CN114606147A - Method for simultaneously enhancing and inhibiting multiple key genes in saccharomyces cerevisiae 7-dehydrocholesterol synthesis - Google Patents

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

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CN114606147A
CN114606147A CN202210253230.3A CN202210253230A CN114606147A CN 114606147 A CN114606147 A CN 114606147A CN 202210253230 A CN202210253230 A CN 202210253230A CN 114606147 A CN114606147 A CN 114606147A
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sterol
reductase
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saccharomyces cerevisiae
synthesis
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CN114606147B (en
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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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Jiangnan University
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Abstract

The invention provides a method for simultaneously enhancing and inhibiting a plurality of key genes in the synthesis of 7-dehydrocholesterol of saccharomyces cerevisiae, belonging to the technical field of biological engineering. The invention realizes the purpose of simultaneous expression and inhibition of 13P 450 enzymes by a dCpf1-VP activation system and a dCas9-RD inhibition system, constructs an engineering yeast for high yield of 7-dehydrocholesterol, and ensures that the yield of 7-DHC reaches 464 mg/L.

Description

Method for simultaneously enhancing and inhibiting multiple key genes in saccharomyces cerevisiae 7-dehydrocholesterol synthesis
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for simultaneously enhancing and inhibiting a plurality of key genes in the synthesis of saccharomyces cerevisiae 7-dehydrocholesterol.
Background
Vitamin D3(Cholecalciferol, VD3) is a hormone precursor in human body, and has the functions of regulating cell differentiation, promoting bone calcification and the like, VD3 is hydroxylated in the liver of human body to form 25-hydroxyvitamin D3(25-hydroxyvitamin D3, 25-OH-VD3), and is converted into 1,25-dihydroxyvitamin D3(1,25-dihydroxyvitamin D3, 1,25- (OH)2-VD3 in the kidney, vitamin D3 in human body is mainly obtained by irradiating 7-dehydrocholesterol (7-dehydrocholestro, 7-DHC) in the bottom layer of skin with ultraviolet rays, and can also be partially obtained by food, but because the dietary structure of each person is different, the effect is poor by supplementing VD3, the detection of the level of VD 363 in China shows more than 60%, and the VD 82 is promoted to be continuously developed by using huge market and market synthetic technology of VD 7-VD 3 and 7-VD 7-VD 3 Alcohol (7-dehydrocholestro, 7-DHC) is an important precursor for synthesizing VD3, and the central problem for producing VD3 is the synthesis of 7-DHC.
The microbial metabolic engineering synthesis method of 7-DHC is mainly based on strains containing natural sterol synthetic pathways, and the proportion of the metabolic flow of the sterol synthetic pathway to the total metabolic flow of the thalli is low, so that the main focus is on gene enhancement and branch path blocking in the known synthetic pathways, for example, Chinese patent CN104988168A enhances the yield of 7-DHC by enhancing the synthesis of a precursor substance acetyl coenzyme A of natural sterol synthesis, but also enhances the expression of genes (acs, adh2, acl and ald6) by replacing a traditional constitutive strong promoter, but the microbial metabolic pressure of the thalli is too high. Chinese patent CN104988168A and chinese patent CN103275997A directly knock out key genes in the ergosterol synthesis pathway: erg5 and erg6, further enhance the synthesis of 7-DHC, but completely block the synthesis of ergosterol, which has irreversible effect on the growth of the strain, and is not suitable for industrial mass production.
The microbial synthesis of VD3 mainly focuses on the screening of strains, such as Chinese patent CN103275997A, Chinese patent CN107075551B and Chinese patent CN 104988168A. Most of the screened strains are applied to VD3 to synthesize active VD3, strains which directly synthesize VD3 are rarely screened, the screening of the strains is time-consuming and labor-consuming, and large-scale later screening and fermentation detection are carried out with a large amount of money, so that the strains with extremely low VD3 yield can be obtained.
The gene regulation tool plays an important role in both basic research and industrial application of saccharomyces cerevisiae. With the research and development of CRISPR systems in recent years, CRISPR systems are widely applied to gene editing and gene regulation of various organisms, for example, chinese patent CN112204147A, plant transcription regulation system based on Cpf1, Cpf1 is used to activate and inhibit the transcription of plants, but when a single Cas protein is used to activate and inhibit, the orthogonality of the system is reduced, and the effect is not obvious. Like Chinese patent CN110468153A, a Cas9 system responding to far-red light is constructed to regulate gene transcription, but the Cas9 system needs to meet additional factors and has a narrow application range.
The current method for synthesizing VD3 mainly comprises the following steps: chemical synthesis, microbial transformation and microbial metabolic synthesis. The chemical synthesis method needs expensive lanolin cholesterol as a raw material, obtains 7-dehydrocholesterol through multi-step group protection and deprotection, and obtains VD3 through illumination reaction, ring opening and isomerization. The microbial transformation method is only used for transforming VD3 into active VD3 at present, and the used microorganisms are microorganisms of streptomyces, mycobacteria and the like generally. With the development of synthetic biology and genetic engineering, the microbial metabolic engineering method has great potential for producing 7-DHC and VD3 by using ergosterol producing strains.
Disclosure of Invention
The existing technology for producing 7-DHC by using saccharomyces cerevisiae utilizes a sterol synthetic pathway of the saccharomyces cerevisiae, wherein the sterol synthetic pathway comprises 13P 450 enzymes, and the sterol anabolism proportion in the saccharomyces cerevisiae is not high, so that if expression is enhanced only by increasing copy number traditionally, yeast metabolic disorder is caused, other excessive byproducts are generated, and the purification difficulty of a target product is increased. If the synthesis of ergosterol is blocked by simply knocking out erg5 and erg6 genes, although a certain amount of 7-DHC is accumulated, the complete blocking of the synthesis of ergosterol will inevitably have adverse effects on the growth of the production strain, and further the yield of 7-DHC and VD3 is influenced. In order to solve the technical problems, the invention provides a method for simultaneously enhancing and inhibiting a plurality of key genes in the synthesis of saccharomyces cerevisiae 7-dehydrocholesterol.
The first object of the present invention is to provide a method for simultaneously enhancing and inhibiting several key genes in the synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae, which comprises simultaneously enhancing the expression of 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 methyl sterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27) and exogenous sterol 24-reductase (Δ dhcr24) in Saccharomyces cerevisiae, and/or simultaneous inhibition of malate synthase (mls1), citrate synthase (cit2), Delta (24) -sterol C-methyltransferase (erg 6).
In one embodiment of the invention, the ethanol dehydrogenase (adh2) is numbered ID: NM-001182812.1, the truncated HMG-CoA reductase (tHMG1) is numbered ID: XM-033912352.1, the isopentenyl diphosphate isomerase (idi1) is numbered ID: NM-001183931.1, the squalene epoxidase (erg1) is numbered ID: NM-001181304.1, the lanosterol 14- α -demethylase (erg11) is numbered ID: NM-001179137.1, the C-14 sterol reductase (erg24) is numbered ID: NM-001183118.1, the C-4 methylsterol oxidase (erg25) is numbered ID: NM-001181189.3, the C-3 sterol dehydrogenase (erg26) is numbered ID: NM-001180866.1, the 3-ketosterol reductase (erg27) is numbered ID: NM-DELTA 5, and the exogenous gene dhNM 24-cr5842 is numbered ID: 001031288.1.
In one embodiment of the invention, the exogenous gene dhcr24 from chicken is used for the codon preference optimization of saccharomyces cerevisiae, and the optimization method comprises the following steps: inputting dhcr24 gene from chicken into websitehttps:// www.genscript.com/tools/gensmart-codon-optimizationAnd optimizing the codon preference of the saccharomyces cerevisiae, and synthesizing the genes after optimization.
In one embodiment of the invention, the malate synthase (mls1) is numbered ID NM-001182955.1, the citrate synthase (cit2) is numbered ID NM-001178718.1, and the Delta (24) -sterol C-methyltransferase (erg6) is numbered ID NM-001182363.1.
In one embodiment of the present invention, the Saccharomyces cerevisiae is Saccharomyces cerevisiae S288C.
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 methyl sterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27), and exogenous gene sterol Δ 24-reductase (dhcr24) are expressed by using inducible promoters gal1p and gal7 p.
In one embodiment of the invention, the strength of the inducible promoters gal1p and gal7p was enhanced using the dCpf1-VP activation system, with an 85% increase in strength of promoter gal1p and a 69% increase in strength of promoter gal7 p.
In one embodiment of the invention, the gene fragment of dCpf1-VP activation system is DPP1-KAN-dCpf1-VP-crRNA1-7-1, and the sequence of DPP1-KAN-dCpf1-VP-crRNA1-7-1 is shown in SEQ ID NO 1.
In one embodiment of the invention, inhibition of malate synthase (mls1), citrate synthase (cit2), Delta (24) -sterol C-methyltransferase (erg6) by the dCas9-RD inhibition system inhibited the erg6, mls1 and cit2 genes by 88%, 84% and 72%, respectively.
In one embodiment of the invention, the gene fragment of dCas9-RD inhibiting system is GAL80-KAN-dCas9-RD-sgRNAEMC, and the sequence of GAL80-KAN-dCas9-RD-sgRNAEMC is shown in SEQ ID NO 2.
A second object of the present invention is to provide an engineered yeast obtained by simultaneously enhancing expression of alcohol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), prenyl diphosphate isomerase (idi1), squalene epoxidase (erg1), lanosterol 14- α -demethylase (erg11), C-14 sterol reductase (erg24), C-4 methyl sterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27), and sterol Δ 24-reductase (dhcr24), which is an exogenous gene, in Saccharomyces cerevisiae, and/or simultaneously inhibiting malate synthase (mls1), citrate synthase (cit2), Delta (24) -sterol C-methyltransferase (erg 6).
The third purpose of the invention is to provide the application of the engineering yeast in synthesizing 7-dehydrocholesterol.
In one embodiment of the invention, the seed liquid of the engineering yeast is cultured in YPD medium at 30 ℃ and 180-. Inoculating 1-5% of YPD liquid culture medium into round bottom shake flask, and culturing.
In one embodiment of the invention, the conditions for culturing the fermentation are: culturing at 30 ℃ and 220rpm for 96-120 h.
In one embodiment of the invention, 50-60% (volume fraction) ethanol is added to replenish the carbon source when the fermentation is carried out for 26 h. After fermentation, the precipitate was centrifuged.
In one embodiment of the invention, the carbon source comprises glucose, sucrose, glycerol, ethanol.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention takes the original Saccharomyces cerevisiae S288C as a starting strain, and dCpf1 and VP (activation domain) are fused to construct an activation system based on dCpf 1. Ethanol dehydrogenase (adh2), truncated HMG-CoA reductase (tHMG1), isopentenyl diphosphate isomerase (idi1), squalene epoxidase (erg1), lanosterol 14-alpha-demethylase (erg11), C-14 sterol reductase (erg24), C-4 methyl sterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27), and exogenous gene sterol delta 24-reductase (dhcr24) in the synthetic pathway of yeast 7-DHC were expressed using inducible promoters gal1p and gal7p, respectively, and the strengths of the promoters gal1p and gal7p were enhanced using a dCpf1-VP activation system to achieve the purpose of increasing the yield 371 of 7-DHC, which is a yield of.5 mg/L. dCas9 is sequentially fused with inhibition domains UME6, MIG1 and TUP1 in series to construct an inhibition system based on dCas9, sgRNA identified by the inhibition system is designed to inhibit malate synthase (mls1), citrate synthase (cit2) and Delta (24) -sterol C-methyltransferase (erg6), so that the yield of 7-DHC is further improved and reaches 464 mg/L.
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In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a schematic diagram showing the construction of the dCpf1-VP activation system of the present invention.
FIG. 2 is a schematic diagram showing the construction of the dCas9-RD inhibiting system of the present invention.
FIG. 3 is a graph of the strength of the promoters gal1p and gal7p enhanced using dCpf 1-VP.
FIG. 4 is a graph showing the effect of inhibition using dCas 9-RD.
FIG. 5 is a graph showing the yield of 7-DHC in the engineered yeast SX-6 and the engineered yeast SX-7 obtained as strains according to the present invention.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
The instrument used for detecting 7-DHC is high performance liquid chromatography, engineering yeast liquid fermented for more than 96 hours is subjected to high-speed centrifugation, supernatant is discarded, sterile water is used for carrying out heavy suspension, glass beads with the diameter of 0.5mm are added, a high-speed homogenizing crusher is used for crushing for 10min, the crushed mixed liquid is taken out, 2% ascorbic acid and 1% HBT are added and mixed evenly, 10% absolute ethyl alcohol and 2% 1.5mol/L potassium hydroxide methanol solution are sequentially added, saponifying in water at 80 deg.C for 2 hr, ultrasonic vibrating the extractive solution (isopropanol: n-hexane 1:3 or petroleum ether or methanol) for 30 min, removing impurities in the lower layer with separating funnel, freeze drying the extractive solution, re-dissolving with methanol or acetonitrile or mixture of methanol and acetonitrile, filtering with 0.55 μm, and analyzing with HPLC. The mobile phase uses methanol and water in a ratio of 90:10 or 95:5 or 98: 2. The detector uses an ultraviolet detector, and the detection wavelength is 265-280 nm.
