Sterol C24-reductase mutant capable of reducing byproducts and saccharomyces cerevisiae capable of producing 7-dehydrocholesterol

Abstract

The invention discloses a sterol C24-reductase mutant capable of reducing byproducts and saccharomyces cerevisiae capable of producing 7-dehydrocholesterol, and belongs to the technical field of biology. In order to solve the problems of byproduct production and low catalytic activity caused by wide substrate spectrum of a key enzyme DHCR24 in the current 7-DHC synthesis process, the sterol C24-reductase mutant DHCR24M is obtained by mutating leucine at the 162nd site and aspartic acid at the 386th site of DHCR24 through enzyme engineering modification exploration. The mutant is introduced into an original strain of saccharomyces cerevisiae, fermentation verification shows that compared with an unmutated strain, byproducts, namely chost-7-en-3-ol and chost-5, 7-dien-3-ol, of the mutated strain are reduced by 31.5% and 46.1% respectively, the target product is increased by 90.2%, a new thought and a new method are provided for industrial production of 7-dehydrocholesterol and active vitamin D3, and the application prospect is broad. And the method has a very wide application prospect.

Description

Technical Field

[0001] The invention relates to a sterol C24-reductase mutant capable of reducing byproducts and a 7-dehydrocholesterol-producing yeast, belonging to the field of biotechnology. Background Art

[0002] 7-Dehydrocholesterol (7-DHC) is a steroid compound with high added value in the medical and industrial fields. It can be used not only for liquid crystal manufacturing and electromagnetic detection, but also has a greater demand in biomedicine and human health. 7-DHC can be converted into vitamin D3 (VD3) by ultraviolet irradiation. Therefore, 7-DHC is the direct precursor of synthetic VD3. VD3 can promote intestinal absorption of calcium to maintain bone calcium balance, promote bone absorption by increasing the number of osteoclasts, maintain the calcium and phosphorus levels required for bone formation, and can also treat osteoporosis and rickets in the elderly, reduce the incidence of common cancers, anti-inflammatory, immunomodulatory, hypothyroidism, improve skin quality, and prevent infection with the new coronavirus. With the gradual improvement of people's health awareness and the intensification of population aging, the demand for VD3 has increased year by year, resulting in a huge market for its direct precursor 7-DHC and broad application prospects.

[0003] There are two main ways to obtain VD3 on the market. One is to extract it from tuna liver oil, and the other is to synthesize it through the photochemical reaction of 7-dehydrocholesterol. Direct extraction will produce allergens and the output is severely limited. Therefore, the method of synthesizing VD3 in industry is to first prepare 7-DHC by chemical synthesis. The mainstream chemical synthesis method uses lanolin as raw material, and obtains 7-DHC through esterification, oxidation, reduction, and hydrolysis reactions, and then obtains VD3 through photoreaction. The chemical synthesis method has the problems of complex process, high energy consumption, harsh reaction conditions and serious pollution, which is not conducive to sustainable development. The method of producing 7-DHC by microbial fermentation is green, environmentally friendly and sustainable.

[0004] At present, the synthesis of 7-dehydrocholesterol by microorganisms is mainly based on Saccharomyces cerevisiae as the chassis cell, and 7-dehydrocholesterol is obtained by introducing heterologous sterol C24-reductase (DHCR24), overexpressing truncated HMG-CoA reductase gene (tHMG1), and squalene epoxidase gene (ERG1), with a yield of 5.5 mg / g cell dry weight (CN 103275997A). Since the synthesis pathway of 7-dehydrocholesterol involves multiple organelles, it may cause difficulties in the transmission of intermediate metabolites. Therefore, the synthesis pathway of 7-dehydrocholesterol is reconstructed and re-fixed in mitochondria, peroxisomes, endoplasmic reticulum, lipid droplets, etc., further improving the yield of 7-dehydrocholesterol (CN 112813129A, CN113151027A, CN116751698A). The above patents all introduce heterologous DHCR24, which is the most critical enzyme in the synthesis pathway of 7-DHC. Without DHCR24, 7-DHC cannot be synthesized.

