换为启动子P COX9并过表达磷酸戊糖途径GND1、TKL1、TAL1和ZWF1基因，供体DNA为PGI1-P COX9-T CYC1-GND1-P THD3-P tHXT7-TKL1-T TDH2-T ADH1-TAL1-P PGK1-P TEF1-ZWF1-T ZWF1-PGI1up；将异柠檬酸脱氢酶IDH2基因启动子P IDH2替换为启动子P GSY1。
本实施例工程菌具体改造方法均可参考文献(Yu et al.,Cell,2018:174:1549-1558e1514.)获得。工程菌发酵过程采用15mL Delft-D发酵培养基/100mL摇瓶，初始pH为5.6，初始OD 600＝0.1，30℃，220rpm条件下发酵96h，测定生物量及香紫苏醇产量。香紫苏醇采用正己烷萃取，并以香紫苏内酯作为内标，利用GC-MS检测。如图3所示，香紫苏醇产量显著提高至26.2mg/L/OD 600。
实施例4 靶点调控对酿酒酵母合成香紫苏醇的影响
工程菌SCX38敲除基因ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c，获得工程菌SCX42。具体实施方法以ROX1基因为例：首先，构建靶向ROX1基因ORF框的sgRNA表达载体；然后，分别扩增ROX1基因上下游各300bp序列并通过融合PCR方法获得完整供体DNA片段；随后，将gRNA表达载体和基因表达盒(各500ng)，通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR验证正确，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株保存备用。其它5个转录因子靶点敲除均遵循上述过程，实现无缝敲除。
工程菌发酵过程采用工程菌发酵过程采用15mL Delft-D发酵培养基/100mL摇瓶，初始pH为5.6，初始OD 600＝0.1，30℃，220rpm条件下发酵96h，测定生物量及香紫苏醇产量。香紫苏醇采用正己烷萃取，并以香紫苏内酯作为内标，利用GC-MS检测。如图3所示，通过对相关靶点基因进一步敲除，香紫苏醇产量达到934mg/L，单位OD 600产量为75.4mg/L/OD 600，是迄今为止的最高产量，证明了酿酒酵母作为细胞工厂生产香紫苏醇的巨大潜力。所得的香紫苏醇峰图如图7所示。
实施例5 香紫苏醇合成的酿酒酵母二倍体菌株构建
将工程菌SCX42由MATa转换为MATα，借助CRISPR/Cas9系统，首先，构建靶向MATa区域的sgRNA表达载体；然后，以MATα基因组为模板，扩增基因组配型区域；随后，将gRNA-HMRa表达载体和供体DNA(各500ng)，通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR扩增后送测序显示配型为α型的转化子，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株命名为SCX42-α。配型转换改造方法可参考专利(天津大学.一种转换酵母交配型的方法:CN201610102424.8[P].2016-04-13.)获得。
将SCX42由缺陷型ura3 -、his3 -转变为ura3 -、met3 -，具体实施方案是：构建靶向MET3基因区域的sgRNA表达载体，然后扩增MET3基因上下游各300bp及HIS3基因ORF框，并通过融合PCR获得供体DNA。随后，将gRNA表达载体和基因表达盒(各500ng)，通过化学转化法转化到酿酒酵母中，涂布到筛选平板于30℃静置培养3天，转化子经液体SD培养基培养后，通过菌落PCR验证正确，涂布于含有5-氟乳清酸的平板进行质粒丢失，质粒丢失后的菌株命名为SCX42*。
将工程菌SCX42-α与SCX42*分别在YPD培养基中单独培养，然后各取0.5mL菌液混合接种于20mL YPD培养基，培养48h。取发酵液离心，水洗两遍后适当稀释并涂布于SD-URA3固体培养基，长出的单菌落即为二倍体。
实施例6