In order to be different from the traditional method for increasing the copy number of genes, a CRISPR-based polygene activation and inhibition system is designed, the key genes of the synthetic pathway of Saccharomyces cerevisiae 7-DHC are activated and inhibited, the growth state of the strain is not influenced as much as possible, the yield of 7-DHC is increased, finally, the key genes are activated and inhibited by the system, the effect is obvious, the growth state of the strain is normal, and the yield of 7-DHC is 464 mg/L.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a construction method of a multiple gene activation and inhibition system based on CRISPR technology;
the invention utilizes the constructed system to regulate and control the synthetic route of the saccharomyces cerevisiae 7-DHC;
among the genes that can be enhanced using the above system are: 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 methyl sterol oxidase (erg25), C-3 sterol dehydrogenase (erg26), 3-ketosterol reductase (erg27), sterol Δ 24-reductase (dhcr 24);
among the genes to be suppressed using the above system are: malate synthase (mls1), citrate synthase (cit2), Delta (24) -sterol C-methyltransferase (erg 6).
The invention also provides application of the engineering yeast in the production of synthesizing 7-dehydrocholesterol.
In one embodiment of the invention, the seed liquid of the engineering yeast is cultured in YPD medium at 30 ℃ and 180-. Inoculating 1-5% of YPD liquid culture medium into round bottom shake flask, and culturing.
In one embodiment of the invention, the conditions for culturing the fermentation are: culturing at 30 ℃ and 220rpm for 96-120 h.
In one embodiment of the invention, ethanol is added as a carbon source to supplement 50-60% (volume fraction) of the time of fermentation to 26 h. After fermentation, the precipitate was centrifuged.
In one embodiment of the invention, the carbon source comprises glucose, sucrose, glycerol, ethanol.
Example 1 construction of Saccharomyces cerevisiae producing 7-DHC and genes related thereto in the enhanced pathway
(a) Taking the genome of Saccharomyces cerevisiae engineering bacteria S288C as a template, amplifying by using primers ADH-F and ADH-R to obtain a gene fragment ADH2, amplifying by using primers IDI-F and IDI-R to obtain a gene fragment IDI1, amplifying by using primers E1-F, E1-R to obtain a gene fragment ERG1, amplifying by using primers E11-F, E11-R to obtain a gene fragment ERG11, amplifying by using primers E24-F, E24-R to obtain a gene fragment ERG24, amplifying by using primers E25-F, E25-R to obtain a gene fragment ERG25, amplifying by using primers E26-F, E26-R to obtain a gene fragment ERG26, amplifying by using primers E27-F, E27-R to obtain a gene fragment ERG27, amplifying by using primers GAL1-F, GAL1-R to obtain a promoter gene fragment GAL1, amplifying by using primers GAL 685 7-F, GAL7-R was amplified to obtain the promoter gene fragment GAL7 p. By gene synthesis, fragments dhcr24 and thmg1 were obtained. Amplifying by using primers 208UP-F and 208UP-R to obtain upstream homology arm fragment 208UP, amplifying by using primers 208Down-F and 208Down-R to obtain downstream homology arm fragment 208Down, amplifying by using primers 1622UP-F and 1622UP-R to obtain upstream homology arm fragment 1622UP, amplifying by using primers 1622Down-F and 1622Down-R to obtain downstream homology arm fragment 1622Down, amplifying by using primers 308UP-F and 308UP-R to obtain upstream homology arm fragment 308UP, amplifying by using primers 308Down-F and 308Down-R to obtain downstream homology arm fragment 308Down, amplifying by using primers 1021UP-F and 1021UP-R to obtain upstream homology arm fragment UP 1021, amplifying by using primers 1021Down-F and 1021Down-R to obtain downstream homology arm fragment 1021Down, amplifying by using primers 911-F and 1021UP-R, 911UP-R is amplified to obtain an upstream homologous arm fragment 911UP, and primers 911Down-F and 911Down-R are adopted to amplify to obtain a downstream homologous arm fragment 911 Down.
(b) Respectively carrying out overlap PCR on the promoter fragments gal1p and gal7p obtained in the above step and the gene fragment obtained in the 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, and 911-gal1p-erg26-gal7p-erg 27.
(c) Plasmid loop P is carried out by taking pML104 plasmid as a template and respectively using primers 208-F and 208-R to obtain a plasmid capable of identifying the insertion sites of the fragments 208-gal1P-dch24-gal7P-adh2, which is named as pML104-1, plasmid loop P is carried out by using primers 1622-F and 1622-R to obtain a plasmid capable of identifying the insertion sites of the fragments 1622-gal1P-thmg1-gal7P-idi1, which is named as pML104-2, plasmid loop P is carried out by using primers 308-F and 308-R to obtain a plasmid capable of identifying the insertion sites of the fragments 308-gal1P-erg1-gal7P-erg11, which is named as pML104-3, plasmid loop P is carried out by using primers 1021-F and 1021-R to obtain a plasmid loop P capable of identifying the fragments 308-gal 1-1021-erg 8934-gal 7-gal P-erg25, which is named as pML 104-2-R, the plasmid loop P was performed with the primers 911-F and 911-R to obtain a plasmid capable of recognizing the insertion site of the fragment 911-gal1P-erg26-gal7P-erg27, which was named pML 104-5.
(d) Respectively pouring the fragments and plasmids obtained in the steps (b) and (c) into saccharomyces cerevisiae, after sequencing verification, drawing lines on a YPD plate of 5-FOA for correct single colonies, and culturing for 2-3 days at 30 ℃ to obtain strains named as SX-1, SX-2, SX-3, SX-4 and SX-5.
The primer sequence is as follows:
ADH-F：aaaacagttgaatattccctcaaaaatgatgtctattccagaaactcaaaaagcc
ADH-R：aagatattcagtttttgtcattgccgtcttatttagaagtgtcaacaacgtatct
IDI-F：gttgaatattccctcaaaaatgatgactgccgacaacaatagtatg
IDI-R：ttacttcttcaattcgtacattatattatagcattctatgaatttg
E1-F：atacctctatactttaacgtcaaggagatgtctgctgttaacgttgcacc
E1-R：gtccatatctttccatagatttttaaccaatcaactcaccaaacaaaaatggg
E11-F：acagttgaatattccctcaaaaatgatgtctgctaccaagtcaatcgttg
E11-R：ctattgtgtaatagaagttagatcttttgttctggatttctcttttccca
E24-F：atactttaacgtcaaggagatggtatcagctttgaatcccag
E24-R：ccgtccatatctttccatagatttttaataaacatatggaatgatcttgtaagga
E25-F：aatattccctcaaaaatgatgtctgccgttttcaacaacg
E25-R：tatttcacagaatgaatttcttagttagtcttcttttgagcattgttttcagc
E26-F：ttaacgtcaaggagatgtcaaagatagattcagttttaattatcggtggtt
E26-R：tccatatctttccatagatttttacaaaccttcgtccatccaggc
E27-F：gaatattccctcaaaaatgatgaacaggaaagtagctatcgtaacgg
E27-R：atttctgttttaaatttaaatgggggttctagtttcaacaatttg
GAL1-F：cggattagaagccgccgagcgggc
GAL1-R：ctccttgacgttaaagtatagaggtata
GAL7-F：aaatctatggaaagatatggacgg
GAL7-R：catttttgagggaatattcaactg
208UP-F：tggtgaattggctaaacatgccgtc
208UP-R：cccgctcggcggcttctaatccggactctgctagtatttctgatt
208Down-F：gacggcaatgacaaaaactgaatat
208Down-R：agacacttgtatcctatactatca
1622UP-F：ctaaatgtgttgctagtattattt
1622UP-R：cgctcggcggcttctaatccgtgtttttgctgttaccttctgcac
1622Down-F：ttcatagaatgctataatataatgtacgaattgaagaagtaaatt
1622Down-R：gtcctctttttaacgcgtctactaa
308UP-F：gatatatgcagagaaggagcaaat
308UP-R：ccgctcggcggcttctaatccgatgttgaaatttcacttatttta
308Down-F：agaacaaaagatctaacttctattacacaatagtttcaatag
308Down-R：tgaattttaattctacgtcaacga
1021UP-F：gagtccgcgtttgaaattgcagtt
1021UP-R：gtcgcccgctcggcggcttctaatccgacactggaaaatttgagtcatgg
1021Down-F：ctcaaaagaagactaactaagaaattcattctgtgaaatatcacg
1021Down-R：ttgtttatttccagatttctatttt
911UP-F：agtttattttcaaactgtattttga
911UP-R：tgtcgcccgctcggcggcttctaatccgtcaacatccgatttttttctataaaat
911Down-F：ttgttgaaactagaacccccatttaaatttaaaacagaaatgaaatgagc
911Down-R：tattaataggaatagtaatcatagtac
208-F：tttctagctctaaaacagatcttttgtttagcggacgatcatttatctttcactgcggag
208-R：gtccgctaaacaaaagatctgttttagagctagaaatagcaagttaaaataaggctagt
1622-F：tttctagctctaaaactttgcgatgtggtggctttagatcatttatctttcactgcggag
1622-R：taaagccaccacatcgcaaagttttagagctagaaatagcaagttaaaataaggctagtcc
308-F：tttctagctctaaaactatattctgtttgacaagtggatcatttatctttcactgcggag
308-R：cacttgtcaaacagaatatagttttagagctagaaatagcaagttaaaataaggctagtcc
1021-F：tttctagctctaaaaccaattaccaccacacagagggatcatttatctttcactgcggag
1021-R：ctctgtgtggtggtaattggttttagagctagaaatagcaagttaaaataaggctagtcc
911-F：tttctagctctaaaacgggaaacaagacaatattacgatcatttatctttcactgcggag
911-R：gtaatattgtcttgtttcccgttttagagctagaaatagcaagttaaaataaggctagtcc
example 2 construction and application of dCpf1-VP activation System
(a) Cpf1 was mutated to dCpf1 using the primer dCpf1Tu-F, dCpf1Tu-R, using plasmid pCSN068 from Addgene as a template. The activation domain VP was synthesized by gene synthesis and ligated to pCSN068 containing dCpf1 using a seamless clone to give plasmid pCSN 068-VP. A promoter gene fragment SNR52p was obtained by amplification with primers 52p-F and 52p-R using plasmid pML104 purchased from Addgene as a template. The fragments gal1p23-1 are synthesized by directly overlapping primers gal1p23-F1 and gal1p23-R1, and the fragments gal7p23-1 are synthesized by directly overlapping primers gal7p23-F1 and gal7p 23-R1. The fragments gal1p23-2 are synthesized by directly overlapping primers gal1p23-F2 and gal1p23-R2, and the fragments gal7p23-2 are synthesized by directly overlapping primers gal7p23-F2 and gal7p 23-R2. The fragments gal1p23-3 are synthesized by directly adopting primers gal1p23-F3 and gal1p23-R3 in an overlapping way, and the fragments gal7p23-3 are synthesized by directly adopting primers gal7p23-F3 and gal7p23-R3 in an overlapping way. Plasmid pFA6a-TRP1-PGAL1-GFP purchased from Addgene is used as a template, a primer pFA6a-F, pFA6a-R is used for amplification to obtain a fragment pFA6a-TRP1-GFP, a saccharomyces cerevisiae SC288C genome is used as a template, a primer gal7pG-F, gal7pG-R is used for amplification to obtain a promoter gene fragment gal7p-G, and the gal7p-G and the pFA6a-TRP1-GFP are connected by seamless cloning to obtain the plasmid pFA6a-TRP1-PGAL 7-GFP. The gene segment DPP1UP is obtained by amplification by using a primer DPP1UP-F and DPP1UP-R and the gene segment DPP1Down-F and DPP1Down-R are obtained by amplification by using the genome of the saccharomyces cerevisiae SC288C as a template.