[0005] DHCR24 is composed of 516 amino acids and can remove the double bond at C24 on the sterol side chain. However, its substrate spectrum is broad and can recognize lanosterol, 4,4-dimethylcholesta-8,14,24-trien-3β-ol, 4,4-dimethylcholesta-8,24-dien-3β-ol, -ol), zymosterol, cholesta-7,24-dien-3β-ol, 7-dehydrodesmosterol, and desmosterol are converted into different substances, thus deriving the Kandutsch-Russell pathway. Through the action of other enzymes in the synthesis pathway, by-products will be produced and accumulated, affecting the yield and purification of the final product 7-DHC.

[0006] Enzyme engineering and synthetic biology have developed rapidly in the past decade, providing new ideas for the green production of high value-added chemicals through strategies such as molecular docking, molecular dynamics simulation and saturation mutation. Using safe, efficient and environmentally friendly yeast as a cell factory, the key heterologous enzyme DHCR24 for synthesizing 7-dehydrocholesterol is transformed through enzyme engineering, hoping to reduce the content of by-products and increase the yield of the target product 7-DHC, providing new ideas and methods for the industrial production of 7-dehydrocholesterol. Summary of the invention

[0007] To solve the above problems, the present invention provides a sterol C24-reductase mutant, which overcomes the defect of a wide substrate spectrum of sterol C24-reductase, reduces the synthesis of by-products, and improves the catalytic efficiency of 7-dehydrodesmosterol. At the same time, the present invention provides a genetically engineered yeast of saccharomyces cerevisiae based on the mutant, which can not only reduce the synthesis of by-products, but also efficiently produce 7-DHC, with a yield increased by 90.2% compared with the non-mutated strain, and also simplifies the subsequent purification process.

[0008] The first object of the present invention is to provide a recombinant Saccharomyces cerevisiae, wherein a sterol C24-reductase mutant is overexpressed in the recombinant Saccharomyces cerevisiae;

[0009] The sterol C24-reductase mutant is obtained by mutating the sterol C24-reductase whose amino acid sequence is shown in SEQ ID NO.1, and the mutation includes:

[0010] Mutate leucine at position 162 to arginine;

[0011] or mutating aspartic acid at position 386 to methionine;

[0012] Or mutate the leucine at position 162 to arginine and mutate the aspartic acid at position 386 to methionine.

[0013] The existing technologies all express chicken / human DHCR24 in yeast cell chassis to synthesize 7-DHC. Although 7-DHC can be synthesized in this way, the introduction of heterologous enzymes with a wide substrate spectrum will inevitably cause other byproducts to be produced in addition to the target product 7-DHC, resulting in a decrease in the catalytic efficiency of DHCR24. The present invention uses enzyme engineering means, which is no longer a simple integration of gene fragments, but through molecular docking, determines the reasonable active center and key residues, mutates the key residues, determines the best mutant by detecting the yield of 7-DHC, and further uses molecular dynamics to illustrate the mechanism of increased yield, providing a reference for the key enzyme transformation of the later synthesis of 7-DHC.

[0014] Furthermore, the recombinant Saccharomyces cerevisiae is based on modified or unmodified Saccharomyces cerevisiae as a starting strain; the modification includes one or more of the following:

[0015] (1) Overexpression of sterol C24-reductase (DHCR24),

[0016] (2) Overexpression of truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1),

[0017] (3) Overexpression of isopentenyl diphosphate delta-isomerase (IDI1),

[0018] (4) Overexpression of squalene epoxidase (ERG1),

[0019] (5) Overexpression of sterol 14α-demethylase (ERG11),

[0020] (6) Overexpression of alcohol dehydrogenase 2 (ADH2),

[0021] (7) Overexpression of lanosterol synthase (ERG7),

[0022] (8) Overexpression of transcription factor UPC2 mutant,

[0023] (9) using a transcriptional activation system to upregulate the expression of any of the genes encoding (1) to (8),

[0024] (10) using a transcriptional repression system to downregulate the expression of any gene encoding sterol C-24 methyltransferase (ERG6), malate synthase (MLS1), or citrate synthase (CIT2),

[0025] (11) Overexpression of Δ14-sterol reductase (ERG24),

[0026] (12) Overexpression of methylsterol monooxygenase (ERG25),

[0027] (13) Overexpression of sterol 4α-carboxylate 3-dehydrogenase (ERG26),

[0028] (14) Overexpression of sterol reductase (ERG27),

[0029] (15) Overexpressing one or more of lanosterol synthase (ERG7), farnesyl pyrophosphate synthase (ERG20), isopentenyl diphosphate delta-isomerase (IDI1), diphosphomevalonate decarboxylase (MVD1), phosphomevalonate kinase (ERG8), and mevalonate kinase (ERG12), and localizing one or more of the enzymes to the endoplasmic reticulum for expression,

[0030] (16) Overexpression of endoplasmic reticulum regulatory factor (INO2).