工程菌转录组测序分析并实施改造获得过表达或敲除靶点
将工程菌CXM01*、CXM17、CXM18(CXM18由工程菌CXM17敲除转录因子ROX1、DOS2、VBA5、YER134c、YNR063w、YGR259c获得)、SCX38和SCX42于基础成分培养基((Delft-D中加入葡萄糖浓度为20g/L)发酵培养，并分别在葡萄糖剩余浓度为10g/L时间点及葡萄糖耗完乙醇利用时期取菌体进行转录组测序，分析获得可能有利于香紫苏醇合成的改造靶点，其中过表达靶点：ANB1，DAN1，HUG1，PYK1，LAC1，CYB5，YCL021W-A，YCL074W，OYE3，OYE2，HSP31，BLI1；敲除靶点：SPS4，YOR314W，SUL1，CDA2，ERV15，ZRT1，DSE1，ANB1。改造靶点均可在Saccharomyces Genome Database(https://www.yeastgenome.org/)中查找。
借助CRISPR/Cas9系统，将基因过表达靶点分别与启动子P GAL7和终止子T ENO2融合，并整合到工程菌XI-6位点。对于基因敲除靶点，则实施ORF无缝敲除。
工程菌发酵过程采用工程菌发酵过程采用15mL Delft-D发酵培养基/100mL摇瓶，初始pH为5.6，初始OD 600＝0.1，30℃，220rpm条件下发酵96h，测定生物量及香紫苏醇产量。香紫苏醇采用正己烷萃取，并以香紫苏内酯作为内标，利用GC-MS检测。
如图4所示，在SCX42中，靶点基因DNA1，HUG1，LAC1，OYE3和OYE2过表达及靶点基因SUL1，CDA2，ERV15，ZRT1，DSE1和ANB1敲除对香紫苏醇合成都能起到促进作用。其中过表达LAC1基因使得香紫苏醇达到最高产量，1.1g/L(图4)。
实施例7
香紫苏醇批式补料发酵
(1)工程菌摇瓶批试补料发酵
摇瓶批试补料发酵采用50mL发酵液/250mL锥形瓶培养体系，选取工程菌SCX42和二倍体。批试发酵时采用基础成分培养基(Delft-A和Delft-D)，体积为50mL，接种OD 600＝0.2，pH为5.6。补料培养时采用3ⅹDelft-A培养基(Delft-A培养基中加入500g/L的葡萄糖)或者3ⅹDelft-D培养基(Delft-D培养基中加入500g/L的葡萄糖)待批试发酵葡萄糖消耗完即添加1mL的3ⅹDelft-A培养基或2mL的3ⅹDelft-D培养基，并且每24h用4M氢氧化钾调pH到5.6。补料时间点(即对应图中的补糖时间点)如下图5A所示，其中，二倍体在黑点和灰点所示的时间点补料，而其他的工程菌仅在黑点所示的时间点补料。在30℃，220rpm条件下发酵8d，测定生物量及香紫苏醇产量。香紫苏醇采用正己烷萃取，并以香紫苏内酯作为内标，利用GC-MS检测。如图5B和5C所示，工程菌 SCX42在补Delft-D时生长情况略微优于Delft-A培养基，而香紫苏醇含量显著优于Delft-A(2.2g/L)，达到4.5g/L；在同样补Delft-A的情况下，SCX42生长较二倍体更快，但二倍体香紫苏醇含量略高于工程菌SCX42，为2.4g/L。以上结果说明Delft-D培养基相比Delft-A培养基更适合用于工程菌发酵生产香紫苏醇。
(2)工程菌平行生物反应器批试补料发酵
为了获得更高产量的工程菌，我们在工程菌SCX42中过表达LAC1和OYE2基因，获得工程菌SCX42-LAC1-OYE2。批试补料发酵采用1L的DasGip平行生物反应器系统，批试发酵体积为0.4L。批试发酵采用基础成分培养基(Delft-D中加入葡萄糖浓度为20g/L)，体积为0.4L，接种OD 600＝0.4，pH为5.6。补料培养基采用4ⅹD培养基(Delft-D中加入葡萄糖浓度为500g/L)。补料方式采用指数补料，μ＝0.05。批试发酵中葡萄糖耗完，即开始指数补料。在发酵过程中存在香紫苏醇颗粒及菌体黏附在发酵罐罐壁、电极等的现象。最终香紫苏醇产量需将所有黏附的颗粒悬浮到发酵液中定量。
如图6所示，工程菌SCX42-LAC1-OYE2最高产量为11.4g/L。
