CN115044603A - Method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism - Google Patents

Method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism Download PDF

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CN115044603A
CN115044603A CN202210488049.0A CN202210488049A CN115044603A CN 115044603 A CN115044603 A CN 115044603A CN 202210488049 A CN202210488049 A CN 202210488049A CN 115044603 A CN115044603 A CN 115044603A
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范超
刘龙
吴文忠
陈坚
吕雪芹
修翔
齐佳琨
洪皓
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Innobio Corp ltd
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Abstract

The invention discloses a method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism, which utilizes a key gene related to acetyl coenzyme A catabolism to reduce the decomposition of acetyl coenzyme A and balance the reducing power in cells, thereby efficiently synthesizing the 7-dehydrocholesterol. The method overcomes the defects that the traditional technology that only acetyl coenzyme A is enhanced to promote 7-dehydrocholesterol is adopted, but genes related to 7-dehydrocholesterol synthesis are provided, and the synthesis of acetyl coenzyme A is enhanced through overexpression and knockout of the genes, and the consumption of acetyl coenzyme A is reduced, and the embodiment proves that the final yield of 7-DHC reaches 269 mg/L; the yield of 7-DHC is improved by 25 times compared with that of SC 001; the purpose of enhancing the synthesis of the 7-dehydrocholesterol in the microorganisms is achieved.

Description

Method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism
Technical Field
The invention relates to a method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism, in particular to a method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism.
Background
Vitamin D 3 (Cholecalciferol,VD 3 ) Is a fat-soluble vitamin necessary for human body, regulates the metabolism of calcium and phosphorus in the human body, and is often used as a medicament for treating rickets, osteomalacia, maintaining calcium homeostasis and the like. Adult healthy human beings can convert cholesterol taken in vivo into VD 3 Without the need of additional VD 3 . However, with the increase of the global aging population, the outdoor activities of the aged are reduced, and with the increase of the age, the reduction of the absorption capacity of the human body and the deterioration of the visceral functions, the VD (vacuum distillation) can be caused 3 Osteoporosis due to deficiency. Not only the old needs to supplement VD 3 The infants and young children are not fully mature in absorption capacity at birth and need to supply nutrients required by the body for rapid growth, so additional VD needs to be supplemented 3 . 7-dehydrocholestrol (7-dehydrocholestrol, 7-DHC) is synthesized VD 3 Which is converted into VD by ultraviolet irradiation 3 .7-DHC is an important steroid hormone drug intermediate, can not synthesize VD3 by light, and is also an intermediate for synthesizing steroid drugs such as androstenedione (4 AD). To satisfy global VD 3 Will be obtained industriallyThe obtained 7-DHC is converted into VD under the irradiation of a high-pressure mercury lamp with the wavelength of 230-300nm 3 Therefore, the core problem in the production of VD3 lies in the synthesis of 7-DHC.
The traditional industrialized preparation method of 7-DHC mainly comprises an extraction method (using cod liver oil as a raw material) and a chemical synthesis method, and the extraction method is easily influenced by anaphylactic reaction, so the extraction method is gradually replaced by the chemical synthesis method at present. The chemical synthesis method is to synthesize the 7-DHC by taking lanolin cholesterol as a raw material through oxidation, bromination, elimination and hydrolysis reactions. Since the quality of lanolin cholesterol as a substrate in the chemical synthesis method directly determines the quality of 7-DHC as a final product, it is generally necessary to perform treatments such as acidification in order to obtain high-quality lanolin cholesterol. Although the chemical synthesis method is a commonly used method in the current industrial production, the energy consumption is high, the reaction conditions are harsh, the post-treatment is complicated, and the environmental pollution is serious. The method for producing 7-DHC by using the microbial fermentation method has the advantages of greenness and sustainability, and the microorganism (such as saccharomyces cerevisiae) is safe and reliable and has huge industrial application potential, and is used for producing 7-DHC and VD 3 Excellent underplate cells.
Microbial synthesis of 7-DHC has focused mainly on gene enhancement and blockage of branching pathways in known synthetic pathways, but genes whose enhancement is desired are also suitable for the production of other products. When 7-DHC is produced by using microorganisms, although the enhanced and blocked genes have certain effects on synthesizing target products, the 7-DHC synthesis involves a plurality of synthesis paths, so that metabolic flux imbalance is caused, and negative effects are brought to production strains. VD 3 The microbial synthesis of (A) mainly focuses on the screening of strains, but the screening of the strains is time-consuming and labor-consuming, and a large amount of money is spent for large-scale later-stage screening and fermentation detection to obtain VD (vitamin D) 3 Strains with very low yields. Such as Chinese patent CN103275997A, US2007/0204059A1, Chinese patent CN107075551B and Chinese patent CN 104988168A.
The Chinese patent CN104988168A is to enhance the synthesis flux of acetyl coenzyme A and further enhance the synthesis of 7-DHC, and the enhanced genes include: acs, adh2, acl, and ald 6. However, the use of a constitutive promoter for enhancing the synthesis of acetyl-CoA results in an excessively high metabolic stress of the bacterial cells. Chinese patent CN104988168A and Chinese patent CN103275997A further enhance the synthesis of 7-DHC by knocking out ERG5 and ERG6 genes, but the knocking out ERG5 and ERG6 can cause the obstruction of the ergosterol synthesis of saccharomyces cerevisiae. Ergosterol is an important substance determining the fluidity and permeability of membranes, and directly blocking its synthesis results in slow cell growth and is not favorable for the synthesis of the target product 7-DHC. In patent CN202110160871, part of enzymes in a 7-DHC synthesis path are positioned in each compartmentalization of Saccharomyces cerevisiae by using an oxidase and a mitochondrial localization tag to form a relatively independent 7-DHC synthesis path, meanwhile, the storage space of a precursor substance required by 7-DHC synthesis is increased, the feedback effect is also reduced, in the same compartment, the conversion efficiency between the enzymes is improved, the loss of an acting substrate is reduced, and finally, the yield of 7-DHC is improved by4 times to reach 53.31mg/L, but the mass transfer efficiency of a product in the same compartment is reduced, and the improvement of the production efficiency is prevented. Chinese patent CN202110562366 designs a dynamic regulation system, realizes that metabolic flux flows to a target product to a greater extent, and simultaneously does not influence the growth and reproduction of thalli, finally the yield of 7-DHC reaches 156mg/L, but the generation of byproducts is not effectively weakened, the yield of 7-DHC cannot be further improved, and product components are not beneficial to the separation process.
From the above analysis, the conventional technology has a certain benefit in enhancing the synthesis of 7-dehydrocholesterol by enhancing acetyl-coa, but actually does not achieve the desired effect, because the enhancement of the precursor substance increases the supply of the precursor substance on the downstream path, and thus cannot enhance the synthesis competitiveness of 7-DHC. This problem is urgently solved by those skilled in the art.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for efficiently synthesizing 7-dehydrocholesterol by reducing the decomposition of acetyl coenzyme A and balancing the oxidation-reduction force in cells by utilizing a key gene related to the catabolism of acetyl coenzyme A. The method overcomes the traditional technology that only acetyl-coenzyme A is enhanced to promote 7-dehydrocholesterol, but provides genes related to 7-dehydrocholesterol synthesis, and enhances the synthesis of the acetyl-coenzyme A by over-expressing and knocking out the genes, and simultaneously reduces the consumption of the acetyl-coenzyme A, thereby achieving the purpose of enhancing the synthesis of the 7-dehydrocholesterol in microorganisms.
The invention provides a method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism, which comprises the steps of utilizing a key gene related to acetyl coenzyme A catabolism to carry out gene recombination in engineering bacteria so as to reduce the acetyl coenzyme A decomposition; thereby enhancing 7-dehydrocholesterol synthesis.