[0031] Furthermore, the transcription activation system includes a CRISPR / dCpf1-mediated transcription activation system, and the transcription repression system includes a CRISPR / dCas9-mediated transcription repression system.

[0032] Further, the amino acid sequence of sterol C24-reductase (DHCR24) is shown in SEQ ID NO.1; the nucleotide sequence of truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) is shown in SEQ ID NO.3; the NCBI number of isopentenyl diphosphate δ-isomerase (IDI1) is NM_001183931.1; the NCBI number of squalene epoxidase gene (ERG1) is NM_001181304.1; the NCBI number of sterol 14α-demethylase (ERG11) is NM_001179137.1; the NCBI number of alcohol dehydrogenase 2 (ADH2) is NM_001182812.1; the NCBI number of lanosterol synthase (ERG7) is NM_001179202.2; the nucleotide sequence of the transcription factor UPC2 mutant is shown in SEQ ID NO.4; the NCBI number of sterol C-24 methyltransferase (ERG6) is NM_001180521.1; the NCBI number of malate synthase (MLS1) is NM_001182955.1; the NCBI number of citrate synthase (CIT2) is NM_001178718.1; the NCBI number of delta-14-sterol reductase (ERG24) is NM_001183118.1; the NCBI number of methylsterol monooxygenase (ERG25) is NM_001181189.3; the NCBI number of sterol 4α-carboxylate 3-dehydrogenase (ERG26) is NM_001180866.1; the NCBI number of sterol reductase (ERG27) is NM_001183118.1; the NCBI number of sterol 4α-carboxylate 3-dehydrogenase (ERG28) is NM_001183118.1; the NCBI number of sterol 4α-carboxylate 3-dehydrogenase (ERG29) is NM_001183118.1; the NCBI number of sterol 4α-carboxylate 3-dehydrogenase (ERG21) is NM_001183118.1; the NCBI number of sterol 4α-carboxylate 3-dehydrogenase (ERG22) is NM_001183118.1; the NCBI number of sterol 4α-carboxylate 3-dehydrogenase (ERG23) is NM_001183118.1; the NCBI number of sterol 4α-carboxylate 3- The CBI number is NM_001181987.1; the NCBI number of lanosterol synthase (ERG7) is NM_001179202.2; the NCBI number of farnesyl pyrophosphate synthase (ERG20) is NM_001181600.1; the NCBI number of diphosphomevalonate decarboxylase (MVD1) is NM_001183220.1; the NCBI number of phosphomevalonate kinase (ERG8) is NM_001182727.1; the NCBI number of mevalonate kinase (ERG12) is XM_033912620.1; the NCBI number or nucleotide sequence of endoplasmic reticulum regulatory factor (INO2) is NM_001180431.1.

[0033] Furthermore, a localization peptide is used to localize the enzyme to the endoplasmic reticulum for expression; the localization peptide includes the localization peptide CNE1, and the amino acid sequence of the localization peptide CNE1 is shown in SEQ ID NO.5.

[0034] Furthermore, the host of the starting strain includes but is not limited to Saccharomyces cerevisiae S288C; the starting strain includes but is not limited to the recombinant strain XG20.

[0035] The second object of the present invention is to provide a method for constructing the recombinant Saccharomyces cerevisiae, comprising the following steps:

[0036] The recombinant plasmid containing the sterol C24-reductase mutant encoding gene is introduced into the starting strain or the sterol C24-reductase mutant encoding gene is integrated into the genome of the starting strain to obtain a recombinant strain expressing the sterol C24-reductase mutant in a free or integrated manner.

[0037] Preferably, the XG20 strain is used as the starting strain.

[0038] The third object of the present invention is to provide the use of the recombinant Saccharomyces cerevisiae in the preparation of 7-dehydrocholesterol and / or active VD3.

[0039] The fourth object of the present invention is to provide a method for producing 7-dehydrocholesterol and reducing the production of by-products, comprising the step of fermenting with the recombinant Saccharomyces cerevisiae.

[0040] Furthermore, the by-products include cholesta-5,7-dien-3-ol, cholest-7-en-3-ol or the like.

