WO2023173565A1 - Method for simultaneously enhancing and inhibiting multiple key genes during synthesis of 7-dehydrocholesterol in saccharomyces cerevisiae - Google Patents

Method for simultaneously enhancing and inhibiting multiple key genes during synthesis of 7-dehydrocholesterol in saccharomyces cerevisiae Download PDF

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WO2023173565A1
WO2023173565A1 PCT/CN2022/092329 CN2022092329W WO2023173565A1 WO 2023173565 A1 WO2023173565 A1 WO 2023173565A1 CN 2022092329 W CN2022092329 W CN 2022092329W WO 2023173565 A1 WO2023173565 A1 WO 2023173565A1
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sterol
reductase
saccharomyces cerevisiae
dehydrocholesterol
synthesis
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Chinese (zh)
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刘龙
陈坚
吕雪芹
堵国成
李江华
刘延峰
修翔
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江南大学
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Definitions

  • the invention belongs to the field of bioengineering technology, and in particular refers to a method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae.
  • Vitamin D3 (Cholecalciferol, VD3) is a hormone precursor in the human body that can regulate cell differentiation and promote bone calcification.
  • VD3 is hydroxylated in the human liver to form 25-hydroxyvitamin D3 (25-hydroxyvitamin D3 , 25-OH-VD3), converted into 1,25-dihydroxyvitamin D3 (1,25-(OH)2-VD3) in the kidneys.
  • the main source of vitamin D3 in the human body is through ultraviolet light It is converted from 7-dehydrocholestero (7-DHC) in the bottom layer of irradiated skin, and part of it can also be obtained through food.
  • 7-DHC 7-dehydrocholestero
  • Chinese patent CN104988168A and Chinese patent CN103275997A further enhance the synthesis of 7-DHC by directly knocking out the key genes in the ergosterol synthesis pathway: erg5 and erg6, but completely blocking the synthesis of ergosterol will have an irreversible impact on the growth of the strain. Not conducive to industrialized mass production.
  • VD3 The microbial synthesis of VD3 mainly focuses on the screening of bacterial strains, such as Chinese patent CN103275997A, Chinese patent CN107075551B, and Chinese patent CN104988168A. Most of the strains screened are used to synthesize active VD3, and strains that directly synthesize VD3 are rarely screened. Moreover, the screening of strains is time-consuming and laborious, and a lot of money is needed for large-scale post-screening and fermentation testing to obtain extremely low VD3 yields. strains.
  • the current methods for synthesizing VD3 mainly include three methods: chemical synthesis, microbial transformation and microbial metabolic synthesis.
  • the chemical synthesis method needs to use the more expensive lanolin cholesterol as raw material. After multi-step group protection and deprotection, 7-dehydrocholesterol is obtained, and then through light reaction, ring opening and isomerization to obtain VD3. Due to the use of multiple For organic reagents, the product separation and purification process is complicated and the product yield is low.
  • Microbial transformation method is currently only used to convert VD3 into active VD3, and the microorganisms used are generally Streptomyces, Mycobacterium and other microorganisms. With the development of synthetic biology and genetic engineering, microbial metabolic engineering methods using ergosterol-producing strains to produce 7-DHC and VD3 have great potential.
  • the existing production technology of 7-DHC using Saccharomyces cerevisiae utilizes the yeast's own sterol synthesis pathway, which includes 13 P450 enzymes, and the proportion of sterol synthesis and metabolism in yeast is not high. Therefore, if only through traditional copy increase Enhancing expression at a high rate will inevitably lead to yeast metabolic disorders and the production of other excess by-products, making it more difficult to purify the target product. If the synthesis of ergosterol is simply blocked by knocking out the erg5 and erg6 genes, although there will be a certain accumulation of 7-DHC, completely blocking the synthesis of ergosterol will inevitably have an adverse effect on the growth of the production strain, and thus affect the 7-DHC. DHC and VD3 production. In order to solve the above technical problems, the present invention provides a method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae.
  • the first object of the present invention is to provide a method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae, including reducing alcohol dehydrogenase (adh2) and truncated HMG-CoA in Saccharomyces cerevisiae Enzyme (tHMG1), isopentenyl diphosphate isomerase (idi1), squalene epoxidase (erg1), lanosterol 14- ⁇ -demethylase (erg11), C-14 sterol reductase (erg24) , C-4 methylsterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27) and exogenous gene sterol ⁇ 24-reductase (dhcr24) to simultaneously enhance expression , and/or simultaneously inhibit malate synthase (m
  • the number of the alcohol dehydrogenase (adh2) is ID: NM_001182812.1
  • the number of the truncated HMG-CoA reductase (tHMG1) is ID:
  • the number of phosphate isomerase (idi1) is ID: NM_001183931.1
  • the number of squalene epoxidase (erg1) is ID: NM_001181304.1
  • the number of lanosterol 14- ⁇ -demethylase (erg11) is ID: NM_001179137.1
  • the number of C-14 sterol reductase (erg24) is ID: NM_001183118.1
  • the number of C-4 methylsterol oxidase (erg25) is ID: NM_001181189.3, C-3 sterol dehydrogenation
  • the number of the enzyme (erg26) is ID: NM_001180866.1
  • the exogenous gene dhcr24 derived from chicken is used to optimize the codon preference of Saccharomyces cerevisiae.
  • the optimization method is: input the dhcr24 gene derived from chicken into the website https://www.genscript. com/tools/gensmart-codon-optimization , optimize the codon preference of Saccharomyces cerevisiae, and then synthesize the gene after optimization.
  • the number of malate synthase is ID: NM_001182955.1
  • the number of citrate synthase is ID: NM_001178718.1
  • Delta (24)-sterol C The number of methyltransferase (erg6) is ID: NM_001182363.1.
  • Saccharomyces cerevisiae is Saccharomyces cerevisiae S288C.
  • the strengths of the inducible promoters gal1p and gal7p are enhanced using the dCpf1-VP activation system, with the strength of promoter gal1p increased by 85% and promoter gal7p increased by 69%.
  • the gene fragment that functions as the dCpf1-VP activation system is DPP1-KAN-dCpf1-VP-crRNA1-7-1, and the DPP1-KAN-dCpf1-VP-crRNA1-7- The sequence of 1 is shown in SEQ ID NO 1.
  • the malate synthase (mls1), citrate synthase (cit2), and Delta(24)-sterol C-methyltransferase (erg6) are inhibited by the dCas9-RD inhibition system , the three genes erg6, mls1 and cit2 were inhibited by 88%, 84% and 72% respectively.
  • the gene fragment that functions as the dCas9-RD inhibition system is GAL80-KAN-dCas9-RD-sgRNAEMC, and the sequence of GAL80-KAN-dCas9-RD-sgRNAEMC is shown in SEQ ID NO 2 .
  • the second object of the present invention is to provide an engineered yeast that combines alcohol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isoform from Saccharomyces cerevisiae.
  • adh2 alcohol dehydrogenase
  • tHMG1 truncated HMG-CoA reductase
  • isopentenyl diphosphate isoform from Saccharomyces cerevisiae.
  • the third object of the present invention is to provide the application of engineered yeast in the synthesis of 7-dehydrocholesterol.
  • the seed liquid of the engineered yeast is cultured in YPD medium at 30° C. and 180-220 rpm for 16-20 hours, and then fermented in a shake flask. Inoculate 1-5% of the inoculum into a round-bottom shake flask containing YPD liquid medium for culture and fermentation.
  • the conditions for culture and fermentation are: 30°C, 180-220 rpm for 96-120 hours.
  • the carbon source includes glucose, sucrose, glycerol, and ethanol.
  • the present invention uses the original Saccharomyces cerevisiae S288C as the starting strain, fuses dCpf1 with VP (activation domain), and constructs an activation system based on dCpf1.
  • dCas9 was serially fused with the inhibition domains UME6, MIG1, and TUP1 to construct a dCas9-based inhibition system.
  • the sgRNA recognized by the system was designed to inhibit malate synthase (mls1), citrate synthase (cit2), and Delta (24).
  • -Sterol C-methyltransferase (erg6) is inhibited to further increase the production of 7-DHC, making the production of 7-DHC reach 464mg/L.
  • Figure 1 is a schematic diagram of the construction of the dCpf1-VP activation system of the present invention.
  • Figure 2 is a schematic diagram of the construction of the dCas9-RD inhibition system of the present invention.
  • Figure 3 is a graph showing the intensity of promoters gal1p and gal7p enhanced using dCpf1-VP.
  • Figure 4 is a diagram of the inhibition effect using dCas9-RD.
  • Figure 5 is a graph showing the production of 7-DHC of the strains engineered yeast SX-6 and engineered yeast SX-7 obtained in the present invention.
  • the instrument used to detect 7-DHC in the present invention is high-performance liquid chromatography.
  • the engineered yeast liquid fermented for more than 96 hours is centrifuged at high speed, the supernatant is discarded, resuspended in sterile water, and 0.5mm glass beads are added.
  • a separatory funnel to remove impurities in the lower layer, and then extract The mixture is freeze-dried.
  • the mobile phase uses methanol and water in a ratio of 90:10 or 95:5 or 98:2.
  • the detector uses a UV detector with a detection wavelength of 265-280nm.
  • a multi-gene activation and inhibition system based on CRISPR was designed to activate and inhibit the key genes of the 7-DHC synthesis pathway of Saccharomyces cerevisiae without affecting the growth status of the strain as much as possible.
  • this system was finally used to activate and inhibit key genes, and the effect was obvious.
  • the strain grew normally, and the production of 7-DHC was 464mg/L.
  • the present invention provides a method for constructing multiple gene activation and inhibition systems based on CRISPR technology
  • the present invention utilizes the above constructed system to regulate the synthesis pathway of 7-DHC in Saccharomyces cerevisiae;
  • genes enhanced using the above system are: alcohol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isomerase (idi1), squalene epoxygenase (erg1) , Lanosterol 14- ⁇ -demethylase (erg11), C-14 sterol reductase (erg24), C-4 methylsterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3- Ketosterol reductase (erg27), sterol ⁇ 24-reductase (dhcr24);
  • malate synthase mls1
  • citrate synthase cit2
  • Delta(24)-sterol C-methyltransferase erg6
  • the present invention also provides the application of engineered yeast in the synthesis and production of 7-dehydrocholesterol.
  • the seed liquid of the engineered yeast is cultured in YPD medium at 30° C. and 180-220 rpm for 16-20 hours, and then fermented in a shake flask. Inoculate 1-5% of the inoculum into a round-bottom shake flask containing YPD liquid medium for culture and fermentation.
  • the conditions for culture and fermentation are: 30°C, 180-220 rpm for 96-120 hours.
  • ethanol when fermentation reaches 26 hours, 50-60% (volume fraction) ethanol is added as a carbon source for supplementation. After the fermentation is completed, centrifuge to remove the precipitate.
  • the carbon source includes glucose, sucrose, glycerol, and ethanol.
  • the upstream homology arm fragment 208UP was amplified using primers 208UP-F and 208UP-R, the downstream homology arm fragment 208Down was amplified using primers 208Down-F and 208Down-R, and the downstream homology arm fragment 208Down was amplified using primers 1622UP-F and 1622UP-R.
  • the upstream homology arm fragment 1622UP was amplified using primers 1622Down-F and 1622Down-R to obtain the downstream homology arm fragment 1622Down.
  • the upstream homology arm fragment 308UP was amplified using primers 308UP-F and 308UP-R.
  • the primer 308Down-F was used to amplify the upstream homology arm fragment 1622UP.
  • 308Down-R amplifies the downstream homology arm fragment 308Down, uses primers 1021UP-F and 1021UP-R to amplify the upstream homology arm fragment 1021UP, uses primers 1021Down-F and 1021Down-R to amplify the downstream homology arm fragment 1021Down, using primers 911UP-F and 911UP-R to amplify the upstream homology arm fragment 911UP, and using primers 911Down-F and 911Down-R to amplify the downstream homology arm fragment 911Down.
  • step (b) Perform overlapping PCR between the promoter fragments gal1p and gal7p obtained above and the gene fragment obtained in step (a) to obtain gene fragments 208-gal1p-dhcr24-gal7p-adh2, 1622-gal1p-thmg1-gal7p-idi1, 308-gal1p-erg1-gal7p-erg11, 1021-gal1p-erg24-gal7p-erg25, 911-gal1p-erg26-gal7p-erg27.
  • (c) Using the pML104 plasmid as a template, use primers 208-F and 208-R respectively to perform plasmid loop P to obtain a plasmid that can recognize the insertion site of the fragment 208-gal1p-dch24-gal7p-adh2, named pML104-1.
  • Use primers 1622-F and 1622-R to perform plasmid loop P, and obtain a plasmid that can recognize the fragment 1622-gal1p-thmg1-gal7p-idi1 insertion site, named pML104-2.
  • Use primers 308-F and 308-R to perform plasmid loop P.
  • pML104-3 a plasmid capable of recognizing the insertion site of the fragment 308-gal1p-erg1-gal7p-erg11 was obtained, named pML104-3.
  • the plasmid of -gal7p-erg25 insertion site is named pML104-4.
  • step (d) Pour the fragments and plasmids obtained in steps (b) and (c) into Saccharomyces cerevisiae respectively. After sequencing verification, streak the correct single colony on a 5-FOA YPD plate and culture it at 30°C for 2- After 3 days, strains were obtained and named SX-1, SX-2, SX-3, SX-4, and SX-5.