Further, a group of key genes involved in acetyl-CoA catabolism that play an important role in 7-DHC synthesis include:
further, a gene aat1 encoding aspartate aminotransferase (SEQ ID No.41), a phosphoserine phosphatase gene ser2 of the phosphoglycerate pathway (SEQ ID No.42), an NADP (+) dependent glutamate dehydrogenase gene gdh1(SEQ ID No.44), an NADP (+) dependent glutamate dehydrogenase gene gdh3(SEQ ID No.43), and a CDP-diacylglycerol-serine O-phosphotidyltransferase gene cho1(SEQ ID No. 45).
Further, the aspartate aminotransferase (EC number of enzyme-EC: 2.6.1.1) is used to catalyze the conversion of oxaloacetate to aspartate; the phosphoserine phosphatase (EC:3.1.3.3) is involved in the biosynthesis of serine and glycine; the glutamate dehydrogenase (EC:1.4.1.4) catalyzes ammonia and alpha-ketoglutaric acid to synthesize glutamic acid; the CDP-diacylglycerol-serine O-phosphotransferase (EC: 2.7.8.8) plays a role in the biosynthesis of phospholipids.
Further, the method of utilizing key genes related to the catabolism of acetyl-CoA is to knock out these genes.
Further, the method for efficiently synthesizing 7-dehydrocholesterol not only comprises the step of utilizing the key genes related to the catabolism of acetyl-CoA and the like, but also comprises the step of utilizing genes for increasing the synthesis of acetyl-CoA to carry out gene recombination in engineering bacteria, thereby enhancing the synthesis of 7-dehydrocholesterol.
Further, the genes for increasing the synthesis of acetyl-CoA include: mitochondrial malate dehydrogenase mae1(SEQ ID No.40), alcohol dehydrogenase II adh2 or D-lactate dehydrogenase dld 1.
Further, the mitochondrial malate dehydrogenase mae1 (EC: 1.1.1.38) is used for catalyzing the oxidative decarboxylation of malate into pyruvate; the D-lactate dehydrogenase dld1 (EC: 1.1.2.4) is used for catalyzing the oxidation of D-lactate into pyruvate; alcohol dehydrogenase II adh 2(EC: 1.1.1.1) was used to catalyze the conversion of ethanol to acetaldehyde.
Further, the method of utilizing a key gene involved in acetyl-CoA synthesis is to enhance expression of a gene that increases acetyl-CoA synthesis.
Further, the expression enhancing means includes the use of the following constitutive promoters: p TDH3 、P TEF1 、P PGK1 、P ENO2 、P TPL1 Or using the following inducible promoter P GAL1 、P GAL10 、P GAL7 . In general, it is meant that, when homologous recombination is carried out in a strain, a constitutive promoter P is used TDH3 、P TEF1 、P PGK1 、P ENO2 、P TPL1 And inducible promoter P GAL1 、P GAL10 、P GAL7 The gene for acetyl-CoA synthesis to be enhanced described above (mitochondrial malate dehydrogenase mae1, alcohol dehydrogenase II adh2 or D-lactate dehydrogenase dld1) was expressed.
Further, the above engineering bacteria are selected from Saccharomyces cerevisiae S288C, Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae Y187, Pichia pastoris x-33, or Candida tropicalis 1798.
Further, the present invention is achieved by overexpressing and knocking out related genes, namely: the genes for increasing the synthesis of acetyl coenzyme A are overexpressed, and key genes related to the catabolism of the acetyl coenzyme A are knocked out, so that the content of the acetyl coenzyme A serving as a precursor for synthesizing 7-DHC is increased, the proportion of the overall cofactor NADH/NAD + in yeast is influenced, and the growth of thalli and the synthesis of 7-dehydrocholesterol are facilitated;
further, by overexpressing and knocking out the relevant genes, the overall cofactor NADH/NAD + ratio in yeast is controlled to be in the range of 0.3 to 0.8, typically in micromolar.
The original saccharomyces cerevisiae S288C is taken as an original strain, a 7-DHC synthesis module is introduced, and a promoter P is used TDH3 The expression of mitochondrial malate dehydrogenase mae1(SEQ ID No.40) was controlled, followed by knock-out of the aat1(SEQ ID No.41), NADP (+) dependent glutamate dehydrogenase gdh1(SEQ ID No.44), NADP (+) dependent glutamate dehydrogenase gdh3(SEQ ID No.43) and CDP-diacylglycerol-serine O-phosphotransferase cho1(SEQ ID No.45) genes encoding aspartate aminotransferase, respectively, using Cas 9.
Further, the 7-DHC synthesis module comprises artificially synthesized gene segments (SEQ ID No.46) of DHCR24 reductase gene of chicken with optimized codon, endogenous C-8 sterol isomerase (ERG2) and endogenous C-5 sterol desaturase (ERG 3).
Further, the C-8 sterol isomerase ERG2(EC:5.3.3.5) is used to catalyze the isomerization of the delta-8 double bond to the delta-7 position during sterol synthesis. The C-5 sterol desaturase, ERG3(EC:1.14.19.20), is used to catalyze the introduction of a C-5 double bond into epi sterols.
Furthermore, the invention takes the original Saccharomyces cerevisiae S288C as an original strain and uses a promoter P TDH3 Controlling the expression of mitochondrial malate dehydrogenase mae1(SEQ ID No.40), then knocking out aat1, NADP (+) dependent glutamate dehydrogenase gdh1(SEQ ID No.44)/gdh3(SEQ ID No.43) and CDP-diacylglycerol-serine O-phosphotransferase cho1(SEQ ID No.45) genes encoding aspartate aminotransferase respectively by using Cas9, detecting the growth state of the strain and the yield of 7-DHC, and finally achieving the yield of 7-DHC of 269 mg/L.
Specifically, the method for knocking out different genes respectively by using Cas9 is a conventional technology in the field, and generally refers to a process of performing experiments according to the operation of a Cas9 gene knock-out kit.
Compared with the prior art, the invention has the following beneficial effects:
1. the present invention identifies key genes involved in acetyl-CoA catabolism that contribute to 7-dehydrocholesterol synthesis;
2. the growth state of the strain is optimized, the burden of redundant genes is reduced, and the yield of 7-DHC is improved under the condition that the biomass of the strain is not reduced;
after the decomposition of acetyl coenzyme A is inhibited, a large amount of metabolic flux flows into a target metabolite, the yield of the target metabolite 7-dehydrocholesterol is remarkably improved to 269mg/L, the biomass is increased, and the subsequent gene modification is facilitated.
3.7-DHC yield, and finally the 7-DHC yield reaches 269 mg/L; the yield of 7-DHC is improved by 25 times compared with that of SC 001;
4. the proportion of the integral cofactor in the path is optimized while the precursor acetyl coenzyme A synthesized by 7-DHC is increased, which is beneficial to the growth of thalli and the improvement of the yield of 7-DHC.
Drawings
FIG. 1 is the overall metabolic pathway;
FIG. 2 shows the production of 7-DHC by the respective strains;
FIG. 3A, B, C is the value of total NADH/NAD + in each strain and a standard curve.
Detailed Description
The following detailed description of the present invention will be given with reference to examples, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
In the present invention, the percentages and percentages are by mass unless otherwise specifically indicated. Unless otherwise specified, the experimental procedures used are conventional, and the materials, reagents and the like used are commercially available.
Example 1
Construction of recombinant Saccharomyces cerevisiae strains for production of 7-DHC
As shown in FIG. 1, the overall metabolic pathway for 7-DHC synthesis is shown. (a) The genome of the saccharomyces cerevisiae engineering bacteria S228C is used as a template, the primers HO-UP-F and HO-UP-R are adopted to amplify to obtain a gene segment HO-UP, the primers HO-Down-F and HO-Down-R are adopted to amplify to obtain a gene segment HO-Down, and the segments HO-UP and HO-Down are respectively used as the upstream and downstream homologous arms of the integrated segment. The promoter fragment TDH3p is obtained by adopting primers TDH3p-F and TDH3p-R for amplification. The gene fragment chicken-derived C-24 reductase (DHCR24: SEQ ID No.46) was synthesized and amplified using primer DH24-F, DH24-R to obtain gene fragment DHCR 24.