[0041] Furthermore, the fermentation includes shake flask fermentation or fed-batch fermentation.

[0042] Furthermore, the fermentation comprises the following steps: inoculating the recombinant brewer's yeast into a fermentation medium for fermentation, and supplementing ethanol at 16-28 hours of fermentation until the fermentation is completed; the final concentration of the ethanol is 1 g / L.

[0043] Further, the culture was performed at 28-32°C and 180-280 rpm.

[0044] Furthermore, the fermentation medium contains the following components: 30-70 g / L soy peptone, 10-40 g / L glucose, 10-40 g / L sucrose, 10-40 g / L glycerol, and 0.1-5 g / L dipotassium hydrogen phosphate.

[0045] The fifth object of the present invention is to provide a sterol C24-reductase mutant, wherein the sterol C24-reductase mutant is obtained by mutating the sterol C24-reductase with an amino acid sequence as shown in SEQ ID NO.1, wherein the mutation comprises:

[0046] Mutate leucine at position 162 to arginine;

[0047] or mutating aspartic acid at position 386 to methionine;

[0048] Or mutate the leucine at position 162 to arginine and mutate the aspartic acid at position 386 to methionine.

[0049] The sixth object of the present invention is to provide a nucleic acid molecule encoding the sterol C24-reductase mutant.

[0050] The seventh object of the present invention is to provide a gene integration expression cassette or a recombinant plasmid carrying the nucleic acid molecule.

[0051] The eighth object of the present invention is to provide a recombinant cell containing the sterol C24-reductase mutant.

[0052] Furthermore, the host cell is a microorganism, such as bacteria or fungi.

[0053] The ninth objective of the present invention is to provide the use of the sterol C24-reductase, nucleic acid molecule, gene expression cassette or recombinant plasmid or recombinant cell in the preparation of 7-dehydrocholesterol.

[0054] Furthermore, the production is carried out using 7-dehydrosterol as a substrate.

[0055] Beneficial effects of the present invention:

[0056] The present invention provides a sterol C24-reductase mutant (DHCR24M) by performing enzyme engineering modification on DHCR24 and mutating leucine at position 162 and aspartic acid at position 386, thereby overcoming the problem of byproduct generation and low catalytic activity caused by the wide substrate spectrum of the key enzyme DHCR24 in 7-DHC production. The mutant is introduced into the starting strain XG20 of Saccharomyces cerevisiae, and after shake flask fermentation, the byproducts cholest-7-en-3-ol and cholesta-5,7-dien-3-ol are reduced by 31.5% and 46.1% respectively, and the target product is increased by 90.2%, reaching 867 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the plasmid map of pY14-TEF1.

[0058] Figure 2 This is a graph showing the yield of 7-DHC and the peak area of ​​by-products.

[0059] Figure 3 This is the docking diagram of DHCR24 / DHCR24M and the substrate 7-dehydrodesmosterol.

[0060] Figure 4 The kinetic results of original DHCR24 and mutant DHCR24M are shown.

[0061] Figure 5 This is the distance diagram between amino acid 162 and the substrate. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0063] The materials and methods involved in the following examples are as follows:

[0064] (1) Sequence:

[0065] The amino acid sequence of wild-type sterol C24-reductase DHCR24 (GgDHCR24) is shown in SEQ ID NO.1.

[0066] The amino acid sequence of the mutant sterol C24-reductase DHCR24M is shown in SEQ ID NO. 2. Specifically, the key amino acid residue sequence of the active center of sterol C24-reductase is underlined:

[0067] MSAVWSLGAGLLLLLLWVRHRGLEAVLVHHRWIFVCFFLMPLSILFDVYYQLRAWAVRRMHSAPRLHGQRVRHIQEQVREWKEEGGRRYMCTGRP G WLTVSLRVGKYKKTHKNIMINL M DVLEVDSERQVVRVEPLVTMGQLTAYLNPMGWTIPVVPELDD R TVGGLIMGTG IE SSSHIYGLFQHTCMAYELVLADGSLVRCSPTENSDLFYAVPWSCGTLGFLVAAEIKMIPAKKYIRLHYEPVRGLRSICEKFTEESKNKEN S FVEGLVYSLEEAVIMTGVLTDEAEPSKI N RIGNYYKPWFFKHVEKYLKANKTGIEYIPSRHYY H RHTRSIFW E LQDIIPFGNNPVFRYLFGWMVPPKISLLKLTQGEAIRKLYEQHHV V Q M MLVPMKSLEKSIQTFHV D LNVYPLWLCPFLLPNNPGMVHPKGDETELYV D IGAYGEPKTKQFEARASMRQMEKFVRSVHGFQML YADCYMTREEFWDMFDGSLYHSLREQ M NCKDAFPEVYDKICKAARH

[0068] The nucleotide sequence of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 encoding gene is shown in SEQ ID NO.3.