  • ADH-F aaaacagttgaatattccctcaaaaatgatgtctattccagaaactcaaaaagcc
  • ADH-R aagatattcagtttttgtcattgccgtcttatttagaagtgtcaacaacgtatct
  • IDI-F gttgaatattccctcaaaaatgatgactgccgacaacaatagtatg
  • IDI-R ttacttcttcaattcgtacattatattatagcattctatgaatttg
  • E1-F atacctctatactttaacgtcaaggagatgtctgctgttaacgttgcacc
  • E1-R gtccatatctttccatagatttttaaccaatcaactcaccaaacaaaatggg
  • E11-F acagttgaatattccctcaaaatgatgtctgctaccaagtcaatcgttg
  • GAL1-F cggattagaagccgccgagcgggc
  • GAL1-R ctccttgacgttaaagtatagaggtata
  • GAL7-F aaatctatggaaagatatggacgg
  • GAL7-R catttttgagggaatattcaactg
  • 911UP-R tgtcgcccgctcggcggcttctaatccgtcaacatccgatttttttctataaaat
  • 911-R gtaatattgtcttgtttcccgttttagagctagaaatagcaagttaaataaggctagtcc
  • the fragment gal1p23-1 was directly synthesized by overlapping primers gal1p23-F1 and gal1p23-R1, and the fragment gal7p23-1 was synthesized directly by overlapping primers gal7p23-F1 and gal7p23-R1.
  • the fragment gal1p23-2 was directly synthesized by overlapping primers gal1p23-F2 and gal1p23-R2, and the fragment gal7p23-2 was synthesized directly by overlapping primers gal7p23-F2 and gal7p23-R2.
  • the fragment gal1p23-3 was directly synthesized by overlapping primers gal1p23-F3 and gal1p23-R3, and the fragment gal7p23-3 was directly synthesized by overlapping primers gal7p23-F3 and gal7p23-R3.
  • the plasmid pFA6a-TRP1-PGAL1-GFP purchased from Addgene was used as the template, and the primers pFA6a-F and pFA6a-R were used to amplify the fragment pFA6a-TRP1-GFP.
  • the Saccharomyces cerevisiae SC288C genome was used as the template, and the primers gal7pG-F and The promoter gene fragment gal7p-G was amplified from gal7pG-R, and gal7p-G was connected to pFA6a-TRP1-GFP using seamless cloning to obtain plasmid pFA6a-TRP1-PGAL7-GFP.
  • the gene fragment DPP1UP was amplified using primers DPP1UP-F and DPP1UP-R, and the gene fragment DPP1Down was amplified using primers DPP1Down-F and DPP1Down-R.
  • step (b) Seamlessly connect the fragments SNR52p and gal1p23-1/gal1p23-2/gal1p23-3 obtained in step (a) with plasmid pCSN068-VP to obtain plasmids pCSN068-dCpf1-VP-crRNA1-1 and pCSN068- dCpf1-VP-crRNA1-2, pCSN068-dCpf1-VP-crRNA1-3, using the above plasmid as a template, using primers CSN-VPG-F and CSN-VPG-R to amplify the gene fragments KAN-dCpf1-VP-gal1p1, KAN-dCpf1-VP-gal1p2, KAN-dCpf1-VP-gal1p3, and the fragments DPP1UP and DPP1Down were subjected to overlapping PCR to obtain fragments DPP1-KAN-dCpf1-VP-gal1p1, DPP1
  • step (c) Seamlessly connect the fragments SNR52p and gal7p23-1/gal7p23-2/gal7p23-3 obtained in step (a) with plasmid pCSN068-VP to obtain plasmids pCSN068-dCpf1-VP-crRNA7-1 and pCSN068- dCpf1-VP-crRNA7-2, pCSN068-dCpf1-VP-crRNA7-3, using this plasmid as a template, using primers CSN-VPG-F and CSN-VPG-R to amplify the gene fragments KAN-dCpf1-VP-gal7p1, KAN-dCpf1-VP-gal7p2, KAN-dCpf1-VP-gal7p3, and the fragments DPP1UP and DPP1Down were subjected to overlapping PCR to obtain the fragments DPP1-KAN-dCpf1-VP-gal7p1, D
  • DPP1-KAN-dCpf1-VP-crRNA1-7-1 fragment sequence (see SEQ ID NO 1):
  • step (e) Introduce the fragment obtained in step (d) into the competent state of the engineering strain SX-5, screen it on the G418 plate, and use the verification primers YZdCpf1-F and YZdCpf1-R to perform colony PCR verification. Verify the correct single colony, streak it on a 5-FOA YPD plate, culture it at 30°C for 2-3 days, screen out the single colony with the G418 tag removed, and name it SX-6.
  • gal1p23-F1 actggtctcaagatagtaatacgcttaactgctcattggtaagaaattgtctg
  • gal1p23-R1 aatgagcagttaagcgtattactatcttgagaccagtgatcatttatctttcactgcggagaag
  • gal7p23-R1 ttgtgagtgatatcaaaagtaacatcttgagaccagtgatcatttatctttcactgcggagaag
  • gal1p23-F2 actggtctcaagattggttatgaagaggaaaattggggtaagaaattgtctg
  • gal1p23-R2 ccaatttttcctcttcataaccaatcttgagaccagtgatcatttatctttcactgcggagaag
  • gal7p23-F2 actggtctcaagatcttaacccaaaataagggaagggtaagaaattgtctg
  • gal1p23-R3 catttatatatctgttaatagatatcttgagaccagtgatcatttatctttcactgcggagaag
  • KAN1-F tgtggtcaataagagcgacgagctccagcttttgttccctttagt
  • the plasmid pFA6a-TRP1-PGAL1-GFP purchased from Addgene was used as the template, and the primers pFA6a-F and pFA6a-R were used to amplify the fragment pFA6a-TRP1-GFP.
  • the Saccharomyces cerevisiae SC288C genome was used as the template, and the primers pMLS-F and pFA6a-R were used.
  • the promoter gene fragment pMLS was obtained by amplifying pMLS-R, using primers pERG6-F and pERG6-R to amplify the promoter gene fragment pERG6, and using primers pCIT-F and pCIT-R to amplify the promoter gene fragment pCIT. Seam cloning was used to connect pMLS, pCIT-F, pERG6 and pFA6a-TRP1-GFP respectively to obtain plasmids pFA6a-TRP1-pMLS-GFP, pFA6a-TRP1-pCIT-GFP, and pFA6a-TRP1-pERG6-GFP.
  • Plasmid pFA6a-TRP1-pMLS-GFP and fragments URA-dCas9-RD-MLS, pFA6a-TRP1-pCIT-GFP and URA-dCas9-RD-CIT, pFA6a-TRP1-pERG6-GFP and URA-dCas9-RD-ERG6 They were introduced into yeast SC288C to obtain strains SG-MLS, SG-CIT, and SG-ERG6. The green fluorescence intensity was detected using a microplate reader. The results are shown in Figure 4.
  • the gene fragment tRNA-Gly was amplified directly using primers MLS-F and MLS-R overlaps the synthetic fragment MLS-20.
  • the fragment CIT2-20 was directly synthesized by overlapping primers CIT-F and CIT-R.
  • the genome of Saccharomyces cerevisiae SC288C was used as a template, and primers GAL80UP-F and GAL80UP-R were used to amplify the gene fragment GAL80UP.
  • Primers GAL80Down-F and GAL80Down- were used to amplify the fragment.
  • R amplified the gene fragment GAL80Down.
  • step (c) Perform overlapping PCR on the fragments obtained in step (b): dCas9-RD-erg6, KAN-Cas, tRNA-Gly, MLS-20, CIT2-20, GAL80UP, and GAL80Down to obtain the fragment GAL80-KAN-dCas9 -RD-sgRNAEMC (see SEQ ID NO 2).
  • GAL80-KAN-dCas9-RD-sgRNAEMC fragment sequence see SEQ ID NO 2:
  • step (d) Introduce the fragment obtained in step (c) into the competent state of the engineering strain SX-6, screen it on the G418 plate, and use the verification primers YZdCas-F and YZdCas-R to perform colony PCR verification. Verify the correct single colony, streak it on a 5-FOA YPD plate, culture it at 30°C for 2-3 days, screen out the single colony with the G418 tag removed, and name it SX-7.
  • CIT-F agctctaaaactttgctttcaatgtttttgagatcatttatctttcactgcggagaag
  • CIT-R tcaaaacattgaaagcaaagttttagagctagaaatagcaagttaaaataagg
  • ERG6-F agctctaaacgtttcggcgttttctggctttgatcatttatctttcactgcggagaag
  • ERG6-R aagccagaaaacgccgaaacgttttagagctagaaatagcaagttaaataagg
  • Cas-RD-R gcaagttaaataaggctagtccgttatcaacttgaaaagtggcaccga
  • KAN2-F caatcaagacacattaccccgccatcgctgcaggtcgacgaattctaccgttcgt
  • KAN2-R tgaaatggcagtattgataatgataccgttcgtatagcatacatt
  • ERG6-F aaagataaatgatcctgctgctctcttttcttttgttttagagctagaaatagcaagttaaataagg
  • CIT-F aaaactttttcttgttactagtatttgcgcaagcccggaatcgaa
  • GAL80UP-F aggcatacctaatgctggggatga
  • GAL80UP-R aattcgtcgacctgcagcgatggcggggtaatgtgtcttgatt
  • GAL80Down-F agctccagcttttgaatgcaaggtttcgatttcgaagg
  • YZdCas-R caaaagctggagctccaccg

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Abstract

Provided is a method for simultaneously enhancing and inhibiting multiple key genes during synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae by means of a dCpf1-VP activation system and a dCas9-RD inhibition system. In order to achieve the aim of simultaneous expression and inhibition of 13 P450 enzymes, an engineered yeast for producing 7-dehydrocholesterol at a high yield is constructed, so that the 7-DHC yield reaches 464 mg/L.

Description

一种同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法A method to simultaneously enhance and inhibit multiple key genes in 7-dehydrocholesterol synthesis in Saccharomyces cerevisiae 技术领域Technical field
本发明属于生物工程技术领域，尤其是指一种同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法。The invention belongs to the field of bioengineering technology, and in particular refers to a method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae.
背景技术Background technique
维生素D3(Cholecalciferol,VD3)是人体中一种激素前体，具有能够调节细胞分化，促进骨质钙化等功能，VD3在人体的肝脏中被羟基化，形成25-羟基维生素D3(25-hydroxyvitamin D3，25-OH-VD3)，在肾脏中转化为1,25-二羟基维生素D3(1,25-dihydroxyvitamin D3，1,25-(OH)2-VD3。人体中的维生素D3主要来源是通过紫外线照射皮肤底层中的7-脱氢胆固醇(7-dehydrocholestero，7-DHC)转化而来的，同时也可以通过食物获取一部分，但由于每个人的饮食结构不同，通过饮食补充VD3效果不佳。我国居民VD3水平检测显示60％以上的居民缺乏VD3，并且VD3的巨大要用价值和广阔市场需求继续推动合成技术的改革和发展。7-脱氢胆固醇(7-dehydrocholestero，7-DHC)是合成VD3的重要前体物质，生产VD3的核心问题在于7-DHC的合成。Vitamin D3 (Cholecalciferol, VD3) is a hormone precursor in the human body that can regulate cell differentiation and promote bone calcification. VD3 is hydroxylated in the human liver to form 25-hydroxyvitamin D3 (25-hydroxyvitamin D3 , 25-OH-VD3), converted into 1,25-dihydroxyvitamin D3 (1,25-(OH)2-VD3) in the kidneys. The main source of vitamin D3 in the human body is through ultraviolet light It is converted from 7-dehydrocholestero (7-DHC) in the bottom layer of irradiated skin, and part of it can also be obtained through food. However, due to the different dietary structure of each person, dietary supplementation of VD3 is not effective. China Testing of resident VD3 levels shows that more than 60% of residents are deficient in VD3, and the huge value and broad market demand of VD3 continue to promote the reform and development of synthetic technology. 7-dehydrocholestero (7-DHC) is a synthetic VD3 An important precursor substance, the core issue in the production of VD3 lies in the synthesis of 7-DHC.
7-DHC的微生物代谢工程合成法大部分是基于含有天然甾醇合成路径的菌株所进行的，由于甾醇合成路径的代谢流占菌体总体代谢流的比例较低，主要集中在已知合成路径中基因的增强和分支路径的阻断，例如中国专利CN104988168A便通过增强天然甾醇合成的前体物质乙酰辅酶A的合成来提高7-DHC的产量，但也是通过传统的更换组成型强启动子，增强基因(acs、adh2、acl和ald6)的表达，但这样会造成菌体代谢压力过高。中国专利CN104988168A和中国专利CN103275997A通过直接敲除麦角甾醇合 成路径中的关键基因：erg5和erg6，进一步增强7-DHC的合成，但是完全阻断麦角甾醇的合成会对菌株的生长产生不可逆的影响，不利于工业化的大规模生产。Most of the microbial metabolic engineering synthesis methods of 7-DHC are based on strains containing natural sterol synthesis pathways. Since the metabolic flow of the sterol synthesis pathway accounts for a low proportion of the overall metabolic flow of the bacteria, it is mainly concentrated in known synthesis pathways. Gene enhancement and branch path blocking. For example, Chinese patent CN104988168A improves the production of 7-DHC by enhancing the synthesis of acetyl-CoA, a precursor for natural sterol synthesis, but it also enhances the production of 7-DHC by replacing the traditional strong constitutive promoter. Expression of genes (acs, adh2, acl and ald6), but this will cause excessive metabolic pressure on the bacteria. Chinese patent CN104988168A and Chinese patent CN103275997A further enhance the synthesis of 7-DHC by directly knocking out the key genes in the ergosterol synthesis pathway: erg5 and erg6, but completely blocking the synthesis of ergosterol will have an irreversible impact on the growth of the strain. Not conducive to industrialized mass production.