(b) Seamlessly connecting the fragment SNR52p, gal1p23-1/gal1p23-2/gal1p23-3 obtained in step (a) with plasmid pCSN068-VP to obtain plasmid pCSN068-dCpf1-VP-crRNA1-1, pCSN068-dCpf1-VP-crRNA1-2, pCSN068-dCpf1-VP-crRNA1-3, amplifying gene fragments KAN-dCpf1-VP-gal1p1, KAN-dCpf1-VP-gal1p2, KAN-dCpf1-VP-gal1p3, and overlapping with fragment DPP 461 Dow 23 and DPP1 n to obtain fragments DPP 1-VP 1-GCP 1-1, and KAN-dCf 1-1, and 1, the three fragments and plasmids pFA6a-TRP1-PGAL1-GFP are respectively introduced into yeast SC288C at the same time to obtain strains SG1-1, SG1-2 and SG1-3, and green fluorescence intensity detection is respectively carried out by using a microplate reader, and the result is shown in figure 3, so that the optimal binding site is determined.
(c) Seamlessly connecting the fragment SNR52p, gal7p23-1/gal7p23-2/gal7p23-3 obtained in step (a) with plasmid pCSN068-VP to obtain plasmid pCSN068-dCpf1-VP-crRNA7-1, pCSN068-dCpf1-VP-crRNA7-2, pCSN068-dCpf1-VP-crRNA7-3, amplifying gene fragments KAN-dCpf1-VP-gal7p1, KAN-dCpf1-VP-gal7p2, KAN-dCpf1-VP-gal7p3 by using the plasmid as a template and using primers CSN-VPG-F and CSN-VPG-R, overlapping with the fragments DPP 461 and DPP1 to obtain fragments DPP 1-dC 23-2/gal7p23-3, and DPP 1-dC 1-VP 1-1, the three fragments and plasmids pFA6a-TRP1-PGAL7-GFP are respectively introduced into yeast SC288C at the same time to obtain strains SG7-1, SG7-2 and SG7-3, and green fluorescence intensity detection is respectively carried out by using a microplate reader, and the optimal activation site is determined according to the result shown in figure 3.
(d) After pCSN068-dCpf1-VP-crRNA1-1 and pCSN068-dCpf1-VP-crRNA7-1 of the optimal binding sites determined in steps (b) and (c) were digested by Eco31I, plasmid pCSN068-dCpf1-VP-crRNA1-7-1 was assembled using golden gate, and PCR was performed using primers crRNA1-7-F1 and crRNA1-7-R1 to obtain fragment Kan-dCpf1-VP-crRNA1-7-1, which was subjected to overlap PCR with the obtained fragments DPP1UP and DPP1Down to obtain fragment DPP1-KAN-dCpf1-VP-crRNA1-7-1 (see SEQ ID NO 1).
DPP1-KAN-dCpf1-VP-crRNA1-7-1 fragment sequence (see SEQ ID NO 1):
actagtactcgatttctggcgcagcaaaaatatagcattatgtccgataaacacagttgtgatctgtcttgtgatcgcatactctgcagataatcagttgaaatagcagcttttaagtgagaatcttattcttagtctacatcgttacattgtatcagtcacaggtacggagagaaattatacttttcgatttcattcaatgtagtttcttttttacattaaatatagttttccagtagtgcactattattaaggcgcttctgtttttagtcaacactttttcagatagtacctttcaggtggttagagtgcgatccctttaaaaaaaagtattcgtcaacgatgacagggtaaagaataaatgcagcacgcctggcgtatactgctataattgtacatcatgttatcggcgttgattctcaattgtttggtgattagcttttatatatagatagaaacccaacgttggataacctcacgactaacttttttgtattttagaaataatttgtcgatcggttgtatatttttgtcatatattatctagaaacgttagggaataaactgttatctagggtccactaacatacgcgcagttcggaaatcagcaaacatcacttaaaggacacctgctataaactgaattgtgtccaatttttcgagtagttagcagttcaataaagggcacgttatcaattgttaaaggcaaagaatcagaattaaatcatagcaaacgaccaaaatgaacagagtttcgtttattaaaacgcctttcaacataggggcgaaatggagattagaagatgtctttttgctcattatcatgatacttcttaactacccagtgtattaccaacaaccgttcgaacgtcagttttacattaacgatctcactatatcgcatccttatgcgaccgctgcaggtcgacgaattctaccgttcgtataatgtatgctatacgaagttatagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtgtcgaaaacgagctcataacttcgtataatgtatgctatacgaacggtacccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccttttctttttttgcggtcacccccatgtggcggggaggcagaggagtaggtagagcaacgaatcctactatttatccaaattagtctaggaactctttttctagattttttagatttgagggcaagcgctgttaacgactcagaaatgtaagcactacggagtagaacgagaaatccgccataggtggaaatcctagcaaaatcttgcttaccctagctagcctcaggtaagctagccttagcctgtcaaatttttttcaaaatttggtaagtttctactagcaaagcaaacacggttcaacaaaccgaaaactccactcattatacgtggaaaccgaaacaaaaaaacaaaaaccaaaatactcgccaatgagaaagttgctgcgtttctactttcgaggaagaggaactgagaggattgactacgaaaggggcaaaaacgagtcgtattctcccattattgtctgctaccacgcggtctagtagaataagcaaccagtcaacgctaagacaggtaatcaaaataccagtctgctggctacgggctagtttttacctcttttagaacccactgtaaaagtccgttgtaaagcccgttctcactgttggcgtttttttttttttggtttagtttcttatttttcatttttttctttcatgaccaaaaacaaacaaatctcgcgatttgtactgcggccactggggcgtggccaaaaaaatgacaaatttagaaaccttagtttctgatttttcctgttatgaggagatatgataaaaaatattactgctttattgttttttttttatctactgaaatagagaaacttacccaaggaggaggcaaaaaaaagagtatatatacagcagctaccattcagattttaatatattcttttctcttcttctacactattattataataattttactatattcatttttagcttaaaacctcatagaatattattcttcagtcactcgcttaaatacttatcaaaaatgtccatctaccaagagtttgtcaacaaatactctttgtctaagactttacgtttcgaattgattccacaaggtaagactttggaaaacattaaggctcgtggtttgatcttggacgacgaaaagagagccaaggattacaagaaggccaagcaaatcatcgataagtaccaccaattcttcattgaagaaatcttatcctctgtctgtatctccgaagatctattgcaaaactactccgatgtctacttcaagttgaaaaagtctgacgatgacaacttgcaaaaggatttcaaatctgccaaggacaccatcaagaaacaaatttctgaatacataaaggactctgaaaaatttaagaacttattcaaccaaaacttgatcgatgctaagaagggtcaagaatctgacttgatcttgtggttgaagcaatctaaggacaacggtatcgaattgttcaaggctaactctgatatcactgacattgacgaagctttggaaatcatcaagtctttcaaaggttggactacctatttcaagggtttccacgaaaaccgtaagaatgtctactcttccaacgacatcccaacttccatcatttacagaatcgttgacgacaatttgccaaagttcctagaaaacaaggccaaatacgaatccttgaaggacaaggctccagaagccattaactacgaacaaatcaagaaggacttggctgaagaattaactttcgacattgactacaagacttctgaagttaaccaaagagttttctctttggacgaagtcttcgagattgctaacttcaacaactacttgaaccaatctggtatcaccaaattcaacaccatcatcggtggtaagttcgtcaacggtgaaaacaccaagagaaagggtatcaacgaatacattaacttgtactctcagcaaatcaacgacaagactttaaagaaatacaagatgtctgttttgttcaagcaaattttgtctgacactgaatccaagtcttttgtcattgataagttggaagatgattccgacgtcgttaccaccatgcaatctttctacgagcaaatcgctgctttcaagaccgttgaagaaaagtctattaaggaaactttgtctttgttgttcgacgatttgaaggctcaaaagttggatttgtccaagatttacttcaagaatgacaagtctttgactgatttgtctcaacaagttttcgatgactactccgttattggtactgctgtcttggaatacatcacccaacaaatcgctcctaagaacttggacaacccatccaagaaggaacaagaattgattgccaagaagaccgaaaaagctaaatacttgtctttggaaaccattaaattggctttagaagagttcaacaagcacagagatattgacaagcaatgtagattcgaagaaattttggctaacttcgctgctatcccaatgatcttcgacgaaattgctcaaaacaaggataacttggctcaaatctccatcaagtaccaaaaccagggtaagaaggatttgttgcaagcctccgctgaagatgacgtcaaggccatcaaagatttattggaccaaactaacaacttgttgcacaagctaaagatcttccacatctctcaatctgaagataaagctaacattttggataaggacgaacacttctacttagttttcgaagaatgttacttcgaactagctaacatcgtcccattgtacaacaagatcagaaactacattactcaaaaaccatactctgatgaaaagttcaagttaaacttcgaaaattctaccttggctaacggttgggacaagaacaaagaaccagacaacaccgccatcttgttcattaaggacgacaagtactacttgggtgtcatgaacaaaaagaacaataagattttcgacgacaaagctatcaaggagaacaagggtgaagggtacaagaagattgtttataagttgttgccaggtgctaacaaaatgttgccaaaggttttcttctccgctaagtctatcaagttctataacccttctgaagacattttgagaatcagaaaccactccacccacaccaagaacggttctccacaaaagggttacgaaaagttcgaattcaacatcgaagactgtagaaagttcatcgatttctacaagcaatccatttccaagcatccagaatggaaggatttcggtttcagattctctgacactcaaagatacaactccattgatgaattctacagagaagtcgaaaaccaaggttacaagttgactttcgaaaacatctctgaatcttacattgattccgtcgttaaccaaggtaagttgtacttgttccaaatctacaacaaggacttctccgcctactccaagggtagaccaaacttgcacaccttgtactggaaggctttgtttgacgaaagaaacttgcaagatgttgtctacaagctgaacggtgaagctgaattgttttacagaaagcaaagtattccaaagaaaatcactcacccagctaaggaagccattgccaacaagaataaagacaaccctaagaaggaatctgtttttgaatacgacttaatcaaggataagagattcaccgaagacaaattcttcttccattgtccaatcaccatcaacttcaagtcctctggagctaacaagttcaacgatgaaatcaacttgttattgaaggaaaaggctaacgatgttcacatcttgtctatcgctcgtggtgaaagacacttggcttactacactttggttgatggtaagggtaacatcattaagcaagacacctttaacattatcggtaacgacagaatgaagaccaactaccacgacaaattggctgctattgaaaaggacagagactctgctagaaaggactggaaaaagatcaataacatcaaggaaatgaaggaaggttacttgtctcaagttgtccatgaaattgctaagttggttatcgaatacaatgctatcgttgtcttcgaggatttgaacttcggttttaagagaggtagattcaaggttgaaaagcaagtttaccaaaaattggaaaagatgttgattgaaaagttgaactacttagtcttcaaggacaatgaatttgacaagactggtggtgtcttgagagcttaccaattgactgctccattcgaaactttcaagaagatgggtaagcaaaccggtatcatctactacgttccagctggtttcacttctaaaatctgtccagttaccggtttcgtcaaccaattgtacccaaagtacgaatccgtttccaagtcccaagaatttttctccaagttcgacaagatctgttacaacttagacaagggttatttcgagttttccttcgattacaaaaactttggtgacaaagccgctaagggtaaatggactatcgcttctttcggttctagattgatcaacttccgtaactccgataagaaccacaactgggacactagagaagtttacccaaccaaggaattagaaaaattgttgaaggactactctattgaatacggtcacggtgaatgtatcaaggctgccatctgtggtgaatctgataagaagttcttcgctaagctaacttccgtcttgaacaccattttgcaaatgagaaactccaagaccggtactgaattggactacttgatttctccagttgccgatgttaacggtaacttcttcgactctagacaagctccaaagaacatgccacaagacgctgatgctaacggtgcctaccacattggtttgaagggtttgatgttgttgggtcgtattaagaacaaccaagaaggtaagaagttgaacctagtcattaagaacgaagaatacttcgaatttgttcaaaacagaaacaactccagagctgacccaaagaagaagagaaaagtcccgaaaaagaaacgcaaagttgggcgcgccgaggccagcggttccggacgggctgacgcattggacgattttgatctggatatgctgggaagtgacgccctcgatgattttgaccttgacatgcttggttcggatgcccttgatgactttgacctcgacatgctcggcagtgacgcccttgatgatttcgacctggacatgctgattaactctagaagttccggatctccgaaaaagaaacgcaaagttggtagccagtacctgcccgacaccgacgaccggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatcatgaagaagtcccccttcagcggccccaccgaccctagacctccacctagaagaatcgccgtgcccagcagatccagcgccagcgtgccaaaacctgccccccagccttaccccttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttccccagcggccagatctctcaggcctctgctctggctccagcccctcctcaggtgctgcctcaggctcctgctcctgcaccagctccagccatggtgtctgcactggctcaggcaccagcacccgtgcctgtgctggctcctggacctccacaggctgtggctccaccagcccctaaacctacacaggccggcgagggcacactgtctgaagctctgctgcagctgcagttcgacgacgaggatctgggagccctgctgggaaacagcaccgatcctgccgtgttcaccgacctggccagcgtggacaacagcgagttccagcagctgctgaaccagggcatccctgtggcccctcacaccaccgagcccatgctgatggaataccccgaggccatcacccggctcgtgacaggcgctcagaggcctcctgatccagctcctgcccctctgggagcaccaggcctgcctaatggactgctgtctggcgacgaggacttcagctctatcgccgatatggatttctcagccttgctgtaatctttgaaaagataatgtatgattatgctttcactcatatttatacagaaacttgatgttttctttcgagtatatacaaggtgattacatgtacgtttgaagtacaactctagattttgtagtgccctcttgggctagcggtaaaggtgcgcattttttcacaccctacaatgttctgttcaaaagattttggtcaaacgctgtagaagtgaaagttggtgcgcatgtttcggcgttcgaaacttctccgcagtgaaagataaatgatcACTGGTCTCAagatagtaatacgcttaactgctcattAGGTaatttctactgttgtagatgttacttttgatatcactcacaaaattTGAGACCAGTtctagagccgttcccacaaataattatacgtatatgcttcttttcgtttactatatatctatatttacaagcctttattcactgatgcaatttgtttccaaatacttttttggagatctcataactagatatcatgatggcgcaacttggcgctatcttaattactctggctgccaggcccgtgtagagggccgcaagaccttctgtacgccatatagtctctaagaacttgaacaagtttctagacctattgccgcctttcggatcgctattgttgcggccgccagctgaagcttcgtacgctgcaggtcgacgaattcttgactgaatcaccgttgatgcctttatggagaaaaatggtggcctttctaccactgttaggagctgcactaattgctctatccagaactcaagattacagacatcatttcgtcgatgtaattttagggtctatgttgggttatataatggcacactttttctacagaagaatcttcccacccattgatgatcctcttccgttcaaaccattgatggacgattcagatgtcaccctggaggaagcagtcacccatcagaggatcccggatgaggaattacatcctttgtccgatgaaggtatgtaagaataaaaaagaatatatactccacatgacatacgaaatatacgtatttattgttctgtatggaataacagcgattacataaagatgacatgttacttctttattcaaattaatcttgacgtgcaagggcctgcttgttatttcatcggacaatcccaacatcactttacacgaaagccttagaagtttattatttgttttaagttggactatagtgatgtaggtagtttcttaggaagcagttgagtagctgatttttgagataagaacctggtgtaatcaatctataaacagcctagaatctttttaagcaaatttacttttacatttatctctatcttctttcttacaagaagttattttcattacaaaaggacattaaatacactaaattttcaatctttacattgttggaaagcctcgttgtcttttaagattttataagcattgattttttttttcaataattttccgttccccttaacacatactatgtataaatgtcattgagtcatctcactttagatcaatattatgaaatacagtgcaacgaacttgaagcgatacgttccatttatatggatgactttactgacttaactaaaagaaagtctagctgggataagcagc
(e) the fragment obtained in step (d) is introduced into the competence of the engineering bacterium SX-5, screening is carried out by a G418 plate, and colony PCR verification is carried out by using verification primers YZDcPcf 1-F and YZDcPcf 1-R. The correct single colony was verified, streaked on a YPD plate of 5-FOA, cultured at 30 ℃ for 2-3d, and the single colony with the G418 tag removed was selected and designated as SX-6.