[0069] The NCBI accession number of isopentenyl diphosphate delta-isomerase IDI1 is NM_001183931.1.

[0070] The NCBI accession number of squalene epoxidase gene ERG1 is NM_001181304.1.

[0071] The NCBI accession number of sterol 14α-demethylase ERG11 is NM_001179137.1.

[0072] The NCBI accession number of alcohol dehydrogenase 2ADH2 is NM_001182812.1.

[0073] The NCBI accession number of lanosterol synthase ERG7 is NM_001179202.2.

[0074] mutated UPC2 G888A )'s nucleotide sequence is shown in SEQ ID NO.4.

[0075] The NCBI accession number of sterol C-24 methyltransferase ERG6 is NM_001180521.1.

[0076] The NCBI accession number of malate synthase MLS1 is NM_001182955.1.

[0077] The NCBI accession number of citrate synthase CIT2 is NM_001178718.1.

[0078] The NCBI accession number of Δ14-sterol reductase ERG24 is NM_001183118.1.

[0079] The NCBI accession number of methylsterol monooxygenase ERG25 is NM_001181189.3.

[0080] The NCBI accession number of sterol 4α-carboxylate 3-dehydrogenase ERG26 is NM_001180866.1.

[0081] The NCBI accession number of sterol reductase ERG27 is NM_001181987.1.

[0082] The NCBI accession number of lanosterol synthase ERG7 is NM_001179202.2.

[0083] The NCBI accession number of farnesyl pyrophosphate synthase ERG20 is NM_001181600.1.

[0084] The NCBI accession number of meglutarate diphosphate decarboxylase MVD1 is NM_001183220.1.

[0085] The NCBI accession number of phosphomevalonate kinase ERG8 is NM_001182727.1.

[0086] The NCBI accession number of mevalonate kinase ERG12 is XM_033912620.1.

[0087] CNE1 is an endoplasmic reticulum localization signal peptide, and the amino acid sequence of CNE1 is shown in SEQ ID NO.5.

[0088] The NCBI accession number of the endoplasmic reticulum regulatory factor INO2 is NM_001180431.1.

[0089] (2) Strains:

[0090] The starting strain Saccharomyces cerevisiae XG20 used in the present invention is described in Modular remodeling of sterolmetabolism for overproduction of 7-dehydrocholesterol in engineered yeast, and the gene modifications involved are shown in Table 1.

[0091] Table 1 XG20 gene modification sites and gene manipulation

[0092]

[0093]

[0094] (3) Culture medium composition:

[0095] Soy peptone fermentation medium: soy peptone 50g / L, glucose 25g / L, sucrose 25g / L, glycerol 25g / L, dipotassium hydrogen phosphate 0.6g / L;

[0096] YPD medium: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L.

[0097] Example 1 Construction of C24-reductase mutant DHCR24M strain

[0098] (a) Using the artificially synthesized gene fragment DHCR24 (codon preference optimization was performed using Saccharomyces cerevisiae as the expression host) as a template, primers Tu162-F1 and Tu162-R1 were used to amplify the DHCR24-162P1 gene fragment, primers Tu162-F2 and Tu162-R2 were used to amplify the DHCR24-162P2 gene fragment, primers Tu386-F1 and Tu386-R1 were used to amplify the DHCR24-386P1 gene fragment, and primers Tu386-F2 and Tu386-R2 were used to amplify the DHCR24-386P2 gene fragment. The pY14-TEF1 plasmid was used as a template. The map of the plasmid is shown in FIG. Figure 1 As shown, primers P14D24-F and P14D24-R were used to circle the plasmid P to obtain the pY14-TEF1 gene fragment, and the DHCR24-162P1 fragment, the DHCR24-162P2 fragment and the pY14-TEF1 fragment were connected by seamless ligase to obtain plasmid pY14-TEF1-DHCR24-162, and the DHCR24-386P1 fragment, the DHCR24-386P2 fragment and the pY14-TEF1 fragment were connected by seamless ligase to obtain plasmid pY14-TEF1-DHCR24-386.