VD3的微生物合成主要集中于菌株的筛选方面，如中国专利CN103275997A、中国专利CN107075551B、中国专利CN104988168A。筛选的菌株大部分都是应用于VD3合成活性VD3，很少筛选直接合成VD3的菌株，并且菌株的筛选费时费力，还需要花费大量金钱进行大规模后期筛选与发酵检测，才能获得VD3产量极低的菌株。The microbial synthesis of VD3 mainly focuses on the screening of bacterial strains, such as Chinese patent CN103275997A, Chinese patent CN107075551B, and Chinese patent CN104988168A. Most of the strains screened are used to synthesize active VD3, and strains that directly synthesize VD3 are rarely screened. Moreover, the screening of strains is time-consuming and laborious, and a lot of money is needed for large-scale post-screening and fermentation testing to obtain extremely low VD3 yields. strains.
基因的调控工具无论是在酿酒酵母的基础研究还是工业应用中，都起着重要的作用。随着近几年CRISPR系统的研究发展，其被广泛的应用于各种生物的基因编辑与基因调控中，如中国专利CN112204147A，基于Cpf1的植物转录调控系统，使用Cpf1对植物进行转录激活与抑制，但是，使用单一的Cas蛋白进行激活与抑制时，会使系统的正交性降低，往往效果不明显。如中国专利CN110468153A，构建一种响应远红光的Cas9系统对基因转录进行调控，但是，其需要满足额外的因素，使用范围较窄。Gene regulation tools play an important role in both basic research and industrial applications of Saccharomyces cerevisiae. With the research and development of CRISPR systems in recent years, it has been widely used in gene editing and gene regulation of various organisms. For example, Chinese patent CN112204147A, a plant transcription regulation system based on Cpf1, uses Cpf1 to activate and inhibit transcription of plants. , however, when using a single Cas protein for activation and inhibition, the orthogonality of the system will be reduced, and the effect is often not obvious. For example, Chinese patent CN110468153A constructs a Cas9 system that responds to far-red light to regulate gene transcription. However, it needs to meet additional factors and has a narrow scope of use.
目前合成VD3的方法主要包括：化学法合成，微生物转化和微生物代谢合成三种方法。化学合成法需要以价格较贵的羊毛脂胆固醇为原料，经过多步基团保护和脱保护，获得7-脱氢胆固醇，再经过光照反应，开环和异构化获得VD3，由于使用多种有机试剂，产物的分离纯化过程复杂，产物收率较低。微生物转化法目前只用于将VD3转化为活性VD3，使用的微生物一般为链霉菌属、分枝杆菌属等微生物。随着合成生物学和基因工程的发展，微生物代谢工程法利用麦角甾醇生产菌株，进行7-DHC和VD3的生产具有巨大的潜力。The current methods for synthesizing VD3 mainly include three methods: chemical synthesis, microbial transformation and microbial metabolic synthesis. The chemical synthesis method needs to use the more expensive lanolin cholesterol as raw material. After multi-step group protection and deprotection, 7-dehydrocholesterol is obtained, and then through light reaction, ring opening and isomerization to obtain VD3. Due to the use of multiple For organic reagents, the product separation and purification process is complicated and the product yield is low. Microbial transformation method is currently only used to convert VD3 into active VD3, and the microorganisms used are generally Streptomyces, Mycobacterium and other microorganisms. With the development of synthetic biology and genetic engineering, microbial metabolic engineering methods using ergosterol-producing strains to produce 7-DHC and VD3 have great potential.
发明内容Contents of the invention
现有利用酿酒酵母进行7-DHC的生产技术利用的是酵母本身的甾醇合成路径，该路径包括13个P450酶，且在酵母中甾醇合成代谢比例不高，因此，如果只通过传统的增加拷贝数进行表达的增强，势必会导致酵母代谢紊 乱，产生其他过量副产物，加大了目标产物的纯化难度。若单纯的通过敲除erg5和erg6基因阻断麦角甾醇的合成，虽然会有一定的7-DHC积累，但完全阻断麦角甾醇的合成势必会对生产菌株的生长产生不良影响，进而影响7-DHC和VD3的产量。为解决上述技术问题，本发明提供一种同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法。The existing production technology of 7-DHC using Saccharomyces cerevisiae utilizes the yeast's own sterol synthesis pathway, which includes 13 P450 enzymes, and the proportion of sterol synthesis and metabolism in yeast is not high. Therefore, if only through traditional copy increase Enhancing expression at a high rate will inevitably lead to yeast metabolic disorders and the production of other excess by-products, making it more difficult to purify the target product. If the synthesis of ergosterol is simply blocked by knocking out the erg5 and erg6 genes, although there will be a certain accumulation of 7-DHC, completely blocking the synthesis of ergosterol will inevitably have an adverse effect on the growth of the production strain, and thus affect the 7-DHC. DHC and VD3 production. In order to solve the above technical problems, the present invention provides a method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae.
本发明的第一个目的在于提供一种同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，包括将酿酒酵母中乙醇脱氢酶(adh2)、截断的HMG-CoA还原酶(tHMG1)、异戊烯二磷酸异构酶(idi1)、角鲨烯环氧酶(erg1)、羊毛甾醇14-α-脱甲基酶(erg11)、C-14甾醇还原酶(erg24)、C-4甲基甾醇氧化酶(erg25)、C-3甾醇脱氢酶(erg26)、3-酮甾醇还原酶(erg27)以及外源基因甾醇△24-还原酶(dhcr24)进行同时增强表达，和/或对苹果酸合酶(mls1)、柠檬酸合酶(cit2)、Delta(24)-甾醇C-甲基转移酶(erg6)进行同时抑制。The first object of the present invention is to provide a method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae, including reducing alcohol dehydrogenase (adh2) and truncated HMG-CoA in Saccharomyces cerevisiae Enzyme (tHMG1), isopentenyl diphosphate isomerase (idi1), squalene epoxidase (erg1), lanosterol 14-α-demethylase (erg11), C-14 sterol reductase (erg24) , C-4 methylsterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27) and exogenous gene sterol Δ24-reductase (dhcr24) to simultaneously enhance expression , and/or simultaneously inhibit malate synthase (mls1), citrate synthase (cit2), and Delta(24)-sterol C-methyltransferase (erg6).
在本发明的一个实施方式中，所述乙醇脱氢酶(adh2)的编号为ID:NM_001182812.1、截断的HMG-CoA还原酶(tHMG1)的编号为ID:XM_033912352.1、异戊烯二磷酸异构酶(idi1)的编号为ID:NM_001183931.1、角鲨烯环氧酶(erg1)的编号为ID:NM_001181304.1、羊毛甾醇14-α-脱甲基酶(erg11)的编号为ID:NM_001179137.1、C-14甾醇还原酶(erg24)的编号为ID:NM_001183118.1、C-4甲基甾醇氧化酶(erg25)的编号为ID:NM_001181189.3、C-3甾醇脱氢酶(erg26)的编号为ID:NM_001180866.1、3-酮甾醇还原酶(erg27)的编号为ID:NM_001181987.1，以及外源基因甾醇△24-还原酶(dhcr24)的编号为ID:NM_001031288.1。In one embodiment of the invention, the number of the alcohol dehydrogenase (adh2) is ID: NM_001182812.1, the number of the truncated HMG-CoA reductase (tHMG1) is ID: The number of phosphate isomerase (idi1) is ID: NM_001183931.1, the number of squalene epoxidase (erg1) is ID: NM_001181304.1, and the number of lanosterol 14-α-demethylase (erg11) is ID: NM_001179137.1, the number of C-14 sterol reductase (erg24) is ID: NM_001183118.1, the number of C-4 methylsterol oxidase (erg25) is ID: NM_001181189.3, C-3 sterol dehydrogenation The number of the enzyme (erg26) is ID: NM_001180866.1, the number of 3-ketosterol reductase (erg27) is ID: NM_001181987.1, and the number of the exogenous gene sterol Δ24-reductase (dhcr24) is ID: NM_001031288 .1.
在本发明的一个实施方式中，所使用来源于鸡的外源基因dhcr24进行了酿酒酵母密码子偏好性优化，优化方法为：将来源于鸡的dhcr24基因输入网站 https://www.genscript.com/tools/gensmart-codon-optimization，进行针对酿酒酵母密码子偏好性优化，优化后进行基因的合成。 In one embodiment of the present invention, the exogenous gene dhcr24 derived from chicken is used to optimize the codon preference of Saccharomyces cerevisiae. The optimization method is: input the dhcr24 gene derived from chicken into the website https://www.genscript. com/tools/gensmart-codon-optimization , optimize the codon preference of Saccharomyces cerevisiae, and then synthesize the gene after optimization.
在本发明的一个实施方式中，所述苹果酸合酶(mls1)的编号为ID: NM_001182955.1、柠檬酸合酶(cit2)的编号为ID:NM_001178718.1、Delta(24)-甾醇C-甲基转移酶(erg6)的编号为ID:NM_001182363.1。In one embodiment of the invention, the number of malate synthase (mls1) is ID: NM_001182955.1, the number of citrate synthase (cit2) is ID: NM_001178718.1, Delta (24)-sterol C -The number of methyltransferase (erg6) is ID: NM_001182363.1.
在本发明的一个实施方式中，所述酿酒酵母为酿酒酵母S288C。In one embodiment of the invention, the Saccharomyces cerevisiae is Saccharomyces cerevisiae S288C.
在本发明的一个实施方式中，所述乙醇脱氢酶(adh2)、截断的HMG-CoA还原酶(tHMG1)、异戊烯二磷酸异构酶(idi1)、角鲨烯环氧酶(erg1)、羊毛甾醇14-α-脱甲基酶(erg11)、C-14甾醇还原酶(erg24)、C-4甲基甾醇氧化酶(erg25)、C-3甾醇脱氢酶(erg26)、3-酮甾醇还原酶(erg27)以及外源基因甾醇△24-还原酶(dhcr24)通过使用诱导型启动子gal1p和gal7p进行表达。In one embodiment of the invention, the alcohol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isomerase (idi1), squalene epoxidase (erg1 ), lanosterol 14-α-demethylase (erg11), C-14 sterol reductase (erg24), C-4 methylsterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3 -Ketosterol reductase (erg27) and exogenous gene sterol Δ24-reductase (dhcr24) are expressed by using inducible promoters gal1p and gal7p.
在本发明的一个实施方式中，所述诱导型启动子gal1p和gal7p的强度是使用dCpf1-VP激活系统进行增强，启动子gal1p的强度增加85％，启动子gal7p增加69％。In one embodiment of the invention, the strengths of the inducible promoters gal1p and gal7p are enhanced using the dCpf1-VP activation system, with the strength of promoter gal1p increased by 85% and promoter gal7p increased by 69%.
在本发明的一个实施方式中，所述dCpf1-VP激活系统发挥功能的基因片段为DPP1-KAN-dCpf1-VP-crRNA1-7-1，所述DPP1-KAN-dCpf1-VP-crRNA1-7-1的序列见SEQ ID NO 1。In one embodiment of the invention, the gene fragment that functions as the dCpf1-VP activation system is DPP1-KAN-dCpf1-VP-crRNA1-7-1, and the DPP1-KAN-dCpf1-VP-crRNA1-7- The sequence of 1 is shown in SEQ ID NO 1.
在本发明的一个实施方式中，所述苹果酸合酶(mls1)、柠檬酸合酶(cit2)、Delta(24)-甾醇C-甲基转移酶(erg6)通过dCas9-RD抑制系统进行抑制，erg6、mls1和cit2三个基因分别被抑制了88％、84％和72％。In one embodiment of the invention, the malate synthase (mls1), citrate synthase (cit2), and Delta(24)-sterol C-methyltransferase (erg6) are inhibited by the dCas9-RD inhibition system , the three genes erg6, mls1 and cit2 were inhibited by 88%, 84% and 72% respectively.
在本发明的一个实施方式中，所述dCas9-RD抑制系统发挥功能的基因片段为GAL80-KAN-dCas9-RD-sgRNAEMC，所述GAL80-KAN-dCas9-RD-sgRNAEMC的序列见SEQ ID NO 2。In one embodiment of the invention, the gene fragment that functions as the dCas9-RD inhibition system is GAL80-KAN-dCas9-RD-sgRNAEMC, and the sequence of GAL80-KAN-dCas9-RD-sgRNAEMC is shown in SEQ ID NO 2 .
本发明的第二个目的在于提供一种工程酵母菌，所述工程酵母菌通过将酿酒酵母中乙醇脱氢酶(adh2)、截断的HMG-CoA还原酶(tHMG1)、异戊烯二磷酸异构酶(idi1)、角鲨烯环氧酶(erg1)、羊毛甾醇14-α-脱甲基酶(erg11)、C-14甾醇还原酶(erg24)、C-4甲基甾醇氧化酶(erg25)、C-3甾醇脱氢酶(erg26)、3-酮甾醇还原酶(erg27)以及外源基因甾醇△24-还原酶(dhcr24)进行同时增强表达，和/或对苹果酸合酶(mls1)、柠檬酸合 酶(cit2)、Delta(24)-甾醇C-甲基转移酶(erg6)进行同时抑制所得。The second object of the present invention is to provide an engineered yeast that combines alcohol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isoform from Saccharomyces cerevisiae. Constructase (idi1), squalene epoxidase (erg1), lanosterol 14-α-demethylase (erg11), C-14 sterol reductase (erg24), C-4 methylsterol oxidase (erg25 ), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27) and exogenous gene sterol Δ24-reductase (dhcr24) to simultaneously enhance expression, and/or malate synthase (mls1 ), citrate synthase (cit2), and Delta(24)-sterol C-methyltransferase (erg6) were simultaneously inhibited.