(b) The design of sgRNA is carried out by using http:// wyrickbioin fo2.smb. wsu. edu/crimpr. html. website, plasmid pML-104 is used as a template, and a primer Cas9-HO-F and Cas9-HO-R are used for carrying out plasmid loop P, so as to obtain pML-104-HO plasmid replacing 20bp sgRNA.
(c) Making original yeast into competence, introducing the plasmids and fragments in the steps (a) and (b) into saccharomyces cerevisiae, screening on a URA-SD plate, culturing at 30 ℃ for 2-3 days, performing colony PCR verification by using YZ-HO-F and YZ-HO-R, performing streak culture on a single colony verified to obtain a YPD solid plate containing 5-FAO, and eliminating pML-104 plasmids contained in the bacteria to obtain the genetically engineered bacteria, which is named as SC 001.
The primer sequences are as follows:
SEQ ID No.1：HO-UP-F：aatgatttcctccctagctgacct
SEQ ID No.2：HO-UP-R：ggcgagtattgataatgataggttagatcccaggcgtagaac
SEQ ID No.3：HO-Down-F：taatttgcggccatagtgcgtttagaacgcttcatca
SEQ ID No.4：HO-Down-R：acgtaggttttgtctcgctaattgc
SEQ ID No.5：TDH3p-F：ttaacctatcattatcaatactcgccatttcaaaga
SEQ ID No.6：TDH3p-R：agaccaaacggcagacattcgaaactaagttctggtgttttaaaact
SEQ ID No.7：DH24-F：agaacttagtttcgaatgtctgccgtttggtct
SEQ ID No.8：DH24-R：agcgttctaaacgcactatggccgcaaattaaagccttcgag
SEQ ID No.9：Cas9-HO-F：tttctagctctaaaactagcatctagcacatactcggatcatttatctttcactgcggag
SEQ ID No.10：Cas9-HO-R：cgagtatgtgctagatgctagttttagagctagaaatagcaagttaaaataaggctagt
SEQ ID No.11：YZ-HO-F：tgacaatttatgacctgcagtaca
SEQ ID No.12：YZ-HO-R：tcctcggtgaatttctcgcagatagac
example 2
Enhanced synthesis of acetyl-CoA
(a) A gene segment mae1(SEQ ID No.40) is obtained by amplification of primers mae1-F and mae1-R by taking a saccharomyces cerevisiae engineering bacterium S228C genome as a template, a gene segment UPERG6 is obtained by amplification of primers UPERG6-F and UPERG6-R, a gene segment DOWNERG6 is obtained by amplification of primers DOWNERG6-F and DOWNERG6-R, and a promoter segment TDH3p containing a mae1 segment overlapping region is obtained by amplification of primers Mae1-TDH3p-F and Mae1-TDH3 p-R.
(b) The design of sgRNA is carried out by using http:// wyrickbioin fo2.smb. wsu. edu/crimpr. html. website, plasmid pML-104 is used as a template, primers Cas9-ERG6-F and Cas9-ERG6-R are used for carrying out plasmid loop P, and pML-104-ERG6 plasmid replacing 20bp sgRNA is obtained.
(c) Making SC001 yeast into competence, introducing the plasmids and fragments in the steps (a) and (b) into competence, screening on a URA-SD plate, culturing at 30 ℃ for 2-3 days, carrying out colony PCR verification by using YZ-ERG6-F and YZ-ERG6-R, carrying out streak culture on a single colony verified to be correct on a YPD solid plate containing 5-FAO, and eliminating pML-104-ERG6 plasmids contained in the bacteria to obtain the genetically engineered bacteria which is named as SC 002.
The primer sequence is as follows:
SEQ ID No.13：mae1-F:aacttagtttcgaatgcttagaaccagactatccgtttccgtt
SEQ ID No.14：mae1-R:gtgcattgatgtcgaagaacactacaattggttggtgtgcacc
SEQ ID No.15：UPERG6-F:cttaccaccggcaactaaaccaac
SEQ ID No.16：UPERG6-R:atggcgagtattgataatgaaagccacacattcctactataacgtc
SEQ ID No.17：DOWNERG6-F:accaaccaattgtagtgttcttcgacatcaatgcactcaaacctg
SEQ ID No.18：DOWNERG6-R:aaactaaaaatggctcgtgttcatgc
SEQ ID No.19：Mae1-TDH3p-F:gtaggaatgtgtggctttcattatcaatactcgccatttcaaaga
SEQ ID No.20：Mae1-TDH3p-R:atagtctggttctaagcattcgaaactaagttctggtgttttaaaact
SEQ ID No.21：Cas9-ERG6-F:ctagctctaaaacaatttctcaagtacttctgagatcatttatctttcactgcggagaa
SEQ ID No.22：Cas9-ERG6-R:cagaagtacttgagaaattgttttagagctagaaatagcaagttaaaataaggctagtcc
SEQ ID No.23：YZ-ERG6-F:tctctcttgctgggcccccaacac
SEQ ID No.24：YZ-ERG6-R:gtcacgggctagtttcttgttgttagt
example 3
Inhibition of acetyl-CoA catabolism and regulation of redox levels in the overall pathway
(a) The design of sgRNA is carried out by using http:// wyrickbioin fo2.smb. wsu. edu/crimpr. html. website, plasmid pML-104 is used as a template, primers Cas9-AAT1-F and Cas9-AAT1-R are used for carrying out plasmid loop P, and pML-104-AAT1 plasmid replacing 20bp sgRNA is obtained. Plasmid loop P was performed using primers Cas9-GDH1-F and Cas9-GDH1-R to obtain pML-104-GDH1 plasmid with 20bp sgRNA replaced. Plasmid loop P was performed using primers Cas9-GDH3-F and Cas9-GDH3-R to obtain pML-104-GDH3 plasmid with 20bp sgRNA replaced. Primers Cas9-CHO1-F and Cas9-CHO1-R are adopted to carry out plasmid loop P, and pML-104-CHO1 plasmid replacing 20bp sgRNA is obtained.
(b) Preparing SC002 yeast into competence, respectively introducing plasmids obtained in step (a) into competence to respectively obtain strains SC003, SC004, SC005 and SC006, then sequentially introducing all plasmids obtained into SC002 to finally obtain engineering bacteria SC007, screening the obtained strains on a URA-SD plate, culturing at 30 ℃ for 2-3d, verifying the SC003 strain by YZ-AAT1-F sequencing, verifying the SC004 strain by YZ-GDH1-F sequencing, verifying the SC005 strain by YZ-GDH3-F sequencing, verifying the SC006 strain by YZ-CHO1-F sequencing, performing streak culture on a YPD solid plate containing 5-FAO, removing pML-104 plasmids contained in the strains to obtain genetically engineered bacteria, which are respectively named SC X, SC004, SC 56300, SC X, SC X and SC003, SC006, SC 563226, SC004X, SC005X, SC006X and SC007X are strains which respectively construct a 7-DHC synthesis module in S228C yeast, knock out aat1 gene of aspartate aminotransferase, gdh1 gene of NADP (+) dependent glutamate dehydrogenase, gdh3 gene of NADP (+) dependent glutamate dehydrogenase and CHO1 gene of CDP-diacylglycerol-serine O-phosphatidyl transferase after enhancing the synthesis of acetyl coenzyme A and knock out the genes.