[0099] (b) Using plasmid pY14-TEF1-DHCR24-162 as a template, primers Tu386-F1 and Tu386-R1 were used to amplify the DHCR24-386P1 gene fragment, and primers Tu386-F2 and Tu386-R2 were used to amplify the DHCR24-386P2 gene fragment. Using pY14-TEF1 plasmid as a template, primers P14D24-F and P14D24-R were used to circle the plasmid to obtain the pY14-TEF1 gene fragment. The DHCR24-386P1 fragment, DHCR24-386P2 fragment and pY14-TEF1 fragment were connected by seamless ligase to obtain plasmid pY14-TEF1-DHCR24-162-386.

[0100] (c) Using plasmid pY14-TEF1-DHCR24-162-386 as a template, primers D24Tu-F and D24Tu-R were used to amplify the DHCR24M gene fragment, using plasmid pY14-TEF1-DHCR24-162 as a template, primers D24Tu-F and D24Tu-R were used to amplify the DHCR24-162 gene fragment, using plasmid pY14-TEF1-DHCR24-386 as a template, primers D24Tu-F and D24Tu-R were used to amplify the DHCR24-162 gene fragment. 4Tu-R amplified DHCR24-386 gene fragment, using pMHyLp-trp plasmid as template, using primers loxTrp-F, loxTrp-R amplified gene fragment lox-trp, using Saccharomyces cerevisiae S228C genome as template, primers mot3UP-F, mot3UP-R amplified gene fragment MOT3UP fragment, primers mot3Down-F, mot3Down-R amplified gene fragment MOT3Down fragment. The obtained fragments DHCR24M or DHCR24-386 or DHCR24-162, MOT3UP, MOT3Down, lox-trp were subjected to overlap extension PCR, and after 1% agarose gel electrophoresis verification, the gel was cut and the fragments were recovered to obtain fusion gene fragments mot3-lox-trp-DHCR24M, mot3-lox-trp-DHCR24-386, mot3-lox-trp-DHCR24-162.

[0101] (d) The gene fragments mot3-lox-trp-DHCR24M, mot3-lox-trp-DHCR24-386, and mot3-lox-trp-DHCR24-162 obtained in step (c) were transformed into the competent state of Saccharomyces cerevisiae XG20 strain (the genetic modification sites and contents involved in the engineering bacteria are listed in Table 1), spread on SD-Trp plates, and cultured at 30° C. for 3 days. The fragments of colony PCR were verified by 1% agarose gel electrophoresis using primers YZ-D24Tu-F and YZ-D24Tu-R. The fragments were cut from the gel and recovered, and sequenced. After verification, strains XG21, XG22, and XG23 were obtained.

[0102] (e) The strains XG21, XG22 and XG23 obtained in step (d) were made competent, and the plasmid PY26-Cre was transformed into Saccharomyces cerevisiae. After a single colony grew on the SD-Ura screening solid plate, it was inoculated into YPD medium and cultured for 12 h, and cultured on a YPD solid plate containing 5-FOA at 30° C. for 3 d. After that, the grown single colonies were transferred to YPD solid plates and SD Ura screening solid plates for comparison and verification. The single colonies that grew normally on the YPD plate but could not grow on the SD Ura screening plate were the correct genetically engineered bacteria and were named XG21M, XG22M and XG23M.

[0103] Primer sequences:

[0104] Tu162-F1:tctaatctaagtttatgtctgccgtttggtctttgg

[0105] Tu162-R1:acggttctatcgtccaattctggg

[0106] Tu162-F2:acgatagaaccgtcggtggattg

[0107] Tu162-R2:acataactaattacatgattaatgcctagcagccttgcaga

[0108] P14D24-F:aagaccaaacggcagacataaacttagattagattgctatgctttctttc

[0109] P14D24-R:tgcaaggctgctaggcattaatcatgtaattagttatgtcacgcttacattc

[0110] Tu386-F1:tctaatctaagtttatgtctgccgtttggtctttgg

[0111] Tu386-R1:aagcatcatttggacgacgtg

[0112] Tu386-F2:tccaaatgatgcttgtccctatgaaatctc

[0113] Tu386-R2:acataactaattacatgattaatgcctagcagccttgcaga

[0114] D24Tu-F:accttggcactggccgtcgttttaggccgcaaattaaagccttcg

[0115] D24Tu-R:acgtcgtccttattcatagcttcaaaatgtttctactccttttttac

[0116] loxTrp-F:agcaatgaatgcggaccatcaccaggaaacagctatgaccatgattacg

[0117] loxTrp-R:aggctttaatttgcggcctaaaacgacggccagtgccaag

[0118] mot3UP-F:gaaagccgacagggacaacg

[0119] mot3UP-R: atggtcatagctgtttcctggtgatggtccgcattcattgctc

[0120] mot3Down-F: tagaaacattttgaagctatgaataaggacgacgttaaacgtg

[0121] mot3Down-R:tccacgttagtggggtgatagc

[0122] YZ-D24Tu-F:agaaccgtcggtggattgatc

[0123] YZ-D24Tu-R:catttggacgacgtggtgct

[0124] Example 2 Fermentation of mutant strains and detection of 7-DHC and its by-products

[0125] Inoculate single colonies of Saccharomyces cerevisiae XG21M, XG22M, and XG23M on solid YPD plates in 5 ml YPD medium, culture at 30°C, 220 rpm for 16-20 hours, then inoculate 10% of the inoculum into a 250 mL round-bottom shake flask containing 25 mL YPD liquid medium, and culture at 30°C, 220 rpm for 60 hours. When fermentation reaches 16-28 hours, add anhydrous ethanol with a final concentration of 1 g / L. 120h after the fermentation, centrifuge at 13000rpm for 30s to remove the supernatant, resuspend the precipitate with distilled water, centrifuge at 13000rpm for 30s, remove the supernatant, add a certain amount of 1.5mol / L potassium hydroxide methanol solution, 0.5mm glass beads, 1g ascorbic acid and 0.5gHBT to the precipitate, mix well, shake 12 cycles of 8m / s in a high-speed homogenizer, saponify the broken liquid in a water bath at 80℃ for 2 hours, shake and extract with petroleum ether after saponification, remove the impurities in the lower layer with a separatory funnel, and distill the petroleum ether layer under reduced pressure, further use methanol or acetonitrile or ethanol or a mixture of methanol and acetonitrile to redissolve the precipitate, and filter at 0.55μm for high performance liquid chromatography analysis. Methanol and water are used as the mobile phase, and a UV detector is used as the detector, a C18 chromatographic column, a column temperature of 40℃, and a detection wavelength of 265nm.

[0126] Gas phase mass spectrometry was used to analyze the by-products. The product was first extracted with ethyl acetate, and the extract was rotary evaporated. Then 50 μL of the derivatization reagent N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added, heated at 60°C for 30 minutes, cooled to room temperature, and subjected to gas chromatography-mass spectrometry. The detection chromatographic column was DB-5, the flow rate was 1.0 mL / min, the injection port temperature was 250°C, the initial temperature was 180°C, maintained for 2 minutes, and then increased to 280°C at a rate of 10°C / min and maintained for 5 minutes.

[0127] After liquid and gas mass spectrometry analysis, the 7-DHC production of the successfully constructed DHCR24M mutant strain XG21M was 867 mg / L, the peak area of ​​the byproduct cholesta-5,7-dien-3-ol was 2460, and the peak area of ​​the byproduct cholest-7-en-3-ol was 2355; the 7-DHC production of the successfully constructed DHCR24-386 mutant strain XG22M was 540 mg / L, the peak area of ​​the byproduct cholesta-5,7-dien-3-ol was 3750, and the peak area of ​​the byproduct cholest-7-en-3-ol was 3100; the 7-DHC production of the successfully constructed DHCR24-162 mutant strain XG23M was 670 mg / L, the peak area of ​​the byproduct cholesta-5,7-dien-3-ol was 3510, and the peak area of ​​the byproduct cholest-7-en-3-ol was 2891 (as shown in Figure 2). Figure 2 shown).