本发明的第三个目的在于提供工程酵母菌在合成7-脱氢胆固醇中的应用。The third object of the present invention is to provide the application of engineered yeast in the synthesis of 7-dehydrocholesterol.
在本发明的一个实施例中，将所述工程酵母菌的种子液在YPD培养基中，在30℃、180-220rpm培养16-20h后，摇瓶发酵。按接种量1-5％接入含有YPD液体培养基的圆底摇瓶内培养发酵。In one embodiment of the present invention, the seed liquid of the engineered yeast is cultured in YPD medium at 30° C. and 180-220 rpm for 16-20 hours, and then fermented in a shake flask. Inoculate 1-5% of the inoculum into a round-bottom shake flask containing YPD liquid medium for culture and fermentation.
在本发明的一个实施例中，培养发酵的条件为：30℃，180-220rpm培养96-120h。In one embodiment of the present invention, the conditions for culture and fermentation are: 30°C, 180-220 rpm for 96-120 hours.
在本发明的一个实施例中，发酵至26h时，加入50-60％(体积分数)乙醇进行碳源的补充。发酵结束后，离心取沉淀。In one embodiment of the present invention, when fermentation reaches 26 hours, 50-60% (volume fraction) ethanol is added to supplement the carbon source. After the fermentation is completed, centrifuge to remove the precipitate.
在本发明的一个实施例中，所述碳源包括葡萄糖、蔗糖、甘油、乙醇。In one embodiment of the invention, the carbon source includes glucose, sucrose, glycerol, and ethanol.
本发明的上述技术方案相比现有技术具有以下优点：The above technical solution of the present invention has the following advantages compared with the existing technology:
本发明以原始的酿酒酵母S288C为出发菌株，将dCpf1与VP(激活域)进行融合，构建以dCpf1为基础的激活系统。将酵母7-DHC合成路径中的乙醇脱氢酶(adh2)、截断的HMG-CoA还原酶(tHMG1)、异戊烯二磷酸异构酶(idi1)、角鲨烯环氧酶(erg1)、羊毛甾醇14-α-脱甲基酶(erg11)、C-14甾醇还原酶(erg24)、C-4甲基甾醇氧化酶(erg25)、C-3甾醇脱氢酶(erg26)、3-酮甾醇还原酶(erg27)以及外源基因甾醇△24-还原酶(dhcr24)分别使用诱导型启动子gal1p和gal7p进行表达，并使用dCpf1-VP激活系统对启动子gal1p和gal7p的强度进行增强，达到提高7-DHC产量的目的，产量为371.5mg/L。将dCas9依次与抑制域UME6、MIG1、TUP1串联融合，构建dCas9为基础的抑制系统，设计该系统识别的sgRNA，对苹果酸合酶(mls1)、柠檬酸合酶(cit2)、Delta(24)-甾醇C-甲基转移酶(erg6)进行抑制，进一步提升7-DHC的产量，使得7-DHC的产量达到464mg/L。The present invention uses the original Saccharomyces cerevisiae S288C as the starting strain, fuses dCpf1 with VP (activation domain), and constructs an activation system based on dCpf1. The alcohol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isomerase (idi1), squalene epoxidase (erg1), and Lanosterol 14-α-demethylase (erg11), C-14 sterol reductase (erg24), C-4 methylsterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-keto Sterol reductase (erg27) and exogenous gene sterol Δ24-reductase (dhcr24) are expressed using inducible promoters gal1p and gal7p respectively, and the dCpf1-VP activation system is used to enhance the strength of promoter gal1p and gal7p to achieve The purpose of increasing the production of 7-DHC is 371.5mg/L. dCas9 was serially fused with the inhibition domains UME6, MIG1, and TUP1 to construct a dCas9-based inhibition system. The sgRNA recognized by the system was designed to inhibit malate synthase (mls1), citrate synthase (cit2), and Delta (24). -Sterol C-methyltransferase (erg6) is inhibited to further increase the production of 7-DHC, making the production of 7-DHC reach 464mg/L.
附图说明Description of the drawings
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施 例并结合附图，对本发明作进一步详细的说明，其中In order to make the content of the present invention easier to understand clearly, the present invention will be further described in detail below based on specific embodiments of the present invention and in conjunction with the accompanying drawings, wherein
图1是本发明dCpf1-VP激活系统的构建示意图。Figure 1 is a schematic diagram of the construction of the dCpf1-VP activation system of the present invention.
图2是本发明dCas9-RD抑制系统的构建示意图。Figure 2 is a schematic diagram of the construction of the dCas9-RD inhibition system of the present invention.
图3是使用dCpf1-VP增强启动子gal1p和gal7p的强度图。Figure 3 is a graph showing the intensity of promoters gal1p and gal7p enhanced using dCpf1-VP.
图4是使用dCas9-RD抑制效果图。Figure 4 is a diagram of the inhibition effect using dCas9-RD.
图5是本发明所得菌株工程酵母菌SX-6和工程酵母菌SX-7的7-DHC的产量图。Figure 5 is a graph showing the production of 7-DHC of the strains engineered yeast SX-6 and engineered yeast SX-7 obtained in the present invention.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.
本发明对7-DHC的检测使用的仪器为高效液相色谱，将发酵96h以上的工程酵母菌液进行高速离心，弃掉上清，使用无菌水进行重悬，加入0.5mm的玻璃珠，使用高速匀浆破碎仪破碎10min，取出破碎后的混合液，加入2％抗坏血酸和1％HBT，混匀，依次加入10％无水乙醇，2％的1.5mol/L的氢氧化钾甲醇溶液，在80℃的水域中皂化2小时，皂化结束后使用萃取液(异丙醇：正己烷＝1:3或石油醚或甲醇)进行超声震荡30分钟，使用分液漏斗除去下层杂质后，将萃取混合液进行冷冻干燥处理，处理结束后使用甲醇或乙腈或甲醇和乙腈的混合物复溶，0.55μm过滤后进行高效液相色谱分析。流动相使用甲醇和水，比例为90:10或者95:5或者98:2。检测器使用紫外检测器，检测波长为265-280nm。The instrument used to detect 7-DHC in the present invention is high-performance liquid chromatography. The engineered yeast liquid fermented for more than 96 hours is centrifuged at high speed, the supernatant is discarded, resuspended in sterile water, and 0.5mm glass beads are added. Use a high-speed homogenizer to crush for 10 minutes. Take out the crushed mixture, add 2% ascorbic acid and 1% HBT, mix well, and add 10% absolute ethanol and 2% 1.5mol/L potassium hydroxide methanol solution in sequence. Saponify in water at 80°C for 2 hours. After the saponification is completed, use the extraction solution (isopropyl alcohol: n-hexane = 1:3 or petroleum ether or methanol) for 30 minutes to conduct ultrasonic vibration. Use a separatory funnel to remove impurities in the lower layer, and then extract The mixture is freeze-dried. After the treatment, it is reconstituted with methanol or acetonitrile or a mixture of methanol and acetonitrile. It is filtered with 0.55 μm and analyzed by high performance liquid chromatography. The mobile phase uses methanol and water in a ratio of 90:10 or 95:5 or 98:2. The detector uses a UV detector with a detection wavelength of 265-280nm.
为了区别于传统的通过增加基因拷贝数的方法，设计基于CRISPR多基因激活与抑制系统，对酿酒酵母7-DHC的合成路径关键基因进行激活与抑制，在尽可能不影响菌株生长状态的同时，提升7-DHC的产量，最终利用该系统对关键基因进行了激活与抑制，并且效果明显，在菌株生长状态正常，并且7-DHC的产量为464mg/L。In order to be different from the traditional method of increasing gene copy number, a multi-gene activation and inhibition system based on CRISPR was designed to activate and inhibit the key genes of the 7-DHC synthesis pathway of Saccharomyces cerevisiae without affecting the growth status of the strain as much as possible. To increase the production of 7-DHC, this system was finally used to activate and inhibit key genes, and the effect was obvious. The strain grew normally, and the production of 7-DHC was 464mg/L.
为了实现本发明的发明目的，本发明采用如下技术方案：In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions:
本发明提供一种基于CRISPR技术的多个基因激活与抑制系统的构建方法；The present invention provides a method for constructing multiple gene activation and inhibition systems based on CRISPR technology;
本发明利用构建的上述系统对酿酒酵母7-DHC的合成路径进行调控；The present invention utilizes the above constructed system to regulate the synthesis pathway of 7-DHC in Saccharomyces cerevisiae;
其中使用上述系统进行增强的基因有：乙醇脱氢酶(adh2)、截断的HMG-CoA还原酶(tHMG1)、异戊烯二磷酸异构酶(idi1)、角鲨烯环氧酶(erg1)、羊毛甾醇14-α-脱甲基酶(erg11)、C-14甾醇还原酶(erg24)、C-4甲基甾醇氧化酶(erg25)、C-3甾醇脱氢酶(erg26)、3-酮甾醇还原酶(erg27)、甾醇△24-还原酶(dhcr24)；Among the genes enhanced using the above system are: alcohol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isomerase (idi1), squalene epoxygenase (erg1) , Lanosterol 14-α-demethylase (erg11), C-14 sterol reductase (erg24), C-4 methylsterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3- Ketosterol reductase (erg27), sterol Δ24-reductase (dhcr24);
其中使用上述系统进行抑制的基因有：苹果酸合酶(mls1)、柠檬酸合酶(cit2)、Delta(24)-甾醇C-甲基转移酶(erg6)。Among the genes inhibited using the above system are: malate synthase (mls1), citrate synthase (cit2), and Delta(24)-sterol C-methyltransferase (erg6).
本发明的还提供工程酵母菌在合成7-脱氢胆固醇生产中的应用。The present invention also provides the application of engineered yeast in the synthesis and production of 7-dehydrocholesterol.
在本发明的一个实施例中，将所述工程酵母菌的种子液在YPD培养基中，在30℃、180-220rpm培养16-20h后，摇瓶发酵。按接种量1-5％接入含有YPD液体培养基的圆底摇瓶内培养发酵。In one embodiment of the present invention, the seed liquid of the engineered yeast is cultured in YPD medium at 30° C. and 180-220 rpm for 16-20 hours, and then fermented in a shake flask. Inoculate 1-5% of the inoculum into a round-bottom shake flask containing YPD liquid medium for culture and fermentation.
在本发明的一个实施例中，培养发酵的条件为：30℃，180-220rpm培养96-120h。In one embodiment of the present invention, the conditions for culture and fermentation are: 30°C, 180-220 rpm for 96-120 hours.
在本发明的一个实施例中，发酵至26h时，加入50-60％(体积分数)乙醇作为碳源进行补充。发酵结束后，离心取沉淀。In one embodiment of the present invention, when fermentation reaches 26 hours, 50-60% (volume fraction) ethanol is added as a carbon source for supplementation. After the fermentation is completed, centrifuge to remove the precipitate.
在本发明的一个实施例中，所述碳源包括葡萄糖、蔗糖、甘油、乙醇。In one embodiment of the invention, the carbon source includes glucose, sucrose, glycerol, and ethanol.
实施例1构建产7-DHC的酿酒酵母菌及强化路径中相关基因Example 1 Construction of 7-DHC-producing Saccharomyces cerevisiae and related genes in the enhancement pathway
(a)以酿酒酵母工程菌S288C基因组为模板，采用引物ADH-F，ADH-R扩增得到基因片段ADH2，采用引物IDI-F，IDI-R扩增得到基因片段IDI1，采用引物E1-F、E1-R扩增得到基因片段ERG1，采用引物E11-F、E11-R扩增得到基因片段ERG11，采用引物E24-F、E24-R扩增得到基因片段ERG24，采用引物E25-F、E25-R扩增得到基因片段ERG25，采用引物E26-F、E26-R 扩增得到基因片段ERG26，采用引物E27-F、E27-R扩增得到基因片段ERG27，采用引物GAL1-F、GAL1-R扩增得到启动子基因片段gal1p，采用引物GAL7-F、GAL7-R扩增得到启动子基因片段gal7p。通过基因合成，获得片段dhcr24和thmg1。采用引物208UP-F、208UP-R扩增得到上游同源臂片段208UP，采用引物208Down-F、208Down-R扩增得到下游同源臂片段208Down，采用引物1622UP-F、1622UP-R扩增得到上游同源臂片段1622UP，采用引物1622Down-F、1622Down-R扩增得到下游同源臂片段1622Down，采用引物308UP-F、308UP-R扩增得到上游同源臂片段308UP，采用引物308Down-F、308Down-R扩增得到下游同源臂片段308Down，采用引物1021UP-F、1021UP-R扩增得到上游同源臂片段1021UP，采用引物1021Down-F、1021Down-R扩增得到下游同源臂片段1021Down，采用引物911UP-F、911UP-R扩增得到上游同源臂片段911UP，采用引物911Down-F、911Down-R扩增得到下游同源臂片段911Down。(a) Using the Saccharomyces cerevisiae engineering strain S288C genome as a template, using primers ADH-F and ADH-R to amplify the gene fragment ADH2, using primers IDI-F and IDI-R to amplify the gene fragment IDI1, and using primers E1-F , E1-R was amplified to obtain gene fragment ERG1, primers E11-F and E11-R were used to amplify gene fragment ERG11, primers E24-F and E24-R were used to amplify gene fragment ERG24, and primers E25-F and E25 were used to amplify gene fragment ERG11. -R amplified gene fragment ERG25, using primers E26-F and E26-R to amplify gene fragment ERG26, using primers E27-F and E27-R to amplify gene fragment ERG27, using primers GAL1-F and GAL1-R The promoter gene fragment gal1p was amplified, and the promoter gene fragment gal7p was amplified using primers GAL7-F and GAL7-R. Through gene synthesis, fragments dhcr24 and thmg1 were obtained. The upstream homology arm fragment 208UP was amplified using primers 208UP-F and 208UP-R, the downstream homology arm fragment 208Down was amplified using primers 208Down-F and 208Down-R, and the downstream homology arm fragment 208Down was amplified using primers 1622UP-F and 1622UP-R. The upstream homology arm fragment 1622UP was amplified using primers 1622Down-F and 1622Down-R to obtain the downstream homology arm fragment 1622Down. The upstream homology arm fragment 308UP was amplified using primers 308UP-F and 308UP-R. The primer 308Down-F was used to amplify the upstream homology arm fragment 1622UP. , 308Down-R amplifies the downstream homology arm fragment 308Down, uses primers 1021UP-F and 1021UP-R to amplify the upstream homology arm fragment 1021UP, uses primers 1021Down-F and 1021Down-R to amplify the downstream homology arm fragment 1021Down, using primers 911UP-F and 911UP-R to amplify the upstream homology arm fragment 911UP, and using primers 911Down-F and 911Down-R to amplify the downstream homology arm fragment 911Down.