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 (14)

  1. 一种工程菌的构建方法，其特征在于，所述构建方法包括以下步骤：
    在宿主菌株中导入DNA片段I，得到所述工程菌；
    所述DNA片段I从上游到下依次包括：启动子、融合基因、终止子；
    所述融合基因从上游到下依次包括：TPS基因、连接肽I的编码基因、LPPS基因；
    所述宿主菌株选自酿酒酵母中的任一种。
  2. 根据权利要求1所述的构建方法，其特征在于，所述融合基因从上游到下依次包括：
    MBP基因、连接肽II的编码基因、TPS基因、连接肽I的编码基因、LPPS基因；
    或
    所述融合基因从上游到下依次包括：
    TPS基因、连接肽I的编码基因、LPPS基因、连接肽II的编码基因、MBP基因。
  3. 根据权利要求1所述的构建方法，其特征在于，所述LPPS基因的核苷酸序列如SEQ ID NO：1所示；
    所述TPS基因的核苷酸序列如SEQ ID NO：2所示；
    所述MBP基因的核苷酸序列如SEQ ID NO：3所示；
    所述连接肽I的编码基因的核苷酸序列如SEQ ID NO：6所示；
    所述连接肽II的编码基因的核苷酸序列如SEQ ID NO：7所示；
    所述启动子选自P eTDH3、P GAL7中的任意一种；
    所述终止子选自T PYX212、T ADH1中的任意一种。
  4. 根据权利要求1所述的构建方法，其特征在于，所述在宿主菌株中导入DNA片段I为将DNA片段I整合到宿主的XI-3基因位点。
  5. 根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括优化MVA的合成途径，包括(a)～(h)中的至少一种：
    (a)敲除宿主菌株的GAL80基因；
    (b)将tHMG1基因导入宿主菌株；
    (c)将SpHMGR基因导入宿主菌株；
    (d)将宿主菌株基因组上ERG20基因突变为ERG20 F96C基因；
    (e)将HMG2 K6R基因和ERG10基因导入宿主菌株；
    (f)将含有2个拷贝HMG2 K6R基因的DNA片段导入宿主菌株；
    (g)将宿主菌株中ERG9基因的启动子替换为P HXT1；
    (h)将含有BTS1和PaGGPPS基因的DNA片段导入宿主菌株；
    所述(a)～(h)不分先后顺序。
  6. 根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括优化MVA的合成途径，包括(a)～(h)中的至少一种：
    (a)敲除宿主菌株的GAL80基因；
    (b)将含有FAA4-T AHD1-P THD3-tHMG1的DNA片段整合到宿主菌株的FAA4基因位点；
    (c)将含有POX1-T TDH2-P tHXT7-SpHMGR的DNA片段整合到宿主菌株的POX1基因位点；
    (d)将宿主菌株基因组上ERG20基因突变为ERG20 F96C基因；
    (e)将含有T ENO2-HMG2 K6R-P GAL10-1-ERG10-T PYK1的DNA片段整合到宿主菌株的XII-2基因位点；
    (f)将含有T PDC1-HMG2 K6R-P GAL10-1-HMG2 K6R-T ENO2的DNA片段整合到宿主菌株的XII-3基因位点；
    (g)将宿主菌株中ERG9基因的启动子替换为P HXT1；
    (h)将含有P GAL7-BTS1-PaGGPPS-T TDH2的DNA片段整合到宿主菌株的XI-2基因位点；
    所述(a)～(h)不分先后顺序。
  7. 根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括优化中心代谢途径，包括(A)～(N)中的至少一种：
    (A)将MmACL基因导入宿主菌株；