The primer sequence is as follows:
SEQ ID No.25：Cas9-AAT1-F:ctagctctaaaacgtagggttgtgacaacacgcgatcatttatctttcactgcggagaa
SEQ ID No.26：Cas9-AAT1-R:gcgtgttgtcacaaccctacgttttagagctagaaatagcaagttaaaataaggctagtcc
SEQ ID No.27：Cas9-GDH1-F:ctagctctaaaacaagaatttcaagatagacaagatcatttatctttcactgcggagaa
SEQ ID No.28：Cas9-GDH1-R:ttgtctatcttgaaattcttgttttagagctagaaatagcaagttaaaataaggctagtcc
SEQ ID No.29：Cas9-GDH3-F:ctagctctaaaacagtgagcgcattcttgaagagatcatttatctttcactgcggagaa
SEQ ID No.30：Cas9-GDH3-R:tcttcaagaatgcgctcactgttttagagctagaaatagcaagttaaaataaggctagtcc
SEQ ID No.31：Cas9-CHO1-F:ctagctctaaaacgtaagcgtaaatctcagacagatcatttatctttcactgcggagaa
SEQ ID No.32：Cas9-CHO1-R:tgtctgagatttacgcttacgttttagagctagaaatagcaagttaaaataaggctagtcc
SEQ ID No.33：YZ-AAT1-F:tggattgagcaattgaaaacctttgc
SEQ ID No.34：YZ-GDH1-F:aagttgctcaaggttacagagtgc
SEQ ID No.35：YZ-GDH3-F:ttcaattcagggtcacgtgggaa
SEQ ID No.36：YZ-CHO1-F:catttcagcatgatgaggaatttg
example 4
Successfully constructed recombinant saccharomyces cerevisiae fermentation level verification
A single colony of saccharomyces cerevisiae SC007X on a solid YPD plate is inoculated in 2mL of YPD medium, cultured for 16-20h at 30 ℃ and 220rpm, and then inoculated into a 250mL of round-bottom shake flask containing 25mL of LYPD liquid medium according to the inoculum size of 2%, and cultured for 96h at 30 ℃ and 220 rpm. When the fermentation time is up to 26h, glucose is added to supplement the carbon source.
Fermenting for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethyl alcohol and 10mL 1.5mol/L potassium hydroxide-methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extracted mixture, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 mu m filtration. 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 nm.
The calculated 7-DHC yield of final SC007X reached 269mg/L (see FIG. 2).
Example 5
Successfully constructed recombinant saccharomyces cerevisiae total intracellular NADH/NAD +
The method comprises the steps of utilizing an NADH/NAD + kit of ATT Biorequest company for detection, firstly centrifuging to obtain thalli, using NAD +/NADH extract for cell lysis, then measuring NADH and NAD + standard substance curves, respectively adding the lysed cells into a 96-well plate, respectively measuring NADH and NAD +, finally comparing with a drawn standard, and calculating the value of total NADH/NAD + in the cells. NADH/NAD + The standard curve and the detection result are shown in FIG. 3, and the data show intracellular NADH/NAD of different constructed strains + Content of intracellular NADH/NAD + The content of (b) is in the range of 0.3-0.8, and a higher yield of 7-DHC can be obtained.
Comparative example 1
Fermentation of the starting Strain Saccharomyces cerevisiae S288C
Inoculating single colony of Saccharomyces cerevisiae S288C on solid YPD plate in 2mLYPD medium, culturing at 30 deg.C and 220rpm for 16-20h, inoculating 2% 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 is up to 26h, glucose is added to supplement the carbon source.
Fermenting for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethyl alcohol and 10mL 1.5mol/L potassium hydroxide-methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extracted mixture, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 mu m filtration. 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 nm.
No production of 7-DHC was detected.
Comparative example 2
Fermentation of recombinant Saccharomyces cerevisiae SC001 for production of 7-DHC
Inoculating SC001 single colony of Saccharomyces cerevisiae on solid YPD plate in 2mLYPD culture medium, culturing at 30 deg.C and 220rpm for 16-20h, inoculating 2% of the culture medium into 250mL round bottom shake flask containing 25mLYPD liquid culture medium, and culturing at 30 deg.C and 220rpm for 96 h. When the fermentation time is up to 26h, glucose is added to supplement the carbon source.
Fermenting for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethyl alcohol and 10mL 1.5mol/L potassium hydroxide-methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extracted mixture, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 mu m filtration. 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 nm.
The final yield of 7-DHC was calculated to be only 10.7 mg/L.
Comparative example 3
Fermentation of enhanced acetyl-coa synthesized recombinant saccharomyces cerevisiae SC002
Inoculating SC002 single colony of Saccharomyces cerevisiae on solid YPD plate in 2mLYPD medium, culturing at 30 deg.C and 220rpm for 16-20h, inoculating 2% of the culture medium 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 is up to 26h, glucose is added to supplement the carbon source.
Fermenting for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethyl alcohol and 10mL 1.5mol/L potassium hydroxide-methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extracted mixture, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 mu m filtration. 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 nm.
The final yield of 7-DHC was calculated to be only 51.6 mg/L.
Comparative example 4
Fermentation of acetyl-CoA knock-out catabolic gene recombinant yeast
Inoculating single colony of Saccharomyces cerevisiae SC003X, SC004X, SC005X and SC006X on solid YPD plate in 2mLYPD culture medium, culturing at 30 deg.C and 220rpm for 16-20h, inoculating 2% of inoculum size into 250mL round bottom shake flask containing 25mLYPD liquid culture medium, and culturing at 30 deg.C and 220rpm for 96 h. When the fermentation time is 26h, glucose is added for supplementing carbon source.
Fermenting for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethyl alcohol and 10mL 1.5mol/L potassium hydroxide-methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extracted mixture, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 mu m filtration. 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 nm.
Calculated final yields of 7-DHC of SC003X, SC004X, SC005X and SC006X respectively reach 80mg/L, 110mg/L, 90mg/L and 76 mg/L.
Comparative example 5
Construction and fermentation of catabolic gene recombinant yeast with other acetyl coenzyme A knocked out
Design of sgRNA was carried out using http:// wyrickbioin fo2.smb. wsu. edu/crishpr. html. website, plasmid pML-104 was used as a template, primers Cas9-ACH1-F and Cas9-ACH1-R were used to carry out plasmid loop P, and pML-104-ACH1 plasmid replacing 20bp sgRNA was obtained.
Preparing SC001 yeast into competence, respectively introducing plasmids obtained in the step (a) into competence, screening obtained strains on a URA-SD plate, culturing at 30 ℃ for 2-3 days, verifying SC001X strain by using YZ-ACH1-F sequencing, and respectively naming the obtained genetically engineered bacteria as SC 001X.
Inoculating single colony of Saccharomyces cerevisiae SC002X on solid YPD plate in 2mLYPD medium, culturing at 30 deg.C and 220rpm for 16-20 hr, inoculating 2% of the strain into 250mL round bottom shake flask containing 25mLYPD liquid medium, and culturing at 30 deg.C and 220rpm for 96 hr. When the fermentation time is up to 26h, glucose is added to supplement the carbon source.
After fermentation for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethanol and 10mL 1.5mol/L potassium hydroxide-methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extract mixture, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 μm filtration. 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 nm.
The final 7-DHC yield was calculated to be only 23 mg/L.
The primer sequence is as follows:
SEQ ID No.37：Cas9-ACH1-F：tttctagctctaaaacctattgtctagtaagacggtgatcatttatctttcactgcggagSEQ ID No.38：Cas9-ACH1-R：accgtcttactagacaataggttttagagctagaaatagcaagttaaaataaggctagtSEQ ID No.39：YZ-ACH1-F：gagctaaggggagcagttacgcaa
comparative example 6
The plasmids pML-104-AAT1, pML-104-GDH1 and pML-104-GDH3 obtained in example 3(a) were introduced into SC002 yeast in this order to obtain strain SC005C, which did not knock out the cho1 gene as compared with the SC007X strain. The obtained SC005X and SC007X were inoculated at 2% inoculum size into 250mL round bottom flasks containing 25mLYPD liquid medium, and cultured at 30 ℃ and 220rpm for 96 h. When the fermentation time is up to 26h, glucose is added to supplement the carbon source.
Fermenting for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethyl alcohol and 10mL 1.5mol/L potassium hydroxide methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extract, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 μm filtration. 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 nm.
The 7-DHC yield of SC007X is calculated to reach 269mg/L, the 7-DHC yield of SC005C is only 115mg/L, and the 7-DHC yield is reduced by 57%.