(b)将上述得到的启动子片段gal1p和gal7p分别与步骤(a)获得的基因片段进行重叠PCR，获得基因片段208-gal1p-dhcr24-gal7p-adh2、1622-gal1p-thmg1-gal7p-idi1、308-gal1p-erg1-gal7p-erg11、1021-gal1p-erg24-gal7p-erg25、911-gal1p-erg26-gal7p-erg27。(b) Perform overlapping PCR between the promoter fragments gal1p and gal7p obtained above and the gene fragment obtained in step (a) to obtain gene fragments 208-gal1p-dhcr24-gal7p-adh2, 1622-gal1p-thmg1-gal7p-idi1, 308-gal1p-erg1-gal7p-erg11, 1021-gal1p-erg24-gal7p-erg25, 911-gal1p-erg26-gal7p-erg27.
(c)以pML104质粒为模板，分别使用引物208-F、208-R进行质粒环P，得到能识别片段208-gal1p-dch24-gal7p-adh2插入位点的质粒，命名为pML104-1，以引物1622-F、1622-R进行质粒环P，得到能识别片段1622-gal1p-thmg1-gal7p-idi1插入位点的质粒，命名为pML104-2，以引物308-F、308-R进行质粒环P，得到能识别片段308-gal1p-erg1-gal7p-erg11插入位点的质粒，命名为pML104-3，以引物1021-F、1021-R进行质粒环P，得到能识别片段1021-gal1p-erg24-gal7p-erg25插入位点的质粒，命名为pML104-4，以引物911-F、911-R进行质粒环P，得到能识别片段911-gal1p-erg26-gal7p-erg27插入位点的质粒，命名为pML104-5。(c) Using the pML104 plasmid as a template, use primers 208-F and 208-R respectively to perform plasmid loop P to obtain a plasmid that can recognize the insertion site of the fragment 208-gal1p-dch24-gal7p-adh2, named pML104-1. Use primers 1622-F and 1622-R to perform plasmid loop P, and obtain a plasmid that can recognize the fragment 1622-gal1p-thmg1-gal7p-idi1 insertion site, named pML104-2. Use primers 308-F and 308-R to perform plasmid loop P. P, a plasmid capable of recognizing the insertion site of the fragment 308-gal1p-erg1-gal7p-erg11 was obtained, named pML104-3. Use primers 1021-F and 1021-R to perform plasmid ring P, and obtain a plasmid capable of recognizing the fragment 1021-gal1p-erg24. The plasmid of -gal7p-erg25 insertion site is named pML104-4. Use primers 911-F and 911-R to perform plasmid ring P to obtain a plasmid that can recognize the insertion site of fragment 911-gal1p-erg26-gal7p-erg27, which is named is pML104-5.
(d)将步骤(b)和(c)获得的片段和质粒分别倒入酿酒酵母中，经过测序验证后，将正确的单菌落在5-FOA的YPD平板上划线，30℃培养2-3天，分别得到菌株命名为SX-1、SX-2、SX-3、SX-4、SX-5。(d) Pour the fragments and plasmids obtained in steps (b) and (c) into Saccharomyces cerevisiae respectively. After sequencing verification, streak the correct single colony on a 5-FOA YPD plate and culture it at 30°C for 2- After 3 days, strains were obtained and named SX-1, SX-2, SX-3, SX-4, and SX-5.
引物序列：Primer sequence:
ADH-F：aaaacagttgaatattccctcaaaaatgatgtctattccagaaactcaaaaagccADH-F: aaaacagttgaatattccctcaaaaatgatgtctattccagaaactcaaaaagcc
ADH-R：aagatattcagtttttgtcattgccgtcttatttagaagtgtcaacaacgtatctADH-R: aagatattcagtttttgtcattgccgtcttatttagaagtgtcaacaacgtatct
IDI-F：gttgaatattccctcaaaaatgatgactgccgacaacaatagtatgIDI-F: gttgaatattccctcaaaaatgatgactgccgacaacaatagtatg
IDI-R：ttacttcttcaattcgtacattatattatagcattctatgaatttgIDI-R: ttacttcttcaattcgtacattatattatagcattctatgaatttg
E1-F：atacctctatactttaacgtcaaggagatgtctgctgttaacgttgcaccE1-F: atacctctatactttaacgtcaaggagatgtctgctgttaacgttgcacc
E1-R：gtccatatctttccatagatttttaaccaatcaactcaccaaacaaaaatgggE1-R: gtccatatctttccatagatttttaaccaatcaactcaccaaacaaaaatggg
E11-F：acagttgaatattccctcaaaaatgatgtctgctaccaagtcaatcgttgE11-F：acagttgaatattccctcaaaaatgatgtctgctaccaagtcaatcgttg
E11-R：ctattgtgtaatagaagttagatcttttgttctggatttctcttttcccaE11-R：ctattgtgtaatagaagttagatcttttgttctggatttctcttttccca
E24-F：atactttaacgtcaaggagatggtatcagctttgaatcccagE24-F：atactttaacgtcaaggagatggtatcagctttgaatcccag
E24-R：ccgtccatatctttccatagatttttaataaacatatggaatgatcttgtaaggaE24-R：ccgtccatatctttccatagatttttaataaacatatggaatgatcttgtaagga
E25-F：aatattccctcaaaaatgatgtctgccgttttcaacaacgE25-F：aatattccctcaaaaatgatgtctgccgttttcaacaacg
E25-R：tatttcacagaatgaatttcttagttagtcttcttttgagcattgttttcagcE25-R：tatttcacagaatgaatttcttagttagtcttcttttgagcattgttttcagc
E26-F：ttaacgtcaaggagatgtcaaagatagattcagttttaattatcggtggttE26-F：ttaacgtcaaggagatgtcaaagatagattcagttttaattatcggtggtt
E26-R：tccatatctttccatagatttttacaaaccttcgtccatccaggcE26-R：tccatatctttccatagatttttacaaaccttcgtccatccaggc
E27-F：gaatattccctcaaaaatgatgaacaggaaagtagctatcgtaacggE27-F：gaatattccctcaaaaatgatgaacaggaaagtagctatcgtaacgg
E27-R：atttctgttttaaatttaaatgggggttctagtttcaacaatttgE27-R：atttctgttttaaatttaaatgggggttctagtttcaacaatttg
GAL1-F：cggattagaagccgccgagcgggcGAL1-F: cggattagaagccgccgagcgggc
GAL1-R：ctccttgacgttaaagtatagaggtataGAL1-R:ctccttgacgttaaagtatagaggtata
GAL7-F：aaatctatggaaagatatggacggGAL7-F: aaatctatggaaagatatggacgg
GAL7-R：catttttgagggaatattcaactgGAL7-R:catttttgagggaatattcaactg
208UP-F：tggtgaattggctaaacatgccgtc208UP-F：tggtgaattggctaaacatgccgtc
208UP-R：cccgctcggcggcttctaatccggactctgctagtatttctgatt208UP-R: cccgctcggcggcttctaatccggactctgctagtatttctgatt
208Down-F：gacggcaatgacaaaaactgaatat208Down-F：gacggcaatgacaaaaactgaatat
208Down-R：agacacttgtatcctatactatca208Down-R：agacacttgtatcctatactatca
1622UP-F：ctaaatgtgttgctagtattattt1622UP-F：ctaaatgtgttgctagtattattt
1622UP-R：cgctcggcggcttctaatccgtgtttttgctgttaccttctgcac1622UP-R：cgctcggcggcttctaatccgtgtttttgctgttaccttctgcac
1622Down-F：ttcatagaatgctataatataatgtacgaattgaagaagtaaatt1622Down-F：ttcatagaatgctataatataatgtacgaattgaagaagtaaatt
1622Down-R：gtcctctttttaacgcgtctactaa1622Down-R：gtcctctttttaacgcgtctactaa
308UP-F：gatatatgcagagaaggagcaaat308UP-F：gatatatgcagagaaggagcaaat
308UP-R：ccgctcggcggcttctaatccgatgttgaaatttcacttatttta308UP-R：ccgctcggcggcttctaatccgatgttgaaatttcacttatttta
308Down-F：agaacaaaagatctaacttctattacacaatagtttcaatag308Down-F：agaacaaaagatctaacttctattacacaatagtttcaatag
308Down-R：tgaattttaattctacgtcaacga308Down-R：tgaattttaattctacgtcaacga
1021UP-F：gagtccgcgtttgaaattgcagtt1021UP-F:gagtccgcgtttgaaattgcagtt
1021UP-R：gtcgcccgctcggcggcttctaatccgacactggaaaatttgagtcatgg1021UP-R:gtcgcccgctcggcggcttctaatccgacactggaaaatttgagtcatgg
1021Down-F：ctcaaaagaagactaactaagaaattcattctgtgaaatatcacg1021Down-F:ctcaaaagaagactaactaagaaattcattctgtgaaatatcacg
1021Down-R：ttgtttatttccagatttctatttt1021Down-R:ttgtttatttccagatttctatttt
911UP-F：agtttattttcaaactgtattttga911UP-F:agtttattttcaaactgtattttga
911UP-R：tgtcgcccgctcggcggcttctaatccgtcaacatccgatttttttctataaaat911UP-R: tgtcgcccgctcggcggcttctaatccgtcaacatccgatttttttctataaaat
911Down-F：ttgttgaaactagaacccccatttaaatttaaaacagaaatgaaatgagc911Down-F:ttgttgaaactagaacccccatttaaatttaaaacagaaatgaaatgagc
911Down-R：tattaataggaatagtaatcatagtac911Down-R:tattaataggaatagtaatcatagtac
208-F：tttctagctctaaaacagatcttttgtttagcggacgatcatttatctttcactgcggag208-F:tttctagctctaaaacagatcttttgtttagcggacgatcatttatctttcactgcggag
208-R：gtccgctaaacaaaagatctgttttagagctagaaatagcaagttaaaataaggctagt208-R: gtccgctaaacaaaagatctgttttagagctagaaatagcaagttaaaataaggctagt
1622-F：tttctagctctaaaactttgcgatgtggtggctttagatcatttatctttcactgcggag1622-F:tttctagctctaaaactttgcgatgtggtggctttagatcatttatctttcactgcggag
1622-R：taaagccaccacatcgcaaagttttagagctagaaatagcaagttaaaataaggctagtcc1622-R: taaagccaccacatcgcaaagttttagagctagaaatagcaagttaaaataaggctagtcc
308-F：tttctagctctaaaactatattctgtttgacaagtggatcatttatctttcactgcggag308-F:tttctagctctaaaactatattctgtttgacaagtggatcatttatctttcactgcggag
308-R：cacttgtcaaacagaatatagttttagagctagaaatagcaagttaaaataaggctagtcc308-R:cacttgtcaaacagaatatagttttagagctagaaatagcaagttaaaataaggctagtcc
1021-F：tttctagctctaaaaccaattaccaccacacagagggatcatttatctttcactgcggag1021-F:tttctagctctaaaaccaattaccaccacacagaggggatcatttatctttcactgcggag
1021-R：ctctgtgtggtggtaattggttttagagctagaaatagcaagttaaaataaggctagtcc1021-R:ctctgtgtggtggtaattggttttagagctagaaatagcaagttaaaataaggctagtcc
911-F：tttctagctctaaaacgggaaacaagacaatattacgatcatttatctttcactgcggag911-F:tttctagctctaaaacgggaaacaagacaatattacgatcatttatctttcactgcggag
911-R：gtaatattgtcttgtttcccgttttagagctagaaatagcaagttaaaataaggctagtcc911-R: gtaatattgtcttgtttcccgttttagagctagaaatagcaagttaaaataaggctagtcc
实施例2dCpf1-VP激活系统的构建与应用Example 2 Construction and application of dCpf1-VP activation system
(a)以购自于Addgene的质粒pCSN068为模板，采用引物dCpf1Tu-F、dCpf1Tu-R将Cpf1突变为dCpf1。基因合成激活结构域VP，使用无缝克隆将激活结构域VP与含有dCpf1的pCSN068进行连接，得到质粒pCSN068-VP。以购自Addgene的质粒pML104为模板，采用引物52p-F和52p-R扩增得到启动子基因片段SNR52p。直接采用引物gal1p23-F1和gal1p23-R1重叠合成片段gal1p23-1，直接采用引物gal7p23-F1和gal7p23-R1重叠合成片段gal7p23-1。直接采用引物gal1p23-F2和gal1p23-R2重叠合成片段gal1p23-2，直接采用引物gal7p23-F2和gal7p23-R2重叠合成片段gal7p23-2。直接采用引物gal1p23-F3和gal1p23-R3重叠合成片段gal1p23-3，直接采用引物gal7p23-F3和gal7p23-R3重叠合成片段gal7p23-3。以购自于Addgene的质粒pFA6a-TRP1-PGAL1-GFP为模板，采用引物pFA6a-F、pFA6a-R扩增得到片段pFA6a-TRP1-GFP，以酿酒酵母SC288C基因组为模板，采用引物gal7pG-F、gal7pG-R扩增得到启动子基因片段gal7p-G，使用无缝克隆将gal7p-G与pFA6a-TRP1-GFP连接，得到质粒pFA6a-TRP1-PGAL7-GFP。以酿酒酵母SC288C的基因组为模板，采用引物DPP1UP-F和DPP1UP-R扩增得到基因片段DPP1UP，采用引物DPP1Down-F和DPP1Down-R扩增得到基因片段DPP1Down。(a) Using plasmid pCSN068 purchased from Addgene as a template, primers dCpf1Tu-F and dCpf1Tu-R were used to mutate Cpf1 into dCpf1. Gene synthesize the activation domain VP, and use seamless cloning to connect the activation domain VP to pCSN068 containing dCpf1 to obtain plasmid pCSN068-VP. Using plasmid pML104 purchased from Addgene as a template, the promoter gene fragment SNR52p was amplified using primers 52p-F and 52p-R. The fragment gal1p23-1 was directly synthesized by overlapping primers gal1p23-F1 and gal1p23-R1, and the fragment gal7p23-1 was synthesized directly by overlapping primers gal7p23-F1 and gal7p23-R1. The fragment gal1p23-2 was directly synthesized by overlapping primers gal1p23-F2 and gal1p23-R2, and the fragment gal7p23-2 was synthesized directly by overlapping primers gal7p23-F2 and gal7p23-R2. The fragment gal1p23-3 was directly synthesized by overlapping primers gal1p23-F3 and gal1p23-R3, and the fragment gal7p23-3 was directly synthesized by overlapping primers gal7p23-F3 and gal7p23-R3. The plasmid pFA6a-TRP1-PGAL1-GFP purchased from Addgene was used as the template, and the primers pFA6a-F and pFA6a-R were used to amplify the fragment pFA6a-TRP1-GFP. The Saccharomyces cerevisiae SC288C genome was used as the template, and the primers gal7pG-F and The promoter gene fragment gal7p-G was amplified from gal7pG-R, and gal7p-G was connected to pFA6a-TRP1-GFP using seamless cloning to obtain plasmid pFA6a-TRP1-PGAL7-GFP. Using the genome of Saccharomyces cerevisiae SC288C as a template, the gene fragment DPP1UP was amplified using primers DPP1UP-F and DPP1UP-R, and the gene fragment DPP1Down was amplified using primers DPP1Down-F and DPP1Down-R.