The primer sequence is as follows:
dCpf1Tu-F:cgatgttcacatcttgtctatcgctcgtggtgaaagacacttggcttac
dCpf1Tu-R:gccaagtgtctttcaccacgagcgatagacaagatgtgaacatcgttagcct
gal1p23-F1:actggtctcaagatagtaatacgcttaactgctcattggtaagaaattgtctg
gal1p23-R1:aatgagcagttaagcgtattactatcttgagaccagtgatcatttatctttcactgcggagaag
gal7p23-F1:actggtctcaagatgttacttttgatatcactcacaaggtaagaaattgtctg
gal7p23-R1:ttgtgagtgatatcaaaagtaacatcttgagaccagtgatcatttatctttcactgcggagaag
gal1p23-F2:actggtctcaagattggttatgaagaggaaaaattggggtaagaaattgtctg
gal1p23-R2:ccaatttttcctcttcataaccaatcttgagaccagtgatcatttatctttcactgcggagaag
gal7p23-F2:actggtctcaagatcttaacccaaaaataagggaaagggtaagaaattgtctg
gal7p23-R2:ctttcccttatttttgggttaagatcttgagaccagtgatcatttatctttcactgcggagaag
gal1p23-F3:actggtctcaagatatctattaacagatatataaatgggtaagaaattgtctg
gal1p23-R3:catttatatatctgttaatagatatcttgagaccagtgatcatttatctttcactgcggagaag
gal7p23-F3:actggtctcaagatgctagcaaagatataaaagcaggggtaagaaattgtctg
gal7p23-R3:cctgcttttatatctttgctagcatcttgagaccagtgatcatttatctttcactgcggagaag
pFA6a-F:tatctttccatagattttacttcaatatagcaatgagcagtt
pFA6a-R:attccctcaaaaatgaaaaaacccggatctcaaa
gal7pG-F:ttgctatattgaagtaaaatctatggaaagatatggacggta
gal7pG-R:gatccgggttttttcatttttgagggaatattca
CSN-VP-F:atgtatgctatacgaacggtacccagtcacgacgttgtaaaacg
CSN-VP-R:atcatacattatcttttcaaagattacagcaaggctgagaaatccat
KAN1-F:tgtggtcaataagagcgacgagctccagcttttgttccctttagt
KAN1-R:ttttttcttcttttacgtaggtacccaattcgccctatagtgagt
52p-F:ggatttctcagccttgctgtaatctttgaaaagataatgtatgat
52p-R:agagggcctatggtgaatcttgagaccagtgatcatttatctttcactgcggagaa
gal1p23-F:actggtctcaagatagtaatacgcttaa
gal1p23-R:actggtctcaacctaatgagcagttaagcgtattactatct
gal7p23-F:actggtctcaaggtaatttctactgttgtagatgttacttttgatat
gal7p23-R:actggtctcaaattttgtgagtgatatcaaaagtaacatctacaacagt
DPP1UP-F:actagtactcgatttctggcgc
DPP1UP-R:aattcgtcgacctgcagcggtcgcataaggatgcgatatagtga
DPP1Down-F:cgctgcaggtcgacgaattcttgactgaatcaccgttgatgcc
DPP1Down-R:gctgcttatcccagctagactttc
CSN-VPG-F:CCCAGTCACGACGTTGTAAAACG
CSN-VPG-R:GCAATTAACCCTCACTAAAGGYZdCpf1-F：agacaagctccaaagaacatgcca
YZdCpf1-R：atgaaataacaagcaggcccttgca
EXAMPLE 3 construction and use of dCas9-RD inhibition System
(a) Cas9 was mutated to dCas9 using primers dCas-F1, dCas-R1, dCas-F2, dCas-R2 using plasmid pML104, available from Addgene, as a template. Synthesizing a suppression domain RD by a gene, connecting the activation domain RD with pML104 by using seamless cloning to obtain a plasmid pML104-RD, taking the constructed plasmid as a template, taking CIT-F and CIT-R as a primer ring P to obtain a plasmid pML104-RD-CIT, taking MLS1-F and MLS1-R as a primer ring P to obtain a plasmid pML104-RD-MLS, and taking ERG6-F and ERG6-R as a primer ring P to obtain a plasmid pML104-RD-ERG 6. Respectively amplifying by using URA-104-F and URA-104-R as primers to obtain fragments URA-dCas9-RD-CIT, URA-dCas9-RD-MLS and URA-dCas9-RD-ERG 6. Plasmid pFA6a-TRP1-PGAL1-GFP purchased from Addgene is taken as a template, primer pFA6a-F, pFA6a-R is used for amplification to obtain fragment pFA6a-TRP1-GFP, Saccharomyces cerevisiae SC288C genome is taken as a template, primer pMLS-F, pMLS-R is used for amplification to obtain promoter gene fragment pMLS, primer pERG6-F, pERG6-R is used for amplification to obtain promoter gene fragment pERG 53, primer pCIT-F, pCIT-R is used for amplification to obtain promoter gene fragment pCIT, and pMLS and pCIT-F, pERG6 are respectively connected with pFA6a-TRP1-GFP by seamless cloning to obtain plasmids pFA6a-TRP1-pMLS-GFP, pFA6a-TRP1-pCIT-GFP and pFA6 a-pERG 1-6-GFP. Plasmids 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 were introduced into yeast SC288C, respectively, to obtain strains SG-MLS, SG-CIT and SG-ERG6, and green fluorescence intensity was measured using a microplate reader, respectively, as shown in FIG. 4.
(b) Plasmid pFA6a-TRP1-pERG6-GFP is taken as a template, a primer Cas-RD-F, Cas-RD-R is adopted for amplification to obtain a gene fragment dCas9-RD-erg6, a plasmid pCSN068 purchased from Addgene is taken as a template, a primer KAN2-F, KAN2-R is adopted for amplification to obtain a gene fragment KAN-Cas, a saccharomyces cerevisiae SC288C genome is taken as a template, a primer tRNA-G-F and tRNA-G-R are adopted for amplification to obtain a gene fragment tRNA-Gly, and primers MLS-F and MLS-R are directly adopted for overlapping to synthesize a fragment MLS-20. Directly overlapping and synthesizing a fragment CIT2-20 by using primers CIT-F and CIT-R, amplifying by using primers GAL80UP-F and GAL80UP-R to obtain a gene fragment GAL80UP by using a genome of saccharomyces cerevisiae SC288C as a template, and amplifying by using primers GAL80Down-F and GAL80Down-R to obtain a gene fragment GAL80 Down.