Comparative example 7
The mae1 gene was enhanced with a promoter gal1p, and the expression of aat1 gene, NADP (+) dependent glutamate dehydrogenase gdh1 gene, NADP (+) dependent glutamate dehydrogenase gdh3 gene, CDP-diacylglycerol-serine O-phosphotransferase cho1 gene of aspartate aminotransferase was down-regulated in sequence with a weak promoter FRS1p to obtain a strain SC 007C. SC007C and SC007X were inoculated at 2% inoculum size into 250mL round bottom flasks containing 25mLYPD liquid medium and cultured at 30 ℃ and 220rpm for 96 h. When the fermentation time is up to 26h, glucose is added to supplement the carbon source.
After fermentation for 90-120h, centrifuging at 5000rpm, resuspending with 10mL sterile water, adding 0.5mm glass beads, crushing for 10min with a high-speed homogenizing crusher, taking out the crushed mixture, adding 1g ascorbic acid and 0.5g HBT, mixing, sequentially adding 20mL absolute ethanol and 10mL 1.5mol/L potassium hydroxide-methanol solution, saponifying for 2h in a water area at 80 ℃, performing ultrasonic treatment for 30min with an extract (isopropanol: 1: 3/petroleum ether) after saponification, removing impurities at the lower layer with a separating funnel, performing freeze drying treatment on the extract mixture, redissolving with methanol or acetonitrile or a mixture of methanol and acetonitrile after the treatment, and performing high performance liquid chromatography after 0.55 μm filtration. 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 nm.
The 7-DHC yield of SC007X was calculated to reach 269mg/L, the 7-DHC yield of SC007C was only 135mg/L, and the 7-DHC yield was reduced by 50%.
In conclusion, the invention enables the strain to generate 7-DHC by constructing the 7-DHC synthesis module, increases the synthesis of acetyl coenzyme A to improve the yield by about 5 times, reduces the decomposition of acetyl coenzyme A to enhance the synthesis of 7-dehydrocholesterol, and greatly improves the yield of 7-DHC (26 times).
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and those skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Daliano Yi biological shares Ltd
<120> method for efficiently synthesizing 7-dehydrocholesterol by regulating/reducing acetyl coenzyme A branch metabolism
<160> 46
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> primer HO-UP-F (none)
<400> 1
aatgatttcc tccctagctg acct 24
<210> 2
<211> 42
<212> DNA
<213> primer HO-UP-R (none)
<400> 2
ggcgagtatt gataatgata ggttagatcc caggcgtaga ac 42
<210> 3
<211> 37
<212> DNA
<213> primer HO-Down-F (none)
<400> 3
taatttgcgg ccatagtgcg tttagaacgc ttcatca 37
<210> 4
<211> 25
<212> DNA
<213> primer HO-Down-R (none)
<400> 4
acgtaggttt tgtctcgcta attgc 25
<210> 5
<211> 36
<212> DNA
<213> TDH3p-F (none)
<400> 5
ttaacctatc attatcaata ctcgccattt caaaga 36
<210> 6
<211> 47
<212> DNA
<213> TDH3p-R (none)
<400> 6
agaccaaacg gcagacattc gaaactaagt tctggtgttt taaaact 47
<210> 7
<211> 33
<212> DNA
<213> DH24-F (none)
<400> 7
agaacttagt ttcgaatgtc tgccgtttgg tct 33
<210> 8
<211> 42
<212> DNA
<213> DH24-R (none)
<400> 8
agcgttctaa acgcactatg gccgcaaatt aaagccttcg ag 42
<210> 9
<211> 60
<212> DNA
<213> Cas9-HO-F (none)
<400> 9
tttctagctc taaaactagc atctagcaca tactcggatc atttatcttt cactgcggag 60
<210> 10
<211> 59
<212> DNA
<213> Cas9-HO-R (none)
<400> 10
cgagtatgtg ctagatgcta gttttagagc tagaaatagc aagttaaaat aaggctagt 59
<210> 11
<211> 24
<212> DNA
<213> YZ-HO-F (none)
<400> 11
tgacaattta tgacctgcag taca 24
<210> 12
<211> 27
<212> DNA
<213> YZ-HO-R (none)
<400> 12
tcctcggtga atttctcgca gatagac 27
<210> 13
<211> 43
<212> DNA
<213> mae1-F (none)
<400> 13
aacttagttt cgaatgctta gaaccagact atccgtttcc gtt 43
<210> 14
<211> 43
<212> DNA
<213> mae1-R (none)
<400> 14
gtgcattgat gtcgaagaac actacaattg gttggtgtgc acc 43
<210> 15
<211> 24
<212> DNA
<213> UPERG6-F (none)
<400> 15
cttaccaccg gcaactaaac caac 24
<210> 16
<211> 46
<212> DNA
<213> UPERG6-R (none)
<400> 16
atggcgagta ttgataatga aagccacaca ttcctactat aacgtc 46
<210> 17
<211> 45
<212> DNA
<213> DOWNERG6-F (none)
<400> 17
accaaccaat tgtagtgttc ttcgacatca atgcactcaa acctg 45
<210> 18
<211> 26
<212> DNA
<213> DOWNERG6-R (none)
<400> 18
aaactaaaaa tggctcgtgt tcatgc 26
<210> 19
<211> 45
<212> DNA
<213> Mae1-TDH3p-F (none)
<400> 19
gtaggaatgt gtggctttca ttatcaatac tcgccatttc aaaga 45
<210> 20
<211> 48
<212> DNA
<213> Mae1-TDH3p-R (none)
<400> 20
atagtctggt tctaagcatt cgaaactaag ttctggtgtt ttaaaact 48
<210> 21
<211> 59
<212> DNA
<213> Cas9-ERG6-F (none)
<400> 21
ctagctctaa aacaatttct caagtacttc tgagatcatt tatctttcac tgcggagaa 59
<210> 22
<211> 60
<212> DNA
<213> Cas9-ERG6-R (none)
<400> 22
cagaagtact tgagaaattg ttttagagct agaaatagca agttaaaata aggctagtcc 60
<210> 23
<211> 24
<212> DNA
<213> YZ-ERG6-F (none)
<400> 23
tctctcttgc tgggccccca acac 24
<210> 24
<211> 27
<212> DNA
<213> YZ-ERG6-R (none)
<400> 24
gtcacgggct agtttcttgt tgttagt 27
<210> 25
<211> 59
<212> DNA
<213> Cas9-AAT1-F (none)
<400> 25
ctagctctaa aacgtagggt tgtgacaaca cgcgatcatt tatctttcac tgcggagaa 59
<210> 26
<211> 61
<212> DNA
<213> Cas9-AAT1-R (none)
<400> 26
gcgtgttgtc acaaccctac gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
c 61
<210> 27
<211> 59
<212> DNA
<213> Cas9-GDH1-F (none)
<400> 27
ctagctctaa aacaagaatt tcaagataga caagatcatt tatctttcac tgcggagaa 59
<210> 28
<211> 61
<212> DNA
<213> Cas9-GDH1-R (none)
<400> 28
ttgtctatct tgaaattctt gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
c 61
<210> 29
<211> 59
<212> DNA
<213> Cas9-GDH3-F (none)
<400> 29
ctagctctaa aacagtgagc gcattcttga agagatcatt tatctttcac tgcggagaa 59
<210> 30
<211> 61
<212> DNA
<213> Cas9-GDH3-R (none)
<400> 30
tcttcaagaa tgcgctcact gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
c 61
<210> 31
<211> 59
<212> DNA
<213> Cas9-CHO1-F (none)
<400> 31