(b)将步骤(a)中得到的片段SNR52p、gal1p23-1/gal1p23-2/gal1p23-3、与质粒pCSN068-VP进行无缝连接，得到质粒pCSN068-dCpf1-VP-crRNA1-1、pCSN068-dCpf1-VP-crRNA1-2、pCSN068-dCpf1-VP-crRNA1-3，以上述质粒为模板，采用引物CSN-VPG-F与CSN-VPG-R扩增得到基因片段KAN-dCpf1-VP-gal1p1、KAN-dCpf1-VP-gal1p2、KAN-dCpf1-VP-gal1p3，与片段DPP1UP、DPP1Down进行重叠PCR，获得片段 DPP1-KAN-dCpf1-VP-gal1p1、DPP1-KAN-dCpf1-VP-gal1p2、DPP1-KAN-dCpf1-VP-gal1p3，将上述三个片段分别与质粒pFA6a-TRP1-PGAL1-GFP同时导入酵母SC288C中，得到菌株SG1-1、SG1-2、SG1-3，分别使用酶标仪进行绿色荧光强度的检测，结果如图3，确定最佳结合位点。(b) Seamlessly connect the fragments SNR52p and gal1p23-1/gal1p23-2/gal1p23-3 obtained in step (a) with plasmid pCSN068-VP to obtain plasmids pCSN068-dCpf1-VP-crRNA1-1 and pCSN068- dCpf1-VP-crRNA1-2, pCSN068-dCpf1-VP-crRNA1-3, using the above plasmid as a template, using primers CSN-VPG-F and CSN-VPG-R to amplify the gene fragments KAN-dCpf1-VP-gal1p1, KAN-dCpf1-VP-gal1p2, KAN-dCpf1-VP-gal1p3, and the fragments DPP1UP and DPP1Down were subjected to overlapping PCR to obtain fragments DPP1-KAN-dCpf1-VP-gal1p1, DPP1-KAN-dCpf1-VP-gal1p2, and DPP1-KAN. -dCpf1-VP-gal1p3, the above three fragments were introduced into yeast SC288C simultaneously with the plasmid pFA6a-TRP1-PGAL1-GFP to obtain strains SG1-1, SG1-2, and SG1-3. Green fluorescence was measured using a microplate reader. Intensity detection, the results are shown in Figure 3, to determine the best binding site.
(c)将步骤(a)中得到的片段SNR52p、gal7p23-1/gal7p23-2/gal7p23-3、与质粒pCSN068-VP进行无缝连接，得到质粒pCSN068-dCpf1-VP-crRNA7-1、pCSN068-dCpf1-VP-crRNA7-2、pCSN068-dCpf1-VP-crRNA7-3，以该质粒为模板，采用引物CSN-VPG-F与CSN-VPG-R扩增得到基因片段KAN-dCpf1-VP-gal7p1、KAN-dCpf1-VP-gal7p2、KAN-dCpf1-VP-gal7p3，与片段DPP1UP、DPP1Down进行重叠PCR，获得片段DPP1-KAN-dCpf1-VP-gal7p1、DPP1-KAN-dCpf1-VP-gal7p2、DPP1-KAN-dCpf1-VP-gal7p3，将上述三个片段分别与质粒pFA6a-TRP1-PGAL7-GFP同时导入酵母SC288C中，得到菌株SG7-1、SG7-2、SG7-3，分别使用酶标仪进行绿色荧光强度的检测，结果如图3，确定最佳激活位点。(c) Seamlessly connect the fragments SNR52p and gal7p23-1/gal7p23-2/gal7p23-3 obtained in step (a) with plasmid pCSN068-VP to obtain plasmids pCSN068-dCpf1-VP-crRNA7-1 and pCSN068- dCpf1-VP-crRNA7-2, pCSN068-dCpf1-VP-crRNA7-3, using this plasmid as a template, using primers CSN-VPG-F and CSN-VPG-R to amplify the gene fragments KAN-dCpf1-VP-gal7p1, KAN-dCpf1-VP-gal7p2, KAN-dCpf1-VP-gal7p3, and the fragments DPP1UP and DPP1Down were subjected to overlapping PCR to obtain the fragments DPP1-KAN-dCpf1-VP-gal7p1, DPP1-KAN-dCpf1-VP-gal7p2, and DPP1-KAN. -dCpf1-VP-gal7p3, the above three fragments were introduced into yeast SC288C simultaneously with the plasmid pFA6a-TRP1-PGAL7-GFP to obtain strains SG7-1, SG7-2, and SG7-3. Green fluorescence was measured using a microplate reader. Intensity detection, the results are shown in Figure 3, to determine the best activation site.
(d)将步骤(b)和(c)中确定的最佳结合位点的pCSN068-dCpf1-VP-crRNA1-1和pCSN068-dCpf1-VP-crRNA7-1通过Eco31I酶切后，使用golden gate组装为质粒pCSN068-dCpf1-VP-crRNA1-7-1，采用引物crRNA1-7-F1和crRNA1-7-R1进行PCR得到片段Kan-dCpf1-VP-crRNA1-7-1，将其与获得的片段DPP1UP、DPP1Down进行重叠PCR，获得片段DPP1-KAN-dCpf1-VP-crRNA1-7-1(见SEQ ID NO 1)。(d) After digesting pCSN068-dCpf1-VP-crRNA1-1 and pCSN068-dCpf1-VP-crRNA7-1 at the best binding sites determined in steps (b) and (c) with Eco31I, assemble using golden gate For plasmid pCSN068-dCpf1-VP-crRNA1-7-1, use primers crRNA1-7-F1 and crRNA1-7-R1 to perform PCR to obtain the fragment Kan-dCpf1-VP-crRNA1-7-1, and combine it with the obtained fragment DPP1UP , DPP1Down was used to perform overlapping PCR, and the fragment DPP1-KAN-dCpf1-VP-crRNA1-7-1 was obtained (see SEQ ID NO 1).
DPP1-KAN-dCpf1-VP-crRNA1-7-1片段序列(见SEQ ID NO 1)：DPP1-KAN-dCpf1-VP-crRNA1-7-1 fragment sequence (see SEQ ID NO 1):
Figure PCTCN2022092329-appb-000001
Figure PCTCN2022092329-appb-000001
Figure PCTCN2022092329-appb-000002
Figure PCTCN2022092329-appb-000002
Figure PCTCN2022092329-appb-000003
Figure PCTCN2022092329-appb-000003
Figure PCTCN2022092329-appb-000004
Figure PCTCN2022092329-appb-000004
Figure PCTCN2022092329-appb-000005
Figure PCTCN2022092329-appb-000005
Figure PCTCN2022092329-appb-000006
Figure PCTCN2022092329-appb-000006
(e)将步骤(d)得到片段，导入工程菌SX-5的感受态中，通过G418平板进行筛选，使用验证引物YZdCpf1-F和YZdCpf1-R进行菌落PCR验证。验证正确的单菌落，将其在5-FOA的YPD平板进行划线，在30℃培养2-3d，筛选去掉G418标签的单菌落，命名为SX-6。(e) Introduce the fragment obtained in step (d) into the competent state of the engineering strain SX-5, screen it on the G418 plate, and use the verification primers YZdCpf1-F and YZdCpf1-R to perform colony PCR verification. Verify the correct single colony, streak it on a 5-FOA YPD plate, culture it at 30°C for 2-3 days, screen out the single colony with the G418 tag removed, and name it SX-6.