(c) Subjecting the fragment obtained in step (b): dCas9-RD-erg6, KAN-Cas, tRNA-Gly, MLS-20, CIT2-20, GAL80UP, GAL80Down, by overlap PCR, to obtain fragment GAL80-KAN-dCas9-RD-sgRNAEMC (see SEQ ID NO 2). GAL80-KAN-dCas 9-RD-sgRNAMC fragment sequence (see SEQ ID NO 2):
aggcatacctaatgctggggatgaaacacgttggacgttttggggcgcctgtctacaggataaagacgggtcggatacctgcacaagcaatttggcacctgcataccccatttccccagtagataacttcaacacacacatcaatgtccctcaccagtttatttccaaaagagacgctttttactacctgactagattttcattttgtttcttttggattgcgcttgcctttgtaggtgtgtcgtttatcctttacgttttgacttggtgctcgaagatgctttcagagatggtgcttatcctcatgtcttttgggtttgtcttcaatacggcagccgttgtcttgcaaacggccgcctctgccatggcaaagaatgctttccatgacgatcatcgtagtgcccaattgggtgcctctatgatgggtatggcttgggcaagtgtctttttatgtatcgtggaatttatcctgctggtcttctggtctgttagggcaaggttggcctctacttactccatcgacaattcaagatacagaacctcctccagatggaatcccttccatagagagaaggagcaagcaactgacccaatattgactgccactggacctgaagacatgcaacaaagtgcaagcatagtggggccttcttccaatgctaatccggtcactgccactgctgctacggaaaaccaacctaaaggtattaacttcttcactataagaaaatcacacgagcgcccggacgatgtctctgtttaaatggcgcaagttttccgctttgtaatatatatttatacccctttcttctctcccctgcaatataatagtttaattctaatattaataatatcctatattttcttcatttaccggcgcactctcgcccgaacgacctcaaaatgtctgctacattcataataaccaaaagctcataacttttttttttgaacctgaatatatatacatcacatatcactgctggtccttgccgaccagcgtatacaatctcgatagttggtttcccgttctttccactcccgtcatggactacaacaagagatcttcggtctcaaccgtgcctaatgcagctcccataagagtcggattcgtcggtctcaacgcagccaaaggatgggcaatcaagacacattaccccgccatcgctgcaggtcgacgaattctaccgttcgtataatgtatgctatacgaagttatagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtgtcgaaaacgagctcataacttcgtataatgtatgctatacgaacggtatcattatcaatactgccatttcaaagaatacgtaaataattaatagtagtgattttcctaactttatttagtcaaaaaattagccttttaattctgctgtaacccgtacatgcccaaaatagggggcgggttacacagaatatataacatcgtaggtgtctgggtgaacagtttattcctggcatccactaaatataatggagcccgctttttaagctggcatccagaaaaaaaaagaatcccagcaccaaaatattgttttcttcaccaaccatcagttcataggtccattctcttagcgcaactacagagaacaggggcacaaacaggcaaaaaacgggcacaacctcaatggagtgatgcaacctgcctggagtaaatgatgacacaaggcaattgacccacgcatgtatctatctcattttcttacaccttctattaccttctgctctctctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattcccctacttgactaataagtatataaagacggtaggtattgattgtaattctgtaaatctatttcttaaacttcttaaattctacttttatagttagtcttttttttagttttaaaacaccaagaacttagtttcgaataaacacacataaacaaacaaaatggacaagaagtattctatcggactggctatcgggactaatagcgtcgggtgggccgtcatcactgacgagtacaaggtgccctctaagaagttcaaggtgctcgggaacaccgaccggcattccatcaagaaaaatctgatcggagctctcctctttgattcaggggagaccgctgaagcaacccgcctcaagcggactgctagacggcggtacaccaggaggaagaaccggatttgttaccttcaagagatattctccaacgaaatggcaaaggtcgacgacagcttcttccataggctggaagaatcattcctcgtggaagaggataagaagcatgaacggcatcccatcttcggtaatatcgtcgacgaggtggcctatcacgagaaatacccaaccatctaccatcttcgcaaaaagctggtggactcaaccgacaaggcagacctccggcttatctacctggccctggcccacatgattaagttcagaggccacttcctgatcgagggcgacctcaatcctgacaatagcgatgtggataaactgttcatccagctggtgcagacttacaaccagctctttgaagagaaccccatcaatgcaagcggagtcgatgccaaggccattctgtcagcccggctgtcaaagagccgcagacttgagaatcttatcgctcagctgccgggtgaaaagaaaaatggactgttcgggaacctgattgctctttcacttgggctgactcccaatttcaagtctaatttcgacctggcagaggatgccaagctgcaactgtccaaggacacctatgatgacgatctcgacaacctcctggcccagatcggtgaccaatacgccgaccttttccttgctgctaagaatctttctgacgccatcctgctgtctgacattctccgcgtgaacactgaaatcaccaaggcccctctttcagcttcaatgattaagcggtatgatgagcaccaccaggacctgaccctgcttaaggcactcgtccggcagcagcttccggagaagtacaaggaaatcttctttgaccagtcaaagaatggatacgccggctacatcgacggaggtgcctcccaagaggaattttataagtttatcaaacctatccttgagaagatggacggcaccgaagagctcctcgtgaaactgaatcgggaggatctgctgcggaagcagcgcactttcgacaatgggagcattccccaccagatccatcttggggagcttcacgccatccttcggcgccaagaggacttctacccctttcttaaggacaacagggagaagattgagaaaattctcactttccgcatcccctactacgtgggacccctcgccagaggaaatagccggtttgcttggatgaccagaaagtcagaagaaactatcactccctggaacttcgaagaggtggtggacaagggagccagcgctcagtcattcatcgaacggatgactaacttcgataagaacctccccaatgagaaggtcctgccgaaacattccctgctctacgagtactttaccgtgtacaacgagctgaccaaggtgaaatatgtcaccgaagggatgaggaagcccgcattcctgtcaggcgaacaaaagaaggcaattgtggaccttctgttcaagaccaatagaaaggtgaccgtgaagcagctgaaggaggactatttcaagaaaattgaatgcttcgactctgtggagattagcggggtcgaagatcggttcaacgcaagcctgggtacctaccatgatctgcttaagatcatcaaggacaaggattttctggacaatgaggagaaagaggacatccttgaggacattgtcctgactctcactctgttcgaggaccgggaaatgatcgaggagaggcttaagacctacgcccatctgttcgacgataaagtgatgaagcaacttaaacggagaagatataccggatggggacgccttagccgcaaactcatcaacggaatccgggacaaacagagcggaaagaccattcttgatttccttaagagcgacggattcgctaatcgcaacttcatgcaacttatccatgatgattccctgacctttaaggaggacatccagaaggcccaagtgtctggacaaggtgactcactgcacgagcatatcgcaaatctggctggttcacccgctattaagaagggtattctccagaccgtgaaagtcgtggacgagctggtcaaggtgatgggtcgccataaaccagagaacattgtcatcgagatggccagggaaaaccagactacccagaagggacagaagaacagcagggagcggatgaaaagaattgaggaagggattaaggagctcgggtcacagatccttaaagagcacccggtggaaaacacccagcttcagaatgagaagctctatctgtactaccttcaaaatggacgcgatatgtatgtggaccaagagcttgatatcaacaggctctcagactacgacgtggacgctatcgtccctcagagcttcctcaaagacgactcaattgacaataaggtgctgactcgctcagacaagaaccggggaaagtcagataacgtgccctcagaggaagtcgtgaaaaagatgaagaactattggcgccagcttctgaacgcaaagctaatcactcagcggaagttcgacaatctcactaaggctgagaggggcggactgagcgaactggacaaagcaggattcattaaacggcaacttgtggagactcggcagattactaaacatgtagcccaaatccttgactcacgcatgaataccaagtacgacgaaaacgacaaacttatccgcgaggtgaaggtgattaccctgaagtccaagctggtcagcgatttcagaaaggactttcaattctacaaagtgcgggagatcaataactatcatcatgctcatgacgcatatctgaatgccgtggtgggaaccgccctaatcaagaagtacccaaagctggaaagcgagttcgtgtacggagactacaaggtctacgacgtgcgcaagatgattgccaaatctgagcaggagatcggaaaggccaccgcaaagtacttcttctacagcaacatcatgaatttcttcaagaccgaaatcacccttgcaaacggtgagatccggaagaggccgctcatcgagactaatggggagactggcgaaatcgtgtgggacaagggcagagatttcgctaccgtgcgcaaagtgctttctatgcctcaagtgaacatcgtgaagaaaaccgaggtgcaaaccggaggcttttctaaggaatcaatcctccccaagcgcaactccgacaagctcattgcaaggaagaaggattgggaccctaagaagtacggcggattcgattcaccaactgtggcttattctgtcctggtcgtggctaaggtggaaaaaggaaagtctaagaagctcaagagcgtgaaggaactgctgggtatcaccattatggagcgcagctccttcgagaagaacccaattgactttctcgaagccaaaggttacaaggaagtcaagaaggaccttatcatcaagctcccaaagtatagcctgttcgaactggagaatgggcggaagcggatgctcgcctccgctggcgaacttcagaagggtaatgagctggctctcccctccaagtacgtgaatttcctctaccttgcaagccattacgagaagctgaaggggagccccgaggacaacgagcaaaagcaactgtttgtggagcagcataagcattatctggacgagatcattgagcagatttccgagttttctaaacgcgtcattctcgctgatgccaacctcgataaagtccttagcgcatacaataagcacagagacaaaccaattcgggagcaggctgagaatatcatccacctgttcaccctcaccaatcttggtgcccctgccgcattcaagtacttcgacaccaccatcgaccggaaacgctatacctccaccaaagaagtgctggacgccaccctcatccaccagagcatcaccggactttacgaaactcggattgacctctcacagctcggaggggatgagggagctcccaagaaaaagcgcaaggtaggtagttccggatccggtagttccaagcttagtggtggaggaagtggcgggtcagggtcgaattctgcatcttcatctaccaaactagacgacgacttgggtacagcagcagcagtgctatcaaacatgagatcatccccatatagaactcatgataaacccatttccaatgtcaatgacatgaataacacaaatgcgctcggtgtgccggctagtaggcctcattcgtcatcttttccatcaaagggtgtcttaagaccaattctgttacgtatccataattccgaacaacaacccattttcgaaagcaacaattctacatcgggaggtggttcgggtggctctggatcagattcacaagttcaagaactggaaacattaccacccataagaagtttaccgttgcccttcccacacatggactcaggcggtggtagtggtgggagcggtagtaagcttggcggcagcggcggcagctacgaagaagagatcaagcacttgaaactagggctggagcaaagagaccatcaaattgcatctttgaccgtccagcaacagcagcaacagcaacagcagcaacagcaacagcagcaacaggtccagcagcatttacaacagcaacaacagcagctagccgctgcatctgcatctgttccagttgcgtaaactctcgaggcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatgccgagcaaatgcctgcaaatcgctccccatttctctagagcggccgtggtatcgtttagattggcaattacagtgtcttagctcacatgcttataactaattacatgactcgaagacataaaaaacaaaaaaagcaccaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaactttttcttgttactagtatttgcgcaagcccggaatcgaaccgggggcccaacgatggcaacgttggattttaccactaaaccacttgcgcggactagccttattttaacttgctatttctagctctaaaacttttcttaattcttttatgttgcgcaagcccggaatcgaaccgggggcccaacgatggcaacgttggattttaccactaaaccacttgcgcggactagccttattttaacttgctatttctagctctaaaacaaagaaaaagagagcagcaggatcatttatctttcactgcggagaagtttcgaacgccgaaacatgcgcaccaactttcacttctacagcgtttgaccaaaatcttttgaacagaacattgtagggtgtgaaaaaatgcgcacctttaccgctagcccaagagggcactacaaaatctagagttgtacttcaaacgtacatgtaatcaccttgtatatactcgaaagaaaacatcaagtttctgtataaatatgagtgaaagcataatcatacattatcttttcaaagattgtccgcggtggagctccagcttttgaatgcaaggtttcgatttcgaaggctttcccaccttgatggatgctctgatattacacaggttaatcgagagcgtttataaaagtaacatgatgggctccacattaaacgttagcaatatctcgcattatagtttataaaagcatcttgccctgtgcttggcccccagtgcagcgaacgttataaaaacgaatactgagtatatatctatgtaaaacaaccatatcatttcttgttctgaactttgtttacctaactagttttaaatttccctttttcgtgcatgcgggtgttcttatttattagcatactacatttgaaatatcaaatttccttagtagaaaagtgagagaaggtgcactgacacaaaaaataaaatgctacgtataactgtcaaaactttgcagcagcgggcatccttccatcatagcttcaaacatattagcgttcctgatcttcatacccgtgctcaaaatgatcaaacaaactgttattgccaagaaataaacgcaaggctgccttcaaaaactgatccattagatcctcatatcaagcttcctcatagaacgcccaattacaataagcatgttttgctgttatcaccgggtgataggtttgctcaaccatggaaggtagcatggaatcataatttggatactaatacaaatcggccatataatgccattagtaaattgcgctcccatttaggtggttctccaggaatactaataaatgcggtgcatttgcaaaatgaatttattccaaggccaaaacaacacgatgaatggctttatttttttgttattcctgacatgaagctttatgtaattaaggaaacggacatcgaggaatttgcatcttttttagatgaaggagctattcaagcaccaaagctatccttccaggattatttaagcggtaaggccaaggcttcccaacaggttcatgaagtgcatca。
(d) and (c) introducing the fragment obtained in the step (c) into the competence of the engineering bacterium SX-6, screening by a G418 plate, and carrying out colony PCR verification by using verification primers YZDcAs-F and YZDcAs-R. The correct single colony was verified, streaked on a YPD plate of 5-FOA, cultured at 30 ℃ for 2-3d, and the single colony with the G418 tag removed was selected and designated as SX-7.