ctagctctaa aacgtaagcg taaatctcag acagatcatt tatctttcac tgcggagaa 59
<210> 32
<211> 61
<212> DNA
<213> Cas9-CHO1-R (none)
<400> 32
tgtctgagat ttacgcttac gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
c 61
<210> 33
<211> 26
<212> DNA
<213> YZ-AAT1-F (none)
<400> 33
tggattgagc aattgaaaac ctttgc 26
<210> 34
<211> 24
<212> DNA
<213> YZ-GDH1-F (none)
<400> 34
aagttgctca aggttacaga gtgc 24
<210> 35
<211> 23
<212> DNA
<213> YZ-GDH3-F (none)
<400> 35
ttcaattcag ggtcacgtgg gaa 23
<210> 36
<211> 24
<212> DNA
<213> YZ-CHO1-F (none)
<400> 36
catttcagca tgatgaggaa tttg 24
<210> 37
<211> 60
<212> DNA
<213> Cas9-ACH1-F (none)
<400> 37
tttctagctc taaaacctat tgtctagtaa gacggtgatc atttatcttt cactgcggag 60
<210> 38
<211> 59
<212> DNA
<213> Cas9-ACH1-R (none)
<400> 38
accgtcttac tagacaatag gttttagagc tagaaatagc aagttaaaat aaggctagt 59
<210> 39
<211> 24
<212> DNA
<213> YZ-ACH1-F (none)
<400> 39
gagctaaggg gagcagttac gcaa 24
<210> 40
<211> 2010
<212> DNA
<213> mae1 sequence (none)
<400> 40
atgcttagaa ccagactatc cgtttccgtt gctgctagat cgcaactaac cagatccttg 60
acagcatcaa ggacagcacc attaagaaga tggcctattc agcaatcgcg tttatattct 120
tctaacacta gatcgcataa agctaccaca acaagagaaa atactttcca aaagccatac 180
agcgacgagg aggtcactaa aacacccgtc ggttctcgcg ccagaaagat cttcgaagct 240
cctcacccac atgccactcg tttgactgta gaaggtgcca tagaatgtcc cttggagagc 300
tttcaacttt taaactctcc tttatttaac aagggttctg catttacaca agaagaaagg 360
gaagcgttta atttagaagc attgctacca ccacaagtga acactttgga cgaacaactg 420
gaaagaagct acaagcagtt atgctatttg aagacgccct tggccaaaaa cgacttcatg 480
acgtctttga gagtacagaa caaagtccta tattttgcat taataaggag acatatcaag 540
gaattagttc ctatcattta caccccaacc gaaggtgatg ctattgctgc ctattcccac 600
aggttcagaa agccagaagg tgtgttttta gacattaccg aacctgattc catcgaatgt 660
agattggcta catacggtgg agacaaagat gtagactaca tcgttgtgtc ggattcggaa 720
ggtattctgg gaattggtga ccaaggtatc ggtggtgtac gtattgctat ctccaaattg 780
gcattgatga cgctgtgcgg tggtattcat cccggccgtg tgctacctgt gtgtttggac 840
gtcggtacta acaacaagaa actagcccgt gacgaattgt acatgggtaa caagttctcc 900
agaatcaggg gtaagcaata tgacgacttc ttggaaaaat tcatcaaggc cgttaagaaa 960
gtgtatccaa gcgccgttct gcatttcgaa gatttcggtg ttaagaacgc tagaagatta 1020
ctagaaaagt acaggtacga attgccatca ttcaacgatg acattcaggg caccggtgcc 1080
gtcgtgatgg cctcgttgat tgctgctttg aaacatacca acagagactt gaaagacacc 1140
agagtgctta tttacggtgc cgggtctgcg ggcctcggta tcgcagatca aattgtgaat 1200
catatggtca cgcacggcgt tgacaaggaa gaagcgcgca agaaaatctt cttgatggac 1260
agacgtgggt taattctaca atcttacgag gctaactcca ctcccgccca acacgtatac 1320
gctaagagtg atgcggaatg ggctggtatc aacacccgct ctttacatga tgtggtggag 1380
aacgtcaaac caacgtgttt ggttggctgc tccacacaag caggcgcatt cactcaagat 1440
gtcgtagaag aaatgcacaa gcacaatcct agaccgatca ttttcccatt atccaaccct 1500
actagactac acgaagccgt tcctgccgat ttaatgaagt ggaccaacaa caacgctctt 1560
gtagctaccg gatctccttt cccacctgtt gatggttacc gtatctcgga gaacaacaat 1620
tgttactctt tcccaggtat cggtttaggt gccgtactat cgcgtgccac caccatcaca 1680
gacaagatga tctccgctgc agtggaccaa ctagccgaat tgtcgccact aagagagggc 1740
gactcgagac ctgggttgct acccggcctg gacaccatca ccaacacttc tgcgcgtcta 1800
gctaccgctg tgatcttgca agcactcgag gagggaaccg cccgtatcga gcaagaacaa 1860
gtaccgggag gagctcccgg cgaaactgtc aaggttcctc gtgactttga cgaatgttta 1920
cagtgggtca aagcccaaat gtgggagcct gtgtacagac ctatgatcaa ggtccaacat 1980
gacccatcgg tgcacaccaa ccaattgtag 2010
<210> 41
<211> 1356
<212> DNA
<213> aat1 sequence (none)
<400> 41
atgttgagga cgaggcttac taattgcagt ctatggaggc cctactacac gtcatcgctt 60
agtagagtac caagagcacc tccagataaa gtcttggggt tatctgaaca cttcaaaaag 120
gtaaaaaatg ttaacaaaat tgacctgacc gtaggaatat ataaagatgg ttggggcaaa 180
gtgacgacgt ttccctccgt tgcaaaagct caaaaattga ttgaatctca tttagagttg 240
aataagaatc tttcatattt accaataaca ggttccaaag aatttcagga aaacgttatg 300
aaatttttgt tcaaggaatc atgtccgcag tttgggccat tttatttagc ccatgataga 360
atcagctttg ttcagacttt gagtggtaca ggcgccctag ctgtagcagc taaattcttg 420
gcattattta tttcaagaga tatttggata cctgacccat catgggcaaa tcataagaac 480
atttttcaga acaatggttt tgaaaatatt taccggtatt cctattataa ggacggtcag 540
atagacatcg acggatggat tgagcaattg aaaacctttg catataacaa ccagcaagaa 600
aacaataaga acccaccttg cataatcttg catgcgtgtt gtcacaaccc tacaggtctc 660
gacccaacta aagagcaatg ggaaaagatt atagatacta tatatgagct aaaaatggta 720
cccattgttg atatggctta tcagggttta gagtctggta acttactaaa ggacgcatat 780
ttattgaggc tatgtctcaa tgtaaataaa tatccaaatt ggagtaacgg tatctttctt 840
tgtcaatctt ttgccaagaa catgggcctt tatggtgaac gagttggttc cttaagcgtt 900
atcacgccgg caactgcgaa caatggaaag ttcaaccctc tacaacagaa aaactcattg 960
cagcaaaata ttgactccca attaaaaaag attgtcagag gtatgtattc ttctccacca 1020
ggatacggtt ctcgtgtggt aaatgtagta ttatcagatt tcaaattgaa acagcaatgg 1080
ttcaaggatg ttgatttcat ggttcagaga ttgcatcacg tcagacagga gatgtttgac 1140
cgtctagggt ggccggatct tgtaaatttc gcacaacagc acggtatgtt ttactataca 1200
aggtttagtc caaagcaagt cgaaatattg agaaacaatt acttcgtcta tttaacaggt 1260
gatggtagat tgtcgcttag cggagtcaat gattcgaacg ttgattactt atgtgaatct 1320
cttgaagcag tctcgaaaat ggacaaactc gcataa 1356
<210> 42
<211> 930
<212> DNA
<213> ser2 sequence (none)
<400> 42
atgtcaaagt ttgttatcac ctgcatagct catggagaaa atctcccaaa agaaaccatc 60
gaccagattg cgaaagaaat tactgaaagc tcggcgaaag atgtatcaat caatggtacc 120
aagaaactat cggccagagc taccgatata ttcattgaag tcgcaggatc gattgttcaa 180
aaagacctca agaataagct gacgaacgtc attgacagtc ataatgatgt tgatgttatt 240
gtttctgtcg acaatgaata tcgtcaagcc aaaaagctct ttgtctttga catggattca 300
acattaatct accaagaggt catcgaattg attgccgctt atgctggtgt tgaagaacaa 360
gtgcacgaga tcacagaaag agccatgaac aatgaacttg atttcaaaga gtctttaaga 420
gaacgcgtta aattattgca aggcctccaa gtcgatacac tatatgatga aataaaacaa 480
aagctagagg tcaccaaggg tgtgccagaa ctatgcaagt tccttcacaa aaaaaattgc 540
aagctcgctg ttttaagcgg tggttttatt cagtttgccg gttttatcaa ggatcagtta 600