引物序列：Primer sequence:
dCpf1Tu-F:cgatgttcacatcttgtctatcgctcgtggtgaaagacacttggcttacdCpf1Tu-F:cgatgttcacatcttgtctatcgctcgtggtgaaagacacttggcttac
dCpf1Tu-R:gccaagtgtctttcaccacgagcgatagacaagatgtgaacatcgttagcctdCpf1Tu-R:gccaagtgtctttcaccacgagcgatagacaagatgtgaacatcgttagcct
gal1p23-F1:actggtctcaagatagtaatacgcttaactgctcattggtaagaaattgtctggal1p23-F1:actggtctcaagatagtaatacgcttaactgctcattggtaagaaattgtctg
gal1p23-R1:aatgagcagttaagcgtattactatcttgagaccagtgatcatttatctttcactgcggagaaggal1p23-R1:aatgagcagttaagcgtattactatcttgagaccagtgatcatttatctttcactgcggagaag
gal7p23-F1:actggtctcaagatgttacttttgatatcactcacaaggtaagaaattgtctggal7p23-F1:actggtctcaagatgttatacttttgatatcactcacaaggtaagaaattgtctg
gal7p23-R1:ttgtgagtgatatcaaaagtaacatcttgagaccagtgatcatttatctttcactgcggagaaggal7p23-R1:ttgtgagtgatatcaaaagtaacatcttgagaccagtgatcatttatctttcactgcggagaag
gal1p23-F2:actggtctcaagattggttatgaagaggaaaaattggggtaagaaattgtctggal1p23-F2: actggtctcaagattggttatgaagaggaaaaattggggtaagaaattgtctg
gal1p23-R2:ccaatttttcctcttcataaccaatcttgagaccagtgatcatttatctttcactgcggagaaggal1p23-R2:ccaatttttcctcttcataaccaatcttgagaccagtgatcatttatctttcactgcggagaag
gal7p23-F2:actggtctcaagatcttaacccaaaaataagggaaagggtaagaaattgtctggal7p23-F2:actggtctcaagatcttaacccaaaaataagggaaagggtaagaaattgtctg
gal7p23-R2:ctttcccttatttttgggttaagatcttgagaccagtgatcatttatctttcactgcggagaaggal7p23-R2:ctttcccttatttttgggttaagatcttgagaccagtgatcatttatctttcactgcggagaag
gal1p23-F3:actggtctcaagatatctattaacagatatataaatgggtaagaaattgtctggal1p23-F3:actggtctcaagatatctattaacagatatataaatgggtaagaaattgtctg
gal1p23-R3:catttatatatctgttaatagatatcttgagaccagtgatcatttatctttcactgcggagaaggal1p23-R3:catttatatatctgttaatagatatcttgagaccagtgatcatttatctttcactgcggagaag
gal7p23-F3:actggtctcaagatgctagcaaagatataaaagcaggggtaagaaattgtctggal7p23-F3:actggtctcaagatgctagcaaagatataaaagcaggggtaagaaattgtctg
gal7p23-R3:cctgcttttatatctttgctagcatcttgagaccagtgatcatttatctttcactgcggagaaggal7p23-R3:cctgcttttatatctttgctagcatcttgagaccagtgatcatttatctttcactgcggagaag
pFA6a-F:tatctttccatagattttacttcaatatagcaatgagcagttpFA6a-F:tatctttccatagattttacttcaatatagcaatgagcagtt
pFA6a-R:attccctcaaaaatgaaaaaacccggatctcaaapFA6a-R:attccctcaaaaatgaaaaaacccggatctcaaa
gal7pG-F:ttgctatattgaagtaaaatctatggaaagatatggacggtagal7pG-F:ttgctatattgaagtaaaatctatggaaagatatggacggta
gal7pG-R:gatccgggttttttcatttttgagggaatattcagal7pG-R:gatccgggttttttcatttttgagggaatattca
CSN-VP-F:atgtatgctatacgaacggtacccagtcacgacgttgtaaaacgCSN-VP-F:atgtatgctatacgaacggtacccagtcacgacgttgtaaaacg
CSN-VP-R:atcatacattatcttttcaaagattacagcaaggctgagaaatccatCSN-VP-R:atcatacattatcttttcaaagattacagcaaggctgagaaatccat
KAN1-F:tgtggtcaataagagcgacgagctccagcttttgttccctttagtKAN1-F:tgtggtcaataagagcgacgagctccagcttttgttccctttagt
KAN1-R:ttttttcttcttttacgtaggtacccaattcgccctatagtgagtKAN1-R:ttttttcttcttttacgtaggtacccaattcgccctatagtgagt
52p-F:ggatttctcagccttgctgtaatctttgaaaagataatgtatgat52p-F:ggatttctcagccttgctgtaatctttgaaaagataatgtatgat
52p-R:agagggcctatggtgaatcttgagaccagtgatcatttatctttcactgcggagaa52p-R:agagggcctatggtgaatcttgagaccagtgatcatttatctttcactgcggagaa
gal1p23-F:actggtctcaagatagtaatacgcttaagal1p23-F:actggtctcaagatagtaatacgcttaa
gal1p23-R:actggtctcaacctaatgagcagttaagcgtattactatctgal1p23-R:actggtctcaacctaatgagcagttaagcgtattactatct
gal7p23-F:actggtctcaaggtaatttctactgttgtagatgttacttttgatatgal7p23-F:actggtctcaaggtaatttctactgttgtagatgttatttttgatat
gal7p23-R:actggtctcaaattttgtgagtgatatcaaaagtaacatctacaacagtgal7p23-R:actggtctcaaattttgtgagtgatatcaaaagtaacatctacaacagt
DPP1UP-F:actagtactcgatttctggcgcDPP1UP-F:actagtactcgatttctggcgc
DPP1UP-R:aattcgtcgacctgcagcggtcgcataaggatgcgatatagtgaDPP1UP-R:aattcgtcgacctgcagcggtcgcataaggatgcgatatagtga
DPP1Down-F:cgctgcaggtcgacgaattcttgactgaatcaccgttgatgccDPP1Down-F:cgctgcaggtcgacgaattcttgactgaatcaccgttgatgcc
DPP1Down-R:gctgcttatcccagctagactttcDPP1Down-R:gctgcttatcccagctagactttc
CSN-VPG-F:CCCAGTCACGACGTTGTAAAACGCSN-VPG-F:CCCAGTCACGACGTTGTTAAAACG
CSN-VPG-R:GCAATTAACCCTCACTAAAGGYZdCpf1-F：agacaagctccaaagaacatgccaCSN-VPG-R:GCAATTAACCCTCACTAAAGGYZdCpf1-F:agacaagctccaaagaacatgcca
YZdCpf1-R：atgaaataacaagcaggcccttgcaYZdCpf1-R：atgaaataacaagcaggcccttgca
实施例3dCas9-RD抑制系统的构建与应用Example 3 Construction and application of dCas9-RD inhibition system
(a)以购自于Addgene的质粒pML104为模板，采用引物dCas-F1、dCas-R1、dCas-F2、dCas-R2将Cas9突变为dCas9。基因合成抑制结构域RD，使用无缝克隆将激活结构域RD与pML104进行连接，得到质粒pML104-RD，以上述构建的质粒为模板，采用CIT-F和CIT-R为引物环P得到质粒pML104-RD-CIT，采用MLS1-F和MLS1-R为引物环P得到质粒pML104-RD-MLS，采用ERG6-F和ERG6-R为引物环P得到质粒pML104-RD-ERG6。分别采用URA-104-F和URA-104-R为引物扩增得到片段URA-dCas9-RD-CIT、URA-dCas9-RD-MLS、URA-dCas9-RD-ERG6。以购自于Addgene的质粒pFA6a-TRP1-PGAL1-GFP为模板，采用引物pFA6a-F、pFA6a-R扩增得到片段pFA6a-TRP1-GFP，以酿酒酵母SC288C基因组为模板，采用引物pMLS-F、pMLS-R扩增得到启动子基因片段pMLS，采用引物pERG6-F、pERG6-R扩增得到启动子基因片段pERG6，采用引物pCIT-F、pCIT-R扩增得到启动子基因片段pCIT，使用无缝克隆分别将pMLS、pCIT-F、 pERG6与pFA6a-TRP1-GFP连接，得到质粒pFA6a-TRP1-pMLS-GFP、pFA6a-TRP1-pCIT-GFP、pFA6a-TRP1-pERG6-GFP。将质粒pFA6a-TRP1-pMLS-GFP与片段URA-dCas9-RD-MLS、pFA6a-TRP1-pCIT-GFP与URA-dCas9-RD-CIT、pFA6a-TRP1-pERG6-GFP与URA-dCas9-RD-ERG6分别导入酵母SC288C中，得到菌株SG-MLS、SG-CIT、SG-ERG6，分别使用酶标仪进行绿色荧光强度的检测，结果如图4。(a) Using plasmid pML104 purchased from Addgene as a template, primers dCas-F1, dCas-R1, dCas-F2, and dCas-R2 were used to mutate Cas9 into dCas9. Gene synthesize the repression domain RD and use seamless cloning to connect the activation domain RD to pML104 to obtain plasmid pML104-RD. Use the plasmid constructed above as a template and use CIT-F and CIT-R as primer loop P to obtain plasmid pML104. -RD-CIT, using MLS1-F and MLS1-R as primer loop P to obtain plasmid pML104-RD-MLS, and using ERG6-F and ERG6-R as primer loop P to obtain plasmid pML104-RD-ERG6. The fragments URA-dCas9-RD-CIT, URA-dCas9-RD-MLS, and URA-dCas9-RD-ERG6 were amplified using URA-104-F and URA-104-R as primers respectively. The plasmid pFA6a-TRP1-PGAL1-GFP purchased from Addgene was used as the template, and the primers pFA6a-F and pFA6a-R were used to amplify the fragment pFA6a-TRP1-GFP. The Saccharomyces cerevisiae SC288C genome was used as the template, and the primers pMLS-F and pFA6a-R were used. The promoter gene fragment pMLS was obtained by amplifying pMLS-R, using primers pERG6-F and pERG6-R to amplify the promoter gene fragment pERG6, and using primers pCIT-F and pCIT-R to amplify the promoter gene fragment pCIT. Seam cloning was used to connect pMLS, pCIT-F, pERG6 and pFA6a-TRP1-GFP respectively to obtain plasmids pFA6a-TRP1-pMLS-GFP, pFA6a-TRP1-pCIT-GFP, and pFA6a-TRP1-pERG6-GFP. Plasmid pFA6a-TRP1-pMLS-GFP and fragments URA-dCas9-RD-MLS, pFA6a-TRP1-pCIT-GFP and URA-dCas9-RD-CIT, pFA6a-TRP1-pERG6-GFP and URA-dCas9-RD-ERG6 They were introduced into yeast SC288C to obtain strains SG-MLS, SG-CIT, and SG-ERG6. The green fluorescence intensity was detected using a microplate reader. The results are shown in Figure 4.
(b)以质粒pFA6a-TRP1-pERG6-GFP为模板，采用引物Cas-RD-F、Cas-RD-R扩增得到基因片段dCas9-RD-erg6，以购自Addgene的质粒pCSN068为模板，采用引物KAN2-F、KAN2-R扩增得到基因片段KAN-Cas，以酿酒酵母SC288C基因组为模板，采用引物tRNA-G-F和tRNA-G-R扩增得到基因片段tRNA-Gly，直接采用引物MLS-F和MLS-R重叠合成片段MLS-20。直接采用引物CIT-F和CIT-R重叠合成片段CIT2-20，以酿酒酵母SC288C的基因组为模板，采用引物GAL80UP-F和GAL80UP-R扩增得到基因片段GAL80UP，采用引物GAL80Down-F和GAL80Down-R扩增得到基因片段GAL80Down。(b) Using plasmid pFA6a-TRP1-pERG6-GFP as a template, use primers Cas-RD-F and Cas-RD-R to amplify the gene fragment dCas9-RD-erg6. Using plasmid pCSN068 purchased from Addgene as a template, use The gene fragment KAN-Cas was amplified by primers KAN2-F and KAN2-R. Using the Saccharomyces cerevisiae SC288C genome as a template, the gene fragment tRNA-Gly was amplified using primers tRNA-G-F and tRNA-G-R. The gene fragment tRNA-Gly was amplified directly using primers MLS-F and MLS-R overlaps the synthetic fragment MLS-20. The fragment CIT2-20 was directly synthesized by overlapping primers CIT-F and CIT-R. The genome of Saccharomyces cerevisiae SC288C was used as a template, and primers GAL80UP-F and GAL80UP-R were used to amplify the gene fragment GAL80UP. Primers GAL80Down-F and GAL80Down- were used to amplify the fragment. R amplified the gene fragment GAL80Down.
(c)将步骤(b)中获得的片段：dCas9-RD-erg6、KAN-Cas、tRNA-Gly、、MLS-20、CIT2-20、GAL80UP、GAL80Down进行重叠PCR，获得片段GAL80-KAN-dCas9-RD-sgRNAEMC(见SEQ ID NO 2)。GAL80-KAN-dCas9-RD-sgRNAEMC片段序列(见SEQ ID NO 2)：(c) Perform overlapping PCR on the fragments obtained in step (b): dCas9-RD-erg6, KAN-Cas, tRNA-Gly, MLS-20, CIT2-20, GAL80UP, and GAL80Down to obtain the fragment GAL80-KAN-dCas9 -RD-sgRNAEMC (see SEQ ID NO 2). GAL80-KAN-dCas9-RD-sgRNAEMC fragment sequence (see SEQ ID NO 2):
Figure PCTCN2022092329-appb-000007
Figure PCTCN2022092329-appb-000007
Figure PCTCN2022092329-appb-000008
Figure PCTCN2022092329-appb-000008
Figure PCTCN2022092329-appb-000009
Figure PCTCN2022092329-appb-000009
Figure PCTCN2022092329-appb-000010
Figure PCTCN2022092329-appb-000010
Figure PCTCN2022092329-appb-000011
Figure PCTCN2022092329-appb-000011
(d)将步骤(c)得到片段，导入工程菌SX-6的感受态中，通过G418平板进行筛选，使用验证引物YZdCas-F和YZdCas-R进行菌落PCR验证。验证正确的单菌落，将其在5-FOA的YPD平板进行划线，在30℃培养2-3d，筛选去掉G418标签的单菌落，命名为SX-7。(d) Introduce the fragment obtained in step (c) into the competent state of the engineering strain SX-6, screen it on the G418 plate, and use the verification primers YZdCas-F and YZdCas-R to perform colony PCR verification. Verify the correct single colony, streak it on a 5-FOA YPD plate, culture it at 30°C for 2-3 days, screen out the single colony with the G418 tag removed, and name it SX-7.