The primer sequence is as follows:
dCas-F1:tattctatcggactggctatcgggactaatagcgtcgggt
dCas-R1:atagccagtccgatagaatacttcttgtccatggtg
dCas-F2:gacgctatcgtccctcagagcttcctcaaag
dCas-R2:ctctgagggacgatagcgtccacgtcgtagtctgagagcc
CIT-F:agctctaaaactttgctttcaatgtttttgagatcatttatctttcactgcggagaag
CIT-R:tcaaaaacattgaaagcaaagttttagagctagaaatagcaagttaaaataagg
MLS1-F:agctctaaaacgctcaaatcagtgggcgtcggatcatttatctttcactgcggagaag
MLS1-R:cgacgcccactgatttgagcgttttagagctagaaatagcaagttaaaataagg
ERG6-F:agctctaaaacgtttcggcgttttctggcttgatcatttatctttcactgcggagaag
ERG6-R:aagccagaaaacgccgaaacgttttagagctagaaatagcaagttaaaataagg
URA-104-F:cggcatcagagcagattgta
URA-104-R:agcgagtcagtgagcgag
pMLS-F:tattgaagtatgtctaatgcgaaggtactttt
pMLS-R:aattcttcagcagttttcttaattcttttatgt
pERG6-F:cattgctatattgaagtattcgggtgttttctcctatcctctgctgct
pERG6-R:ttttttcttatgctgcctactatattattatt
pCIT-F:tatattgaagtaaattggtgacgttaatctaaa
pCIT-R:ccgggttttttttttcttgttactagtattatt
Cas-RD-F：tcgtataatgtatgctatacgaacggtatcattatcaatactgccatttc
Cas-RD-R：gcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccga
KAN2-F:caatcaagacacattaccccgccatcgctgcaggtcgacgaattctaccgttcgt
KAN2-R:tgaaatggcagtattgataatgataccgttcgtatagcatacatt
tRNA-G-F:tgcgcaagcccggaatcgaa
tRNA-G-R:gcgcaagtggtttagtggtaaaatcc
ERG6-F:aaagataaatgatcctgctgctctctttttctttgttttagagctagaaatagcaagttaaaataagg
ERG6-R:ctagctctaaaacaaagaaaaagagagcagcaggatcatttatctttcactgcggagaa
MLS-F：aaaacttttcttaattcttttatgttgcgcaagcccggaatcgaa
MLS-R：tccgggcttgcgcaacataaaagaattaagaaaagttttagagctagaaat
CIT-F：aaaactttttcttgttactagtatttgcgcaagcccggaatcgaa
CIT-R：tccgggcttgcgcaaatactagtaacaagaaaaagttttagagctagaaat
GAL80UP-F：aggcatacctaatgctggggatga
GAL80UP-R：aattcgtcgacctgcagcgatggcggggtaatgtgtcttgatt
GAL80Down-F：agctccagcttttgaatgcaaggtttcgatttcgaagg
GAL80Down-R：tgatgcacttcatgaacctgttgg
YZdCas-F：cctcatccaccagagcatcac
YZdCas-R：caaaagctggagctccaccg
example 4 fermentation culture of successfully constructed engineered Yeast
Inoculating single colonies of engineering yeast SX-6 and SX-7 on solid YPD plate in 2mLYPD medium, culturing at 30 deg.C and 220rpm for 16-20h, inoculating 1% of the strain into 250mL round bottom shake flask containing 25mLYPD liquid medium, and culturing at 30 deg.C and 220rpm for 96 h. When the fermentation time reached 26h, 50% (volume concentration) ethanol was used as a carbon source for supplementation. After fermentation, the precipitate was centrifuged. The yield of 7-DHC reaches 371.5mg/L and 464mg/L respectively.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> a method for simultaneously enhancing and inhibiting a plurality of key genes in saccharomyces cerevisiae 7-dehydrocholesterol synthesis
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 10119
<212> DNA
<213> (Artificial Synthesis)
<400> 1
actagtactc gatttctggc gcagcaaaaa tatagcatta tgtccgataa acacagttgt 60
gatctgtctt gtgatcgcat actctgcaga taatcagttg aaatagcagc ttttaagtga 120
gaatcttatt cttagtctac atcgttacat tgtatcagtc acaggtacgg agagaaatta 180
tacttttcga tttcattcaa tgtagtttct tttttacatt aaatatagtt ttccagtagt 240
gcactattat taaggcgctt ctgtttttag tcaacacttt ttcagatagt acctttcagg 300
tggttagagt gcgatccctt taaaaaaaag tattcgtcaa cgatgacagg gtaaagaata 360
aatgcagcac gcctggcgta tactgctata attgtacatc atgttatcgg cgttgattct 420
caattgtttg gtgattagct tttatatata gatagaaacc caacgttgga taacctcacg 480
actaactttt ttgtatttta gaaataattt gtcgatcggt tgtatatttt tgtcatatat 540
tatctagaaa cgttagggaa taaactgtta tctagggtcc actaacatac gcgcagttcg 600
gaaatcagca aacatcactt aaaggacacc tgctataaac tgaattgtgt ccaatttttc 660
gagtagttag cagttcaata aagggcacgt tatcaattgt taaaggcaaa gaatcagaat 720
taaatcatag caaacgacca aaatgaacag agtttcgttt attaaaacgc ctttcaacat 780
aggggcgaaa tggagattag aagatgtctt tttgctcatt atcatgatac ttcttaacta 840
cccagtgtat taccaacaac cgttcgaacg tcagttttac attaacgatc tcactatatc 900
gcatccttat gcgaccgctg caggtcgacg aattctaccg ttcgtataat gtatgctata 960
cgaagttata gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg 1020
gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac 1080
tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt 1140
gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga 1200
aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata 1260
aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta 1320
ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa aagactcacg 1380
tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc 1440
gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc 1500
cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg 1560
tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta 1620
ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat 1680
tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc 1740
ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg 1800
ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc 1860
gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac 1920
cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga 1980
aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg 2040
ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa 2100
aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt 2160
ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata 2220
gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt 2280
tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt 2340
caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc 2400
agtgtcgaaa acgagctcat aacttcgtat aatgtatgct atacgaacgg tacccagtca 2460
cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg 2520
ggtacctttt ctttttttgc ggtcaccccc atgtggcggg gaggcagagg agtaggtaga 2580
gcaacgaatc ctactattta tccaaattag tctaggaact ctttttctag attttttaga 2640
tttgagggca agcgctgtta acgactcaga aatgtaagca ctacggagta gaacgagaaa 2700
tccgccatag gtggaaatcc tagcaaaatc ttgcttaccc tagctagcct caggtaagct 2760
agccttagcc tgtcaaattt ttttcaaaat ttggtaagtt tctactagca aagcaaacac 2820
ggttcaacaa accgaaaact ccactcatta tacgtggaaa ccgaaacaaa aaaacaaaaa 2880
ccaaaatact cgccaatgag aaagttgctg cgtttctact ttcgaggaag aggaactgag 2940
aggattgact acgaaagggg caaaaacgag tcgtattctc ccattattgt ctgctaccac 3000
gcggtctagt agaataagca accagtcaac gctaagacag gtaatcaaaa taccagtctg 3060
ctggctacgg gctagttttt acctctttta gaacccactg taaaagtccg ttgtaaagcc 3120
cgttctcact gttggcgttt tttttttttt ggtttagttt cttatttttc atttttttct 3180
ttcatgacca aaaacaaaca aatctcgcga tttgtactgc ggccactggg gcgtggccaa 3240
aaaaatgaca aatttagaaa ccttagtttc tgatttttcc tgttatgagg agatatgata 3300
aaaaatatta ctgctttatt gttttttttt tatctactga aatagagaaa cttacccaag 3360
gaggaggcaa aaaaaagagt atatatacag cagctaccat tcagatttta atatattctt 3420
ttctcttctt ctacactatt attataataa ttttactata ttcattttta gcttaaaacc 3480
tcatagaata ttattcttca gtcactcgct taaatactta tcaaaaatgt ccatctacca 3540
agagtttgtc aacaaatact ctttgtctaa gactttacgt ttcgaattga ttccacaagg 3600
taagactttg gaaaacatta aggctcgtgg tttgatcttg gacgacgaaa agagagccaa 3660
ggattacaag aaggccaagc aaatcatcga taagtaccac caattcttca ttgaagaaat 3720
cttatcctct gtctgtatct ccgaagatct attgcaaaac tactccgatg tctacttcaa 3780
gttgaaaaag tctgacgatg acaacttgca aaaggatttc aaatctgcca aggacaccat 3840
caagaaacaa atttctgaat acataaagga ctctgaaaaa tttaagaact tattcaacca 3900
aaacttgatc gatgctaaga agggtcaaga atctgacttg atcttgtggt tgaagcaatc 3960
taaggacaac ggtatcgaat tgttcaaggc taactctgat atcactgaca ttgacgaagc 4020
tttggaaatc atcaagtctt tcaaaggttg gactacctat ttcaagggtt tccacgaaaa 4080
ccgtaagaat gtctactctt ccaacgacat cccaacttcc atcatttaca gaatcgttga 4140
cgacaatttg ccaaagttcc tagaaaacaa ggccaaatac gaatccttga aggacaaggc 4200
tccagaagcc attaactacg aacaaatcaa gaaggacttg gctgaagaat taactttcga 4260
cattgactac aagacttctg aagttaacca aagagttttc tctttggacg aagtcttcga 4320
gattgctaac ttcaacaact acttgaacca atctggtatc accaaattca acaccatcat 4380
cggtggtaag ttcgtcaacg gtgaaaacac caagagaaag ggtatcaacg aatacattaa 4440
cttgtactct cagcaaatca acgacaagac tttaaagaaa tacaagatgt ctgttttgtt 4500
caagcaaatt ttgtctgaca ctgaatccaa gtcttttgtc attgataagt tggaagatga 4560
ttccgacgtc gttaccacca tgcaatcttt ctacgagcaa atcgctgctt tcaagaccgt 4620
tgaagaaaag tctattaagg aaactttgtc tttgttgttc gacgatttga aggctcaaaa 4680
gttggatttg tccaagattt acttcaagaa tgacaagtct ttgactgatt tgtctcaaca 4740
agttttcgat gactactccg ttattggtac tgctgtcttg gaatacatca cccaacaaat 4800
cgctcctaag aacttggaca acccatccaa gaaggaacaa gaattgattg ccaagaagac 4860
cgaaaaagct aaatacttgt ctttggaaac cattaaattg gctttagaag agttcaacaa 4920
gcacagagat attgacaagc aatgtagatt cgaagaaatt ttggctaact tcgctgctat 4980
cccaatgatc ttcgacgaaa ttgctcaaaa caaggataac ttggctcaaa tctccatcaa 5040
gtaccaaaac cagggtaaga aggatttgtt gcaagcctcc gctgaagatg acgtcaaggc 5100
catcaaagat ttattggacc aaactaacaa cttgttgcac aagctaaaga tcttccacat 5160
ctctcaatct gaagataaag ctaacatttt ggataaggac gaacacttct acttagtttt 5220
cgaagaatgt tacttcgaac tagctaacat cgtcccattg tacaacaaga tcagaaacta 5280
cattactcaa aaaccatact ctgatgaaaa gttcaagtta aacttcgaaa attctacctt 5340
ggctaacggt tgggacaaga acaaagaacc agacaacacc gccatcttgt tcattaagga 5400
cgacaagtac tacttgggtg tcatgaacaa aaagaacaat aagattttcg acgacaaagc 5460
tatcaaggag aacaagggtg aagggtacaa gaagattgtt tataagttgt tgccaggtgc 5520
taacaaaatg ttgccaaagg ttttcttctc cgctaagtct atcaagttct ataacccttc 5580
tgaagacatt ttgagaatca gaaaccactc cacccacacc aagaacggtt ctccacaaaa 5640
gggttacgaa aagttcgaat tcaacatcga agactgtaga aagttcatcg atttctacaa 5700
gcaatccatt tccaagcatc cagaatggaa ggatttcggt ttcagattct ctgacactca 5760
aagatacaac tccattgatg aattctacag agaagtcgaa aaccaaggtt acaagttgac 5820
tttcgaaaac atctctgaat cttacattga ttccgtcgtt aaccaaggta agttgtactt 5880
gttccaaatc tacaacaagg acttctccgc ctactccaag ggtagaccaa acttgcacac 5940
cttgtactgg aaggctttgt ttgacgaaag aaacttgcaa gatgttgtct acaagctgaa 6000
cggtgaagct gaattgtttt acagaaagca aagtattcca aagaaaatca ctcacccagc 6060
taaggaagcc attgccaaca agaataaaga caaccctaag aaggaatctg tttttgaata 6120
cgacttaatc aaggataaga gattcaccga agacaaattc ttcttccatt gtccaatcac 6180
catcaacttc aagtcctctg gagctaacaa gttcaacgat gaaatcaact tgttattgaa 6240
ggaaaaggct aacgatgttc acatcttgtc tatcgctcgt ggtgaaagac acttggctta 6300
ctacactttg gttgatggta agggtaacat cattaagcaa gacaccttta acattatcgg 6360
taacgacaga atgaagacca actaccacga caaattggct gctattgaaa aggacagaga 6420
ctctgctaga aaggactgga aaaagatcaa taacatcaag gaaatgaagg aaggttactt 6480
gtctcaagtt gtccatgaaa ttgctaagtt ggttatcgaa tacaatgcta tcgttgtctt 6540