    (B)将RtME基因导入宿主菌株；
    (C)将‘MDH3基因导入宿主菌株；
    (D)将CTP1基因导入宿主菌株；
    (E)将宿主菌株的PYC1基因启动子P PYC1替换为启动子P TEF1；
    (F)将MPC1基因导入宿主菌株；
    (G)将MPC3基因导入宿主菌株；
    (H)将AnACLa基因、AnACLb基因导入宿主菌株；
    (I)将RtCIT1基因导入宿主菌株；
    (J)将IDP2基因导入宿主菌株；
    (K)将YHM2基因导入宿主菌株；
    (L)将宿主菌株的PGI1基因的启动子替换为启动子P COX9；
    (M)将GND1基因、TKL1基因、TAL1基因、ZWF1基因导入宿主菌株；
    (N)将宿主菌株的IDH2基因的启动子替换为启动子P GSY1；
    所述(A)～(N)不分先后顺序。
  8. 根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括优化中心代谢途径，所述构建方法还包括优化中心代谢途径，包括(A)～(G)中的至少一种：
    (A)将含有HIS3-P TPI1-MmACL-T FBA1-P TDH3-RtME-T CYC1-P tHXT7-’MDH3-T TDH2-P PGK1-CTP1-T ADH1的DNA片段整合到宿主菌株的HIS3基因位点；
    (B)将宿主菌株的PYC1基因的启动子P PYC1替换为启动子P TEF1；
    (C)将含有P tHXT7-MPC3-T DIT1-T MPC1-MPC1-P TPI1的DNA片段整合到宿主菌株的XI-4位点；
    (D)将含有T CYC1-AnACLa-P GAL1-P GAL10-AnACLb-T ADH1的DNA片段整合到宿主菌株的X2位点；
    (E)将含有P TPI1-RtCIT1-T FBA1-T CYC1-IDP2-P THD3-P TEF1-YHM2的DNA片段整合到宿主菌株的GAL1、GAL7、GAL10位点；
    (F)将含有P COX9-T CYC1-GND1-P THD3-P tHXT7-TKL1-T TDH2-T ADH1-TAL1-P PGK1-P TEF1-ZWF1-T ZWF1的DNA片段整合到宿主菌株的PGI1位点；
    (G)将宿主菌株的IDH2基因的启动子替换为启动子P GSY1；
    所述(A)～(G)不分先后顺序。
  9. 根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括靶点调控，包括：
    敲除宿主菌株的ROX1基因、DOS2基因、VBA5基因、YER134c基因、YNR063w基因、YGR259c基因中的至少一种。
  10. 根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括以下的至少一种：
    (I)在宿主菌株中导入ANB1基因、DAN1基因、HUG1基因、PYK1基因、LAC1基因、CYB5基因、YCL021W-A基因、YCL074W基因、OYE3基因、OYE2基因、HSP31基因、BLI1基因中的至少一种；
    和/或
    (II)敲除宿主菌株的SPS4基因、YOR314W基因、SUL1基因、CDA2基因、ERV15基因、ZRT1基因、DSE1基因、ANB1基因中的至少一种。
  11. 根据权利要求1所述的构建方法，其特征在于，所述构建方法通过采用CRISPR/Cas9技术进行构建。
  12. 根据权利要求1所述的构建方法，其特征在于，所述构建方法还包括细胞融合形成双倍体。
  13. 根据权利要求1～12任一项所述的构建方法构建得到的工程菌。
  14. 根据权利要求1～12任一项所述的构建方法构建得到的工程菌、权利要求13所述的工程菌中的至少一种在制备香紫苏醇中的应用。
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