引物序列：Primer sequence:
dCas-F1:tattctatcggactggctatcgggactaatagcgtcgggtdCas-F1:tattctatcggactggctatcgggactaatagcgtcgggt
dCas-R1:atagccagtccgatagaatacttcttgtccatggtgdCas-R1:atagccagtccgatagaatacttcttgtccatggtg
dCas-F2:gacgctatcgtccctcagagcttcctcaaagdCas-F2:gacgctatcgtccctcagagcttcctcaaag
dCas-R2:ctctgagggacgatagcgtccacgtcgtagtctgagagccdCas-R2:ctctgagggacgatagcgtccacgtcgtagtctgagagcc
CIT-F:agctctaaaactttgctttcaatgtttttgagatcatttatctttcactgcggagaagCIT-F:agctctaaaactttgctttcaatgtttttgagatcatttatctttcactgcggagaag
CIT-R:tcaaaaacattgaaagcaaagttttagagctagaaatagcaagttaaaataaggCIT-R:tcaaaaacattgaaagcaaagttttagagctagaaatagcaagttaaaataagg
MLS1-F:agctctaaaacgctcaaatcagtgggcgtcggatcatttatctttcactgcggagaagMLS1-F:agctctaaaacgctcaaatcagtgggcgtcggatcatttatctttcactgcggagaag
MLS1-R:cgacgcccactgatttgagcgttttagagctagaaatagcaagttaaaataaggMLS1-R:cgacgcccactgatttgagcgttttagagctagaaatagcaagttaaaataagg
ERG6-F:agctctaaaacgtttcggcgttttctggcttgatcatttatctttcactgcggagaagERG6-F:agctctaaaacgtttcggcgttttctggcttgatcatttatctttcactgcggagaag
ERG6-R:aagccagaaaacgccgaaacgttttagagctagaaatagcaagttaaaataaggERG6-R:aagccagaaaacgccgaaacgttttagagctagaaatagcaagttaaaataagg
URA-104-F:cggcatcagagcagattgtaURA-104-F:cggcatcagagcagattgta
URA-104-R:agcgagtcagtgagcgagURA-104-R:agcgagtcagtgagcgag
pMLS-F:tattgaagtatgtctaatgcgaaggtacttttpMLS-F:tattgaagtatgtctaatgcgaaggtactttt
pMLS-R:aattcttcagcagttttcttaattcttttatgtpMLS-R:aattcttcagcagttttcttaattcttttatgt
pERG6-F:cattgctatattgaagtattcgggtgttttctcctatcctctgctgctpERG6-F:cattgctatattgaagtattcgggtgttttctcctatcctctgctgct
pERG6-R:ttttttcttatgctgcctactatattattattpERG6-R:ttttttcttatgctgcctactatattattatt
pCIT-F:tatattgaagtaaattggtgacgttaatctaaapCIT-F:tatattgaagtaaattggtgacgttaatctaaa
pCIT-R:ccgggttttttttttcttgttactagtattattpCIT-R:ccgggttttttttttcttgttactagtattatt
Cas-RD-F：tcgtataatgtatgctatacgaacggtatcattatcaatactgccatttcCas-RD-F：tcgtataatgtatgctatacgaacggtatcattatcaatactgccatttc
Cas-RD-R：gcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgaCas-RD-R:gcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccga
KAN2-F:caatcaagacacattaccccgccatcgctgcaggtcgacgaattctaccgttcgtKAN2-F:caatcaagacacattaccccgccatcgctgcaggtcgacgaattctaccgttcgt
KAN2-R:tgaaatggcagtattgataatgataccgttcgtatagcatacattKAN2-R:tgaaatggcagtattgataatgataccgttcgtatagcatacatt
tRNA-G-F:tgcgcaagcccggaatcgaatRNA-G-F:tgcgcaagcccggaatcgaa
tRNA-G-R:gcgcaagtggtttagtggtaaaatcctRNA-G-R:gcgcaagtggtttagtggtaaaatcc
ERG6-F:aaagataaatgatcctgctgctctctttttctttgttttagagctagaaatagcaagttaaaataaggERG6-F:aaagataaatgatcctgctgctctctttttctttgttttagagctagaaatagcaagttaaaataagg
ERG6-R:ctagctctaaaacaaagaaaaagagagcagcaggatcatttatctttcactgcggagaaERG6-R:ctagctctaaaacaaagaaaaagagagcagcaggatcatttatctttcactgcggagaa
MLS-F：aaaacttttcttaattcttttatgttgcgcaagcccggaatcgaaMLS-F:aaaacttttcttaattcttttatgttgcgcaagcccggaatcgaa
MLS-R：tccgggcttgcgcaacataaaagaattaagaaaagttttagagctagaaatMLS-R:tccgggcttgcgcaacataaaagaattaagaaaagttttagagctagaaat
CIT-F：aaaactttttcttgttactagtatttgcgcaagcccggaatcgaaCIT-F: aaaactttttcttgttactagtatttgcgcaagcccggaatcgaa
CIT-R：tccgggcttgcgcaaatactagtaacaagaaaaagttttagagctagaaatCIT-R:tccgggcttgcgcaaatactagtaacaagaaaaagttttagagctagaaat
GAL80UP-F：aggcatacctaatgctggggatgaGAL80UP-F：aggcatacctaatgctggggatga
GAL80UP-R：aattcgtcgacctgcagcgatggcggggtaatgtgtcttgattGAL80UP-R: aattcgtcgacctgcagcgatggcggggtaatgtgtcttgatt
GAL80Down-F：agctccagcttttgaatgcaaggtttcgatttcgaaggGAL80Down-F：agctccagcttttgaatgcaaggtttcgatttcgaagg
GAL80Down-R：tgatgcacttcatgaacctgttggGAL80Down-R:tgatgcacttcatgaacctgttgg
YZdCas-F：cctcatccaccagagcatcacYZdCas-F:cctcatccaccagagcatcac
YZdCas-R：caaaagctggagctccaccgYZdCas-R: caaaagctggagctccaccg
实施例4构建成功的工程酵母菌进行发酵培养Example 4 Successfully constructed engineering yeast for fermentation culture
在2mLYPD培养基中接种固体YPD平板上的工程酵母菌SX-6和SX-7单菌落，30℃，220rpm培养16-20h后，按接种量1％接入含有25mLYPD液体培养基的250mL圆底摇瓶内，30℃，220rpm培养96h。发酵至26h时，使用50％(体积浓度)乙醇作为碳源，进行补充。发酵结束后，离心取沉淀。使得7-DHC的产量分别达到371.5mg/L、464mg/L。Inoculate a single colony of engineered yeast SX-6 and SX-7 on a solid YPD plate into 2 mL LYPD medium. After culturing for 16-20 hours at 30°C and 220 rpm, insert 1% of the inoculum into a 250 mL round bottom containing 25 mL LYPD liquid medium. In the shake flask, culture at 30℃, 220rpm for 96h. When fermentation reaches 26 h, use 50% (volume concentration) ethanol as the carbon source for supplementation. After the fermentation is completed, centrifuge to remove the precipitate. The output of 7-DHC reached 371.5mg/L and 464mg/L respectively.
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications may be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (10)

  1. 一种同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，其特征在于，包括将酿酒酵母中乙醇脱氢酶adh2、截断的HMG-CoA还原酶tHMG1、异戊烯二磷酸异构酶idi1、角鲨烯环氧酶erg1、羊毛甾醇14-α-脱甲基酶erg11、C-14甾醇还原酶erg24、C-4甲基甾醇氧化酶erg25、C-3甾醇脱氢酶erg26、3-酮甾醇还原酶erg27以及经过密码子优化的外源基因甾醇△24-还原酶dhcr24进行同时增强表达，和/或对苹果酸合酶mls1、柠檬酸合酶cit2、Delta(24)-甾醇C-甲基转移酶erg6进行同时抑制。A method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae, which is characterized in that it includes alcohol dehydrogenase adh2, truncated HMG-CoA reductase tHMG1, isoprenyl dihydrogenase in Saccharomyces cerevisiae. Phosphate isomerase idi1, squalene epoxidase erg1, lanosterol 14-α-demethylase erg11, C-14 sterol reductase erg24, C-4 methylsterol oxidase erg25, C-3 sterol dehydrogenation The enzyme erg26, 3-ketosterol reductase erg27 and the codon-optimized exogenous gene sterol Δ24-reductase dhcr24 are simultaneously enhanced to express, and/or malate synthase mls1, citrate synthase cit2, Delta (24 )-sterol C-methyltransferase erg6 for simultaneous inhibition.
  2. 根据权利要求1中所述的同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，其特征在于，所述乙醇脱氢酶adh2的编号为ID:NM_001182812.1、截断的HMG-CoA还原酶tHMG1的编号为ID:XM_033912352.1、异戊烯二磷酸异构酶idi1的编号为ID:NM_001183931.1、角鲨烯环氧酶erg1的编号为ID:NM_001181304.1、羊毛甾醇14-α-脱甲基酶erg11的编号为ID:NM_001179137.1、C-14甾醇还原酶erg24的编号为ID:NM_001183118.1、C-4甲基甾醇氧化酶erg25的编号为ID:NM_001181189.3、C-3甾醇脱氢酶erg26的编号为ID:NM_001180866.1、3-酮甾醇还原酶erg27的编号为ID:NM_001181987.1以及外源基因甾醇△24-还原酶dhcr24的编号为ID:NM_001031288.1、苹果酸合酶mls1的编号为ID:NM_001182955.1、柠檬酸合酶cit2的编号为ID:NM_001178718.1、Delta(24)-甾醇C-甲基转移酶erg6的编号为ID:NM_001182363.1。The method of simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae according to claim 1, wherein the number of the alcohol dehydrogenase adh2 is ID: NM_001182812.1, truncated The number of HMG-CoA reductase tHMG1 is ID: The number of sterol 14-α-demethylase erg11 is ID: NM_001179137.1, the number of C-14 sterol reductase erg24 is ID: NM_001183118.1, and the number of C-4 methylsterol oxidase erg25 is ID: NM_001181189 .3. The number of C-3 sterol dehydrogenase erg26 is ID: NM_001180866.1, the number of 3-ketosterol reductase erg27 is ID: NM_001181987.1, and the number of exogenous gene sterol △24-reductase dhcr24 is ID :NM_001031288.1, the number of malate synthase mls1 is ID: NM_001182955.1, the number of citrate synthase cit2 is ID: NM_001178718.1, the number of Delta(24)-sterol C-methyltransferase erg6 is ID :NM_001182363.1.
  3. 根据权利要求1所述的同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，其特征在于，所述乙醇脱氢酶adh2、截断的HMG-CoA还原酶tHMG1、异戊烯二磷酸异构酶idi1、角鲨烯环氧酶erg1、羊毛甾醇14-α-脱甲基酶erg11、C-14甾醇还原酶erg24、C-4甲基甾醇氧化酶erg25、C-3甾醇脱氢酶erg26、3-酮甾醇还原酶erg27以及经过密码子优化的外源基因甾醇△24-还原酶dhcr24通过使用诱导型启动子gal1p和gal7p进行表达。The method of simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae according to claim 1, characterized in that the alcohol dehydrogenase adh2, truncated HMG-CoA reductase tHMG1, isopentyl Endenediphosphate isomerase idi1, squalene epoxidase erg1, lanosterol 14-α-demethylase erg11, C-14 sterol reductase erg24, C-4 methylsterol oxidase erg25, C-3 sterol Dehydrogenase erg26, 3-ketosterol reductase erg27, and codon-optimized foreign gene sterol Δ24-reductase dhcr24 were expressed by using inducible promoters gal1p and gal7p.
  4. 根据权利要求3所述的同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，其特征在于，所述诱导型启动子gal1p和gal7p的强度是使用dCpf1-VP激活系统进行增强。The method of simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae according to claim 3, characterized in that the intensity of the inducible promoters gal1p and gal7p is carried out using a dCpf1-VP activation system. Enhance.
  5. 根据权利要求4所述的同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，其特征在于，所述dCpf1-VP激活系统发挥功能的基因片段为DPP1-KAN-dCpf1-VP-crRNA1-7-1，所述DPP1-KAN-dCpf1-VP-crRNA1-7-1的序列见SEQ ID NO 1。The method of simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae according to claim 4, characterized in that the gene fragment that functions in the dCpf1-VP activation system is DPP1-KAN-dCpf1- VP-crRNA1-7-1, the sequence of DPP1-KAN-dCpf1-VP-crRNA1-7-1 is shown in SEQ ID NO 1.
  6. 根据权利要求1中所述的同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，其特征在于，所述苹果酸合酶mls1、柠檬酸合酶cit2、Delta(24)-甾醇C-甲基转移酶erg6通过dCas9-RD抑制系统进行抑制。The method of simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae according to claim 1, characterized in that the malate synthase mls1, citrate synthase cit2, and Delta (24) -Sterol C-methyltransferase erg6 is inhibited by the dCas9-RD inhibition system.
  7. 根据权利要求6中所述的同时增强和抑制酿酒酵母7-脱氢胆固醇合成中多个关键基因的方法，其特征在于，所述dCas9-RD抑制系统发挥功能的基因片段为GAL80-KAN-dCas9-RD-sgRNAEMC，所述GAL80-KAN-dCas9-RD-sgRNAEMC的序列见SEQ ID NO 2。The method of simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae according to claim 6, characterized in that the gene fragment that functions as the dCas9-RD inhibition system is GAL80-KAN-dCas9 -RD-sgRNAEMC, the sequence of GAL80-KAN-dCas9-RD-sgRNAEMC is shown in SEQ ID NO 2.
  8. 一种工程酵母菌，其特征在于，所述工程酵母菌通过将酿酒酵母中乙醇脱氢酶adh2、截断的HMG-CoA还原酶tHMG1、异戊烯二磷酸异构酶idi1、角鲨烯环氧酶erg1、羊毛甾醇14-α-脱甲基酶erg11、C-14甾醇还原酶erg24、C-4甲基甾醇氧化酶erg25、C-3甾醇脱氢酶erg26、3-酮甾醇还原酶erg27以及经过密码子优化的外源基因甾醇△24-还原酶dhcr24进行同时增强表达，和/或对苹果酸合酶mls1、柠檬酸合酶cit2、Delta(24)-甾醇C-甲基转移酶erg6进行同时抑制所得。An engineered yeast, characterized in that the engineered yeast combines alcohol dehydrogenase adh2, truncated HMG-CoA reductase tHMG1, isopentenyl diphosphate isomerase idi1, squalene epoxy in Saccharomyces cerevisiae. Enzyme erg1, lanosterol 14-α-demethylase erg11, C-14 sterol reductase erg24, C-4 methylsterol oxidase erg25, C-3 sterol dehydrogenase erg26, 3-ketosterol reductase erg27, and The codon-optimized exogenous gene sterol Δ24-reductase dhcr24 is simultaneously enhanced for expression, and/or malate synthase mls1, citrate synthase cit2, and Delta(24)-sterol C-methyltransferase erg6 are while suppressing gains.
  9. 权利要求8中所述的工程酵母菌在合成7-脱氢胆固醇中的应用。Application of the engineered yeast described in claim 8 in the synthesis of 7-dehydrocholesterol.
  10. 根据权利要求9中所述的应用，其特征在于，将所述工程酵母菌的种子液在培养基中摇瓶发酵。The application according to claim 9, characterized in that the seed liquid of the engineered yeast is fermented in a shaking flask in a culture medium.
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