ggtttagatt tttgtaaggc aaacttgttg gaagttgaca ctgatggaaa attaaccggt 660
aaaacactgg gtcctatcgt agacggacag tgcaagagtg aaacccttct gcaactatgt 720
aatgactaca atgttccagt tgaagcaagt tgtatggtgg gtgacggtgg taacgacctg 780
ccagccatgg ctaccgccgg gtttgggatc gcatggaacg ccaagccaaa ggtgcagaaa 840
gctgcacctt gtaagttgaa taccaagagc atgactgaca ttttatacat tcttggttac 900
accgatgatg aaatatacaa tagacaatga 930
<210> 43
<211> 1374
<212> DNA
<213> gdh3 sequence (none)
<400> 43
atgacaagcg aaccagagtt tcagcaggct tacgatgaga tcgtttcttc tgtggaggat 60
tccaaaattt ttgaaaaatt cccacagtat aaaaaagtgt tacctattgt ttctgtcccg 120
gagaggatca ttcaattcag ggtcacgtgg gaaaatgata atggcgagca agaagtggct 180
caaggataca gggtgcagtt caattcagcc aagggccctt acaagggtgg cctacgcttc 240
cacccatcag tgaacctgtc tatcctaaaa tttttgggtt ttgaacagat cttcaagaat 300
gcgctcactg ggctagatat gggcggtggt aagggtggcc tgtgtgtgga cttgaaaggc 360
aagtctgaca acgagatcag aaggatttgt tatgcgttca tgagagaact gagcaggcat 420
attggtaagg acacagacgt gcccgcagga gatattggtg tcggtggccg tgaaattggc 480
tacctattcg gcgcttacag atcatacaag aactcctggg aaggtgtgtt gactggtaag 540
ggtttaaact ggggtggctc acttatcagg ccggaggcca ccgggttcgg cttagtttac 600
tatacgcaag caatgatcga ttatgcaaca aacggcaagg agtcgtttga gggcaaacgt 660
gtgacaatct ccggaagtgg caatgttgcg caatatgcag ctttgaaagt gatcgagctg 720
ggtggtattg tggtgtcttt atccgattcg aaggggtgca tcatctctga gacgggcatt 780
acttctgagc aaattcacga tatcgcttcc gccaagatcc gtttcaagtc gttagaggaa 840
atcgttgatg aatactctac tttcagcgaa agtaagatga agtacgttgc aggagcacgc 900
ccatggacgc atgtgagcaa cgtcgacatt gccttgccct gtgccaccca aaacgaggtc 960
agtggtgacg aagccaaggc cctagtggca tctggcgtta agttcgttgc cgaaggtgct 1020
aacatgggtt ctacacccga ggctatttct gttttcgaaa cagcgcgtag cactgcaacc 1080
aatgcaaagg atgcagtttg gtttgggcca ccaaaggcag ctaacctggg cggcgtggca 1140
gtatccggtc tggaaatggc tcagaattct caaaaagtaa cttggactgc cgagcgggtc 1200
gatcaagaac taaagaagat aatgatcaac tgcttcaacg actgcataca ggccgcacaa 1260
gagtactcta cggaaaaaaa tacaaacacc ttgccatcat tggtcaaggg ggccaacatt 1320
gccagcttcg tcatggtggc tgacgcaatg cttgaccagg gagacgtttt ttag 1374
<210> 44
<211> 1365
<212> DNA
<213> gdh1 sequence (none)
<400> 44
atgtcagagc cagaatttca acaagcttac gaagaagttg tctcctcttt ggaagactct 60
actcttttcg aacaacaccc agaatacaga aaggttttgc caattgtttc tgttccagaa 120
agaatcatac aattcagagt cacctgggaa aatgacaagg gtgaacaaga agttgctcaa 180
ggttacagag tgcaatataa ctccgccaag ggtccataca agggtggtct acgtttccat 240
ccttccgtga acttgtctat cttgaaattc ttgggtttcg aacaaatctt caagaactcc 300
ttgaccggcc tagacatggg tggtggtaaa ggtggtctat gtgtggactt gaagggaaga 360
tctaataacg aaatcagaag aatctgttat gctttcatga gagaattgag cagacacatt 420
ggtcaagaca ctgacgtgcc agctggtgat atcggtgttg gtggtcgtga aattggttac 480
ctgttcggtg cttacagatc atacaagaac tcctgggaag gtgtcttaac cggtaagggt 540
ttgaactggg gtggttcttt gatcagacca gaagccactg gttacggttt agtttactat 600
actcaagcta tgatcgacta tgccacaaac ggtaaggaat ctttcgaagg taagcgcgtc 660
accatctctg gtagtggtaa cgttgctcaa tacgctgcct tgaaggttat tgagctaggt 720
ggtactgtcg tttccctatc tgactccaag ggttgtatca tctctgaaac tggtatcacc 780
tccgaacaag tcgctgatat ttccagtgct aaggtcaact tcaagtcctt ggaacaaatc 840
gtcaacgaat actctacttt ctccgaaaac aaagtgcaat acattgctgg tgctcgtcca 900
tggacccacg tccaaaaggt cgacattgct ttgccatgtg ccacccaaaa tgaagtcagc 960
ggtgaagaag ccaaggcctt ggttgctcaa ggtgtcaagt ttattgccga aggttccaac 1020
atgggttcca ctccagaagc tattgccgtc tttgaaactg ctcgttccac cgccactgga 1080
ccaagcgaag ctgtttggta cggtccacca aaggctgcta acttgggtgg tgttgctgtt 1140
tctggtttag aaatggcaca aaactctcaa agaatcacat ggactagcga aagagttgac 1200
caagagttga agagaattat gatcaactgt ttcaatgaat gtatcgacta tgccaagaag 1260
tacactaagg acggtaaggt cttgccatct ttggtcaaag gtgctaatat cgcaagtttc 1320
atcaaggtct ctgatgctat gtttgaccaa ggtgatgtat tttaa 1365
<210> 45
<211> 831
<212> DNA
<213> cho1 sequence (none)
<400> 45
atggttgaat cagatgaaga tttcgcacct caagaattcc cacacacgga cacagacgtt 60
atcgtaaatg aacacagaga cgaaaatgac gggtatgcct cagatgaagt tggtggcaca 120
ttaagcagaa gggcctcaag tatattttct ataaatacaa ctccattggc cccccctaat 180
gctactgata tccaaaaatt tacaagtgac gaacatcatt tcagcatgat gaggaatttg 240
catatggcag attacattac tatgctgaat ggattttctg ggttttactc tattgtaagt 300
tgtctgagat ttacgcttac aggtaaacct cattacgtcc agcgtgccca tttctttatt 360
ttattgggta tgtgtttcga tttccttgac gggagagtag cacgtctgag aaataggtct 420
tccttaatgg gtcaagaact ggactctttg gccgacttgg tttcctttgg tgtggctccg 480
gctgcaattg cttttgccat tggatttcaa actacattcg atgtcatgat tttatctttc 540
tttgtccttt gcggcttagc gagattagca aggtttaatg tcaccgttgc tcaattaccc 600
aaagattcta gcacaggtaa atctaaatat tttgagggat tgcctatgcc aaccactcta 660
gctttggtgt tgggtatggc atattgtgtc agaaagggtt taatttttga taacatccca 720
ttcggcattt ttagagaaga ccaaatattg gaatttcatc caattattct agtatttttt 780
atccatgggt gtggaatgat ttcgaagagc ttgaaaattc caaagccata g 831
<210> 46
<211> 1800
<212> DNA
<213> Synthesis of DHCR24 sequence (none)
<400> 46
atgtctgccg tttggtcttt gggtgccggt ttgttgttgc ttttgctttg ggttaggcac 60
agaggtttgg aggctgtctt ggttcaccat agatggatct tcgtctgctt cttccttatg 120
ccactttcta ttctttttga cgtctactac cagttgaggg cttgggccgt tagaaggatg 180
cacagtgctc caaggttgca tggtcaaagg gtcagacaca tccaagaaca agtcagagaa 240
tggaaggagg agggaggaag aaggtatatg tgcaccggaa ggcccggttg gcttaccgtc 300
tctcttaggg ttggtaagta taaaaagact cataaaaata ttatgattaa ccttatggac 360
gtccttgagg tcgacagtga gagacaagtt gttagggtcg agcctcttgt taccatgggt 420
cagttgaccg cctacttgaa tcctatgggt tggaccatcc cagtcgtccc agaattggac 480
gatcttaccg tcggtggatt gatcatggga accggaatcg agagttcttc tcacatctac 540
ggtcttttcc aacacacttg tatggcctac gagttggtct tggccgacgg atctcttgtt 600
aggtgcagtc ctaccgagaa ctctgacttg ttctacgccg tcccatggtc ttgcggtacc 660
cttggattct tggttgccgc cgagatcaag atgattccag ccaagaagta cattagattg 720
cactacgagc cagttagagg attgaggtct atctgcgaga aattcaccga ggagtctaaa 780
aataaagaga acagtttcgt cgagggtctt gtctacagtc ttgaggaggc cgtcattatg 840