cgaggatttg aacttcggtt ttaagagagg tagattcaag gttgaaaagc aagtttacca 6600
aaaattggaa aagatgttga ttgaaaagtt gaactactta gtcttcaagg acaatgaatt 6660
tgacaagact ggtggtgtct tgagagctta ccaattgact gctccattcg aaactttcaa 6720
gaagatgggt aagcaaaccg gtatcatcta ctacgttcca gctggtttca cttctaaaat 6780
ctgtccagtt accggtttcg tcaaccaatt gtacccaaag tacgaatccg tttccaagtc 6840
ccaagaattt ttctccaagt tcgacaagat ctgttacaac ttagacaagg gttatttcga 6900
gttttccttc gattacaaaa actttggtga caaagccgct aagggtaaat ggactatcgc 6960
ttctttcggt tctagattga tcaacttccg taactccgat aagaaccaca actgggacac 7020
tagagaagtt tacccaacca aggaattaga aaaattgttg aaggactact ctattgaata 7080
cggtcacggt gaatgtatca aggctgccat ctgtggtgaa tctgataaga agttcttcgc 7140
taagctaact tccgtcttga acaccatttt gcaaatgaga aactccaaga ccggtactga 7200
attggactac ttgatttctc cagttgccga tgttaacggt aacttcttcg actctagaca 7260
agctccaaag aacatgccac aagacgctga tgctaacggt gcctaccaca ttggtttgaa 7320
gggtttgatg ttgttgggtc gtattaagaa caaccaagaa ggtaagaagt tgaacctagt 7380
cattaagaac gaagaatact tcgaatttgt tcaaaacaga aacaactcca gagctgaccc 7440
aaagaagaag agaaaagtcc cgaaaaagaa acgcaaagtt gggcgcgccg aggccagcgg 7500
ttccggacgg gctgacgcat tggacgattt tgatctggat atgctgggaa gtgacgccct 7560
cgatgatttt gaccttgaca tgcttggttc ggatgccctt gatgactttg acctcgacat 7620
gctcggcagt gacgcccttg atgatttcga cctggacatg ctgattaact ctagaagttc 7680
cggatctccg aaaaagaaac gcaaagttgg tagccagtac ctgcccgaca ccgacgaccg 7740
gcaccggatc gaggaaaagc ggaagcggac ctacgagaca ttcaagagca tcatgaagaa 7800
gtcccccttc agcggcccca ccgaccctag acctccacct agaagaatcg ccgtgcccag 7860
cagatccagc gccagcgtgc caaaacctgc cccccagcct taccccttca ccagcagcct 7920
gagcaccatc aactacgacg agttccctac catggtgttc cccagcggcc agatctctca 7980
ggcctctgct ctggctccag cccctcctca ggtgctgcct caggctcctg ctcctgcacc 8040
agctccagcc atggtgtctg cactggctca ggcaccagca cccgtgcctg tgctggctcc 8100
tggacctcca caggctgtgg ctccaccagc ccctaaacct acacaggccg gcgagggcac 8160
actgtctgaa gctctgctgc agctgcagtt cgacgacgag gatctgggag ccctgctggg 8220
aaacagcacc gatcctgccg tgttcaccga cctggccagc gtggacaaca gcgagttcca 8280
gcagctgctg aaccagggca tccctgtggc ccctcacacc accgagccca tgctgatgga 8340
ataccccgag gccatcaccc ggctcgtgac aggcgctcag aggcctcctg atccagctcc 8400
tgcccctctg ggagcaccag gcctgcctaa tggactgctg tctggcgacg aggacttcag 8460
ctctatcgcc gatatggatt tctcagcctt gctgtaatct ttgaaaagat aatgtatgat 8520
tatgctttca ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg 8580
attacatgta cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta 8640
aaggtgcgca ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg 8700
tagaagtgaa agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa 8760
atgatcactg gtctcaagat agtaatacgc ttaactgctc attaggtaat ttctactgtt 8820
gtagatgtta cttttgatat cactcacaaa atttgagacc agttctagag ccgttcccac 8880
aaataattat acgtatatgc ttcttttcgt ttactatata tctatattta caagccttta 8940
ttcactgatg caatttgttt ccaaatactt ttttggagat ctcataacta gatatcatga 9000
tggcgcaact tggcgctatc ttaattactc tggctgccag gcccgtgtag agggccgcaa 9060
gaccttctgt acgccatata gtctctaaga acttgaacaa gtttctagac ctattgccgc 9120
ctttcggatc gctattgttg cggccgccag ctgaagcttc gtacgctgca ggtcgacgaa 9180
ttcttgactg aatcaccgtt gatgccttta tggagaaaaa tggtggcctt tctaccactg 9240
ttaggagctg cactaattgc tctatccaga actcaagatt acagacatca tttcgtcgat 9300
gtaattttag ggtctatgtt gggttatata atggcacact ttttctacag aagaatcttc 9360
ccacccattg atgatcctct tccgttcaaa ccattgatgg acgattcaga tgtcaccctg 9420
gaggaagcag tcacccatca gaggatcccg gatgaggaat tacatccttt gtccgatgaa 9480
ggtatgtaag aataaaaaag aatatatact ccacatgaca tacgaaatat acgtatttat 9540
tgttctgtat ggaataacag cgattacata aagatgacat gttacttctt tattcaaatt 9600
aatcttgacg tgcaagggcc tgcttgttat ttcatcggac aatcccaaca tcactttaca 9660
cgaaagcctt agaagtttat tatttgtttt aagttggact atagtgatgt aggtagtttc 9720
ttaggaagca gttgagtagc tgatttttga gataagaacc tggtgtaatc aatctataaa 9780
cagcctagaa tctttttaag caaatttact tttacattta tctctatctt ctttcttaca 9840
agaagttatt ttcattacaa aaggacatta aatacactaa attttcaatc tttacattgt 9900
tggaaagcct cgttgtcttt taagatttta taagcattga tttttttttt caataatttt 9960
ccgttcccct taacacatac tatgtataaa tgtcattgag tcatctcact ttagatcaat 10020
attatgaaat acagtgcaac gaacttgaag cgatacgttc catttatatg gatgacttta 10080
ctgacttaac taaaagaaag tctagctggg ataagcagc 10119
<210> 2
<211> 10119
<212> DNA
<213> (Artificial Synthesis)
<400> 2
aggcatacct aatgctgggg atgaaacacg ttggacgttt tggggcgcct gtctacagga 60
taaagacggg tcggatacct gcacaagcaa tttggcacct gcatacccca tttccccagt 120
agataacttc aacacacaca tcaatgtccc tcaccagttt atttccaaaa gagacgcttt 180
ttactacctg actagatttt cattttgttt cttttggatt gcgcttgcct ttgtaggtgt 240
gtcgtttatc ctttacgttt tgacttggtg ctcgaagatg ctttcagaga tggtgcttat 300
cctcatgtct tttgggtttg tcttcaatac ggcagccgtt gtcttgcaaa cggccgcctc 360
tgccatggca aagaatgctt tccatgacga tcatcgtagt gcccaattgg gtgcctctat 420
gatgggtatg gcttgggcaa gtgtcttttt atgtatcgtg gaatttatcc tgctggtctt 480
ctggtctgtt agggcaaggt tggcctctac ttactccatc gacaattcaa gatacagaac 540
ctcctccaga tggaatccct tccatagaga gaaggagcaa gcaactgacc caatattgac 600
tgccactgga cctgaagaca tgcaacaaag tgcaagcata gtggggcctt cttccaatgc 660
taatccggtc actgccactg ctgctacgga aaaccaacct aaaggtatta acttcttcac 720
tataagaaaa tcacacgagc gcccggacga tgtctctgtt taaatggcgc aagttttccg 780
ctttgtaata tatatttata cccctttctt ctctcccctg caatataata gtttaattct 840
aatattaata atatcctata ttttcttcat ttaccggcgc actctcgccc gaacgacctc 900
aaaatgtctg ctacattcat aataaccaaa agctcataac tttttttttt gaacctgaat 960
atatatacat cacatatcac tgctggtcct tgccgaccag cgtatacaat ctcgatagtt 1020
ggtttcccgt tctttccact cccgtcatgg actacaacaa gagatcttcg gtctcaaccg 1080
tgcctaatgc agctcccata agagtcggat tcgtcggtct caacgcagcc aaaggatggg 1140
caatcaagac acattacccc gccatcgctg caggtcgacg aattctaccg ttcgtataat 1200
gtatgctata cgaagttata gatctgttta gcttgcctcg tccccgccgg gtcacccggc 1260
cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc 1320
atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc 1380
catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg 1440
cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta 1500
gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa 1560
aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa 1620
aagactcacg tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat 1680
aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag 1740
cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca 1800
gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat 1860
tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca 1920
ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg 1980
ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta 2040
tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt 2100
gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg 2160
ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt 2220
gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac 2280
caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg 2340
ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg 2400
ctcgatgagt ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt 2460
catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt 2520
atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa 2580
tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac 2640
gccgccatcc agtgtcgaaa acgagctcat aacttcgtat aatgtatgct atacgaacgg 2700
tatcattatc aatactgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc 2760
taactttatt tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa 2820
atagggggcg ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc 2880
ctggcatcca ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga 2940
atcccagcac caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta 3000
gcgcaactac agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt 3060
gatgcaacct gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc 3120
tcattttctt acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa 3180
aaggttgaaa ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg 3240
gtaggtattg attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat 3300
agttagtctt ttttttagtt ttaaaacacc aagaacttag tttcgaataa acacacataa 3360
acaaacaaaa tggacaagaa gtattctatc ggactggcta tcgggactaa tagcgtcggg 3420
tgggccgtca tcactgacga gtacaaggtg ccctctaaga agttcaaggt gctcgggaac 3480
accgaccggc attccatcaa gaaaaatctg atcggagctc tcctctttga ttcaggggag 3540
accgctgaag caacccgcct caagcggact gctagacggc ggtacaccag gaggaagaac 3600
cggatttgtt accttcaaga gatattctcc aacgaaatgg caaaggtcga cgacagcttc 3660
ttccataggc tggaagaatc attcctcgtg gaagaggata agaagcatga acggcatccc 3720
atcttcggta atatcgtcga cgaggtggcc tatcacgaga aatacccaac catctaccat 3780
cttcgcaaaa agctggtgga ctcaaccgac aaggcagacc tccggcttat ctacctggcc 3840
ctggcccaca tgattaagtt cagaggccac ttcctgatcg agggcgacct caatcctgac 3900
aatagcgatg tggataaact gttcatccag ctggtgcaga cttacaacca gctctttgaa 3960
gagaacccca tcaatgcaag cggagtcgat gccaaggcca ttctgtcagc ccggctgtca 4020
aagagccgca gacttgagaa tcttatcgct cagctgccgg gtgaaaagaa aaatggactg 4080
ttcgggaacc tgattgctct ttcacttggg ctgactccca atttcaagtc taatttcgac 4140
ctggcagagg atgccaagct gcaactgtcc aaggacacct atgatgacga tctcgacaac 4200
ctcctggccc agatcggtga ccaatacgcc gaccttttcc ttgctgctaa gaatctttct 4260
gacgccatcc tgctgtctga cattctccgc gtgaacactg aaatcaccaa ggcccctctt 4320
tcagcttcaa tgattaagcg gtatgatgag caccaccagg acctgaccct gcttaaggca 4380
ctcgtccggc agcagcttcc ggagaagtac aaggaaatct tctttgacca gtcaaagaat 4440
ggatacgccg gctacatcga cggaggtgcc tcccaagagg aattttataa gtttatcaaa 4500
cctatccttg agaagatgga cggcaccgaa gagctcctcg tgaaactgaa tcgggaggat 4560
ctgctgcgga agcagcgcac tttcgacaat gggagcattc cccaccagat ccatcttggg 4620
gagcttcacg ccatccttcg gcgccaagag gacttctacc cctttcttaa ggacaacagg 4680
gagaagattg agaaaattct cactttccgc atcccctact acgtgggacc cctcgccaga 4740
ggaaatagcc ggtttgcttg gatgaccaga aagtcagaag aaactatcac tccctggaac 4800
ttcgaagagg tggtggacaa gggagccagc gctcagtcat tcatcgaacg gatgactaac 4860
ttcgataaga acctccccaa tgagaaggtc ctgccgaaac attccctgct ctacgagtac 4920
tttaccgtgt acaacgagct gaccaaggtg aaatatgtca ccgaagggat gaggaagccc 4980
gcattcctgt caggcgaaca aaagaaggca attgtggacc ttctgttcaa gaccaataga 5040
aaggtgaccg tgaagcagct gaaggaggac tatttcaaga aaattgaatg cttcgactct 5100
gtggagatta gcggggtcga agatcggttc aacgcaagcc tgggtaccta ccatgatctg 5160
cttaagatca tcaaggacaa ggattttctg gacaatgagg agaaagagga catccttgag 5220
gacattgtcc tgactctcac tctgttcgag gaccgggaaa tgatcgagga gaggcttaag 5280
acctacgccc atctgttcga cgataaagtg atgaagcaac ttaaacggag aagatatacc 5340
ggatggggac gccttagccg caaactcatc aacggaatcc gggacaaaca gagcggaaag 5400
accattcttg atttccttaa gagcgacgga ttcgctaatc gcaacttcat gcaacttatc 5460
catgatgatt ccctgacctt taaggaggac atccagaagg cccaagtgtc tggacaaggt 5520
gactcactgc acgagcatat cgcaaatctg gctggttcac ccgctattaa gaagggtatt 5580
ctccagaccg tgaaagtcgt ggacgagctg gtcaaggtga tgggtcgcca taaaccagag 5640
aacattgtca tcgagatggc cagggaaaac cagactaccc agaagggaca gaagaacagc 5700
agggagcgga tgaaaagaat tgaggaaggg attaaggagc tcgggtcaca gatccttaaa 5760
gagcacccgg tggaaaacac ccagcttcag aatgagaagc tctatctgta ctaccttcaa 5820
aatggacgcg atatgtatgt ggaccaagag cttgatatca acaggctctc agactacgac 5880
gtggacgcta tcgtccctca gagcttcctc aaagacgact caattgacaa taaggtgctg 5940
actcgctcag acaagaaccg gggaaagtca gataacgtgc cctcagagga agtcgtgaaa 6000
aagatgaaga actattggcg ccagcttctg aacgcaaagc taatcactca gcggaagttc 6060
gacaatctca ctaaggctga gaggggcgga ctgagcgaac tggacaaagc aggattcatt 6120
aaacggcaac ttgtggagac tcggcagatt actaaacatg tagcccaaat ccttgactca 6180
cgcatgaata ccaagtacga cgaaaacgac aaacttatcc gcgaggtgaa ggtgattacc 6240
ctgaagtcca agctggtcag cgatttcaga aaggactttc aattctacaa agtgcgggag 6300
atcaataact atcatcatgc tcatgacgca tatctgaatg ccgtggtggg aaccgcccta 6360
atcaagaagt acccaaagct ggaaagcgag ttcgtgtacg gagactacaa ggtctacgac 6420
gtgcgcaaga tgattgccaa atctgagcag gagatcggaa aggccaccgc aaagtacttc 6480
ttctacagca acatcatgaa tttcttcaag accgaaatca cccttgcaaa cggtgagatc 6540
cggaagaggc cgctcatcga gactaatggg gagactggcg aaatcgtgtg ggacaagggc 6600
agagatttcg ctaccgtgcg caaagtgctt tctatgcctc aagtgaacat cgtgaagaaa 6660
accgaggtgc aaaccggagg cttttctaag gaatcaatcc tccccaagcg caactccgac 6720
aagctcattg caaggaagaa ggattgggac cctaagaagt acggcggatt cgattcacca 6780
actgtggctt attctgtcct ggtcgtggct aaggtggaaa aaggaaagtc taagaagctc 6840
aagagcgtga aggaactgct gggtatcacc attatggagc gcagctcctt cgagaagaac 6900
ccaattgact ttctcgaagc caaaggttac aaggaagtca agaaggacct tatcatcaag 6960
ctcccaaagt atagcctgtt cgaactggag aatgggcgga agcggatgct cgcctccgct 7020
ggcgaacttc agaagggtaa tgagctggct ctcccctcca agtacgtgaa tttcctctac 7080
cttgcaagcc attacgagaa gctgaagggg agccccgagg acaacgagca aaagcaactg 7140
tttgtggagc agcataagca ttatctggac gagatcattg agcagatttc cgagttttct 7200
aaacgcgtca ttctcgctga tgccaacctc gataaagtcc ttagcgcata caataagcac 7260
agagacaaac caattcggga gcaggctgag aatatcatcc acctgttcac cctcaccaat 7320
cttggtgccc ctgccgcatt caagtacttc gacaccacca tcgaccggaa acgctatacc 7380
tccaccaaag aagtgctgga cgccaccctc atccaccaga gcatcaccgg actttacgaa 7440
actcggattg acctctcaca gctcggaggg gatgagggag ctcccaagaa aaagcgcaag 7500
gtaggtagtt ccggatccgg tagttccaag cttagtggtg gaggaagtgg cgggtcaggg 7560
tcgaattctg catcttcatc taccaaacta gacgacgact tgggtacagc agcagcagtg 7620
ctatcaaaca tgagatcatc cccatataga actcatgata aacccatttc caatgtcaat 7680
gacatgaata acacaaatgc gctcggtgtg ccggctagta ggcctcattc gtcatctttt 7740
ccatcaaagg gtgtcttaag accaattctg ttacgtatcc ataattccga acaacaaccc 7800
attttcgaaa gcaacaattc tacatcggga ggtggttcgg gtggctctgg atcagattca 7860
caagttcaag aactggaaac attaccaccc ataagaagtt taccgttgcc cttcccacac 7920
atggactcag gcggtggtag tggtgggagc ggtagtaagc ttggcggcag cggcggcagc 7980
tacgaagaag agatcaagca cttgaaacta gggctggagc aaagagacca tcaaattgca 8040
tctttgaccg tccagcaaca gcagcaacag caacagcagc aacagcaaca gcagcaacag 8100
gtccagcagc atttacaaca gcaacaacag cagctagccg ctgcatctgc atctgttcca 8160
gttgcgtaaa ctctcgaggc gaatttctta tgatttatga tttttattat taaataagtt 8220
ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc 8280
ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc 8340
gctcttattg accacacctc taccggcatg ccgagcaaat gcctgcaaat cgctccccat 8400
ttctctagag cggccgtggt atcgtttaga ttggcaatta cagtgtctta gctcacatgc 8460
ttataactaa ttacatgact cgaagacata aaaaacaaaa aaagcaccac cgactcggtg 8520
ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa 8580
actttttctt gttactagta tttgcgcaag cccggaatcg aaccgggggc ccaacgatgg 8640
caacgttgga ttttaccact aaaccacttg cgcggactag ccttatttta acttgctatt 8700
tctagctcta aaacttttct taattctttt atgttgcgca agcccggaat cgaaccgggg 8760
gcccaacgat ggcaacgttg gattttacca ctaaaccact tgcgcggact agccttattt 8820
taacttgcta tttctagctc taaaacaaag aaaaagagag cagcaggatc atttatcttt 8880
cactgcggag aagtttcgaa cgccgaaaca tgcgcaccaa ctttcacttc tacagcgttt 8940
gaccaaaatc ttttgaacag aacattgtag ggtgtgaaaa aatgcgcacc tttaccgcta 9000
gcccaagagg gcactacaaa atctagagtt gtacttcaaa cgtacatgta atcaccttgt 9060
atatactcga aagaaaacat caagtttctg tataaatatg agtgaaagca taatcataca 9120
ttatcttttc aaagattgtc cgcggtggag ctccagcttt tgaatgcaag gtttcgattt 9180
cgaaggcttt cccaccttga tggatgctct gatattacac aggttaatcg agagcgttta 9240
taaaagtaac atgatgggct ccacattaaa cgttagcaat atctcgcatt atagtttata 9300
aaagcatctt gccctgtgct tggcccccag tgcagcgaac gttataaaaa cgaatactga 9360
gtatatatct atgtaaaaca accatatcat ttcttgttct gaactttgtt tacctaacta 9420
gttttaaatt tccctttttc gtgcatgcgg gtgttcttat ttattagcat actacatttg 9480
aaatatcaaa tttccttagt agaaaagtga gagaaggtgc actgacacaa aaaataaaat 9540
gctacgtata actgtcaaaa ctttgcagca gcgggcatcc ttccatcata gcttcaaaca 9600
tattagcgtt cctgatcttc atacccgtgc tcaaaatgat caaacaaact gttattgcca 9660
agaaataaac gcaaggctgc cttcaaaaac tgatccatta gatcctcata tcaagcttcc 9720
tcatagaacg cccaattaca ataagcatgt tttgctgtta tcaccgggtg ataggtttgc 9780
tcaaccatgg aaggtagcat ggaatcataa tttggatact aatacaaatc ggccatataa 9840
tgccattagt aaattgcgct cccatttagg tggttctcca ggaatactaa taaatgcggt 9900
gcatttgcaa aatgaattta ttccaaggcc aaaacaacac gatgaatggc tttatttttt 9960
tgttattcct gacatgaagc tttatgtaat taaggaaacg gacatcgagg aatttgcatc 10020
ttttttagat gaaggagcta ttcaagcacc aaagctatcc ttccaggatt atttaagcgg 10080
taaggccaag gcttcccaac aggttcatga agtgcatca 10119

Claims (10)

1. A method for simultaneously enhancing and inhibiting a plurality of key genes in the synthesis of 7-dehydrocholesterol of saccharomyces cerevisiae, which is characterized by simultaneously enhancing the expression of ethanol dehydrogenase adh2, truncated HMG-CoA reductase tHMG1, isopentenyl diphosphate isomerase idi1, squalene epoxidase erg1, lanosterol 14-alpha-demethylase erg11, C-14 sterol reductase erg24, C-4 methyl sterol oxidase erg25, C-3 sterol dehydrogenase erg26, 3-ketosterol reductase erg27 and codon-optimized exogenous gene sterol Delta 24-reductase dhcr24 in saccharomyces cerevisiae and/or simultaneously inhibiting malate synthase mls1, citrate synthase cit2 and Delta (24) -sterol C-methyltransferase erg 6.
2. The method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol of Saccharomyces cerevisiae according to claim 1, wherein the ethanol dehydrogenase adh2 is numbered NM _001182812.1, the truncated HMG-CoA reductase tHMG1 is numbered XM _033912352.1, the isopentenyl diphosphate isomerase idi1 is numbered NM _001183931.1, the squalene epoxidase erg1 is numbered NM _001181304.1, the lanosterol 14- α -demethylase erg11 is numbered NM _001179137.1, the C-14 sterol reductase erg24 is numbered ID NM _001183118.1, the C-4 methyl sterol oxidase erg25 is numbered ID: NM _001181189.3, the C-3 sterol dehydrogenase erg26 is numbered NM _001180866.1, the 3-ketosterol reductase erg27 is numbered NM _001181987.1, and the exogenous gene reductase 24-dhidr 24 is numbered NM _001031288.1, The malate synthase mls1 is numbered ID NM-001182955.1, the citrate synthase cit2 is numbered ID NM-001178718.1, and the Delta (24) -sterol C-methyltransferase erg6 is numbered ID NM-001182363.1.
3. The method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of saccharomyces cerevisiae 7-dehydrocholesterol as claimed in claim 1, wherein the alcohol dehydrogenase adh2, truncated HMG-CoA reductase tmg 1, isopentenyl diphosphate isomerase idi1, squalene epoxidase erg1, lanosterol 14- α -demethylase erg11, C-14 sterol reductase erg24, C-4 methyl sterol oxidase erg25, C-3 sterol dehydrogenase erg26, 3-ketosterol reductase erg27 and codon optimized exogenous gene sterol Δ 24-reductase dhcr24 are expressed by using inducible promoters gal1p and gal7 p.
4. The method for simultaneously enhancing and suppressing multiple key genes in the synthesis of 7-dehydrocholesterol of Saccharomyces cerevisiae as claimed in claim 3, wherein the strength of the inducible promoters gal1p and gal7p is enhanced using dCpf1-VP activation system.
5. The method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol of saccharomyces cerevisiae according to claim 4, wherein the gene segment for the function of the dCpf1-VP activation system is DPP1-KAN-dCpf1-VP-crRNA1-7-1, and the sequence of the DPP1-KAN-dCpf1-VP-crRNA1-7-1 is shown in SEQ ID NO 1.
6. The method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol of saccharomyces cerevisiae as claimed in claim 1, wherein the malate synthase mls1, citrate synthase cit2, Delta (24) -sterol C-methyltransferase erg6 are inhibited by dCas9-RD inhibition system.
7. The method for simultaneously enhancing and inhibiting multiple key genes in the synthesis of 7-dehydrocholesterol of saccharomyces cerevisiae according to claim 6, wherein the gene segment in which the dCas9-RD inhibition system functions is GAL80-KAN-dCas 9-RD-sgrnamc, and the sequence of GAL80-KAN-dCas 9-RD-sgrnamc is shown in SEQ ID NO 2.
8. An engineered yeast obtained by simultaneously enhancing expression of alcohol dehydrogenase adh2, truncated HMG-CoA reductase tmgh 1, isopentenyl diphosphate isomerase idi1, squalene epoxidase erg1, lanosterol 14- α -demethylase erg11, C-14 sterol reductase erg24, C-4 methyl sterol oxidase erg25, C-3 sterol dehydrogenase erg26, 3-ketosterol reductase erg27, and codon-optimized exogenous gene sterol Δ 24-reductase dhcr24 in saccharomyces cerevisiae, and/or simultaneously inhibiting malate synthase mls1, citrate synthase cit2, Delta (24) -sterol C-methyltransferase erg 6.
9. The use of the engineered yeast of claim 8 in the synthesis of 7-dehydrocholesterol.
10. The use according to claim 9, wherein the seed liquid of the engineered yeast is subjected to shake flask fermentation in a culture medium.
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