actggagtct tgaccgacga ggctgagcca tctaagatta acagaattgg taattattat 900
aaaccatggt tttttaagca cgttgagaaa tacttgaagg ccaataaaac cggtattgaa 960
tatatccctt ctaggcacta ctaccataga cacactagga gtatcttctg ggagcttcaa 1020
gatatcatcc ctttcggaaa caatccagtc tttagatatc ttttcggatg gatggttcca 1080
ccaaagatct ctttgcttaa gttgacccaa ggtgaagcca ttagaaaatt gtacgagcag 1140
caccacgtcg tccaagacat gcttgtccct atgaaatctc ttgagaagag tatccagacc 1200
ttccacgtcg acttgaacgt ctacccattg tggctttgcc ctttccttct tcctaacaac 1260
cccggtatgg tccaccctaa gggagacgaa accgaacttt acgtcgacat cggagcctac 1320
ggagagccaa aaaccaagca gttcgaggct agagccagta tgaggcagat ggagaagttc 1380
gttagaagtg tccatggttt ccagatgctt tacgccgact gctacatgac tagagaggaa 1440
ttctgggaca tgttcgacgg ttctctttat cattctttga gggagcaaat gaactgtaag 1500
gacgccttcc cagaggtcta tgacaagatc tgcaaggctg ctaggcattc atgtaattag 1560
ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag 1620
ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa 1680
cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca 1740
ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcggcc 1800

Claims (10)

1. A method for regulating/reducing acetyl coenzyme A branch metabolism to efficiently synthesize 7-dehydrocholesterol is characterized by comprising the steps of utilizing a key gene related to acetyl coenzyme A catabolism to carry out gene recombination in engineering bacteria so as to reduce the acetyl coenzyme A catabolism; thereby enhancing 7-dehydrocholesterol synthesis; the key genes related to the catabolism of acetyl-CoA in 7-DHC synthesis include:
aat1 gene encoding aspartate aminotransferase having a nucleotide sequence as set forth in SEQ ID No.41, phosphoserine phosphatase ser2 gene of the phosphoglycerate pathway having a nucleotide sequence as set forth in SEQ ID No.42, NADP (+) dependent glutamate dehydrogenase gdh1 gene having a nucleotide sequence as set forth in SEQ ID No.44, NADP (+) dependent glutamate dehydrogenase gdh3 gene having a nucleotide sequence as set forth in SEQ ID No.43, CDP-diacylglycerol-serine O-phosphotransferase cho1 gene having a nucleotide sequence as set forth in SEQ ID No. 45.
2. The method according to claim 1, wherein the method of using the key genes involved in the catabolism of acetyl-CoA is a knock-out of these genes.
3. The method of claim 1, wherein the method comprises not only using the above-mentioned group of key genes related to the catabolism of acetyl-CoA, but also using genes that increase the synthesis of acetyl-CoA for gene recombination in the engineered bacterium.
4. The method of claim 1, wherein said genes that increase acetyl-coa synthesis comprise: the nucleotide sequence is shown in SEQ ID No.40, the mitochondrial malate dehydrogenase mae1 gene, the alcohol dehydrogenase II adh2 gene or the D-lactate dehydrogenase dld1 gene.
5. The method according to claim 1, wherein the method of utilizing a key gene related to acetyl-CoA synthesis is enhanced expression of a gene that increases acetyl-CoA synthesis; the expression enhancing means includes the use of constitutive promoters: p TDH3 、P TEF1 、P PGK1 、P ENO2 、P TPL1 Or using the following inducible promoter P GAL1 、P GAL10 、P GAL7 。
6. The method of claim 1, wherein the engineering bacteria is selected from the group consisting of Saccharomyces cerevisiae S288C, Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae Y187, Pichia pastoris x-33, and Candida tropicalis 1798.
7. The method of claim 1, wherein the ratio of the cofactor NADH/NAD + as a whole in the yeast is controlled in the range of 0.3 to 0.8 by overexpression and knock-out of the relevant gene.
8. The method of claim 1, wherein the 7-DHC synthesis module is introduced using S288C as starting strain and promoter P is used TDH3 Controlling the expression of mitochondrial malate dehydrogenase mae1 having a nucleotide sequence as set forth in SEQ ID No.40,cas9 was then used to knock out the genes aat1 encoding aspartate aminotransferase, having the nucleotide sequence set forth in SEQ ID No.41, NADP (+) dependent glutamate dehydrogenase gdh1, having the nucleotide sequence set forth in SEQ ID No.44, NADP (+) dependent glutamate dehydrogenase gdh3, having the nucleotide sequence set forth in SEQ ID No.43, and CDP-diacylglycerol-serine O-phosphatidyl transferase cho1, having the nucleotide sequence set forth in SEQ ID No.45, respectively.
9. The method of claim 8, wherein the 7-DHC synthesis module is a codon optimized DHCR24 reductase gene derived from chicken comprising the nucleotide sequence set forth in SEQ ID No.46, endogenous C-8 sterol isomerase ERG2, endogenous C-5 sterol desaturase ERG 3.
10. The method of claim 1, wherein the original s.cerevisiae S288C is used as the starting strain, and the promoter P is used TDH3 Controlling the expression of mitochondrial malate dehydrogenase mae1 having a nucleotide sequence as set forth in SEQ ID No.40, and then knocking out the aat1 encoding aspartate aminotransferase having a nucleotide sequence as set forth in SEQ ID No.41, the NADP (+) dependent glutamate dehydrogenase gdh1 having a nucleotide sequence as set forth in SEQ ID No.44, the gdh3 having a nucleotide sequence as set forth in SEQ ID No.43, and the CDP-diacylglycerol-serine O-phosphatidyltransferase cho1 gene having a nucleotide sequence as set forth in SEQ ID No.45, respectively, using Cas 9.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112813129A (en) * 2021-02-05 2021-05-18 江南大学 Method for increasing 7-dehydrocholesterol yield in yeast by compartmentalization
CN112813129B (en) * 2021-02-05 2023-09-08 江南大学 Method for improving yield of 7-dehydrocholesterol in saccharomycetes by utilizing compartmentalization

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