[0128] Example 3 Molecular Docking and Analysis Kinetic Analysis of Mutant DHCR24M

[0129] First, the structure of DHCR24M was modeled using Alphafold, and then the protein structure was dehydrated and hydrogenated, and the structure of the substrate 7-dehydrodesmosterol was optimized and energy minimized. The semi-flexible docking module LibDock in Discovery Studio was used for molecular docking, and the best docking result was determined according to the scoring function of libdock, such as Figure 3 Select the substrate pocket The molecular dynamics was performed using the Desmond module in Mastro, with the temperature set to 303.15 K and other parameters kept to default. The total duration was 100 ns. The dynamics results are shown in the figure below. Figure 4 As shown in Figure 2, it can be seen that the energy of binding between the entire protein and the substrate is lower and more stable, and the interaction force between the residues in the active center and the substrate becomes more, and the binding is tighter. Figure 5 As shown, the distance between the key amino acid and the substrate is Became It is more conducive to the reaction.

[0130] Comparative Example 1

[0131] (1) Construction of the original C24-reductase DHCR24 strain.

[0132] (a) Artificially synthesized gene fragment DHCR24 (codon preference optimization was performed using Saccharomyces cerevisiae as the expression host), using the Saccharomyces cerevisiae S228C genome as a template, primers mot3UP-F and mot3UP-R to amplify the gene fragment MOT3UP fragment, and primers mot3Down-F and mot3Down-R to amplify the gene fragment MOT3Down fragment. Using the pMHyLp-trp plasmid as a template, primers loxTrp-F and loxTrp-R were used to amplify the gene fragment lox-trp, and the obtained fragments DHCR24, MOT3UP, MOT3Down, and lox-trp were subjected to overlap extension PCR. After verification by 1% agarose gel electrophoresis, the gel was cut and the fragments were recovered to obtain the fusion gene fragment mot3-lox-trp-DHCR24.

[0133] (b) The gene fragment mot3-lox-trp-DHCR24 obtained in step (a) was transformed into the competent state of Saccharomyces cerevisiae XG20 strain, spread on SD-Trp plates, and cultured at 30° C. for 3 days. The fragments of colony PCR were subjected to 1% agarose gel electrophoresis using primers YZ-D24-F and YZ-D24-R to verify that they were correct. The fragments were then cut from the gel and recovered, and sequenced. After verification, strain XG24 was obtained.

[0134] (c) The strain XG24 obtained in step (b) was made competent, and the plasmid PY26-Cre was transformed into Saccharomyces cerevisiae. After a single colony grew on the SD-Ura screening solid plate, it was inoculated into YPD medium and cultured for 12 h, and then cultured on a YPD solid plate containing 5-FOA at 30° C. for 3 d. After that, the grown single colony was transferred to a YPD solid plate and a SD Ura screening solid plate for comparison and verification. The single colony that grew normally on the YPD plate but could not grow on the SD Ura screening plate was the correct genetically engineered bacterium and was named XG24Y.

[0135] Primer sequences:

[0136] YZ-D24-F:attaaaagtaaaactacgtacgcggcg

[0137] YZ-D24-R:aggccatacaagtgtgttggaaaa

[0138] (2) Fermentation of strain XG24Y and detection of 7-DHC and its by-products

[0139] The strain XG24Y was fermented according to the method of Example 2, and the fermentation product was detected according to the same detection method. The 7-DHC yield of the successfully constructed strain XG24Y was 455.6 mg / L, the peak area of ​​the byproduct cholesta-5,7-dien-3-ol was 4572, and the peak area of ​​the byproduct cholest-7-en-3-ol was 3440. Figure 2 shown.

[0140] (3) Molecular docking and kinetic analysis of DHCR24

[0141] First, the structure of DHCR24 was modeled using Alphafold, and then the protein structure was dehydrated and hydrogenated, and the structure of the substrate 7-dehydrodesmosterol was optimized and energy minimized. Molecular docking was performed using the semi-flexible docking module LibDock in DiscoveryStudio, and the best docking result was determined based on the scoring function of libdock. The key amino acid residues in the active center were determined to be: D386, E338, V384, L162, Y471, I173, D404, M119, H329, S265, M497, G96, D436, E174, D294. Figure 3 The molecular dynamics was performed using the Desmond module in Mastro. The temperature was set to 303.15 K, and the others were kept as default. The total duration was 100 ns. The dynamics results are shown in Figure 4 As shown in the figure, it can be seen that the energy of the entire protein binding to the substrate and the interaction force between the residues in the active center and the substrate. Figure 5 As shown, the distance between the key amino acid and the substrate is

[0142] In summary, the 7-DHC production of the mutant strains with D386 and / or L162 mutations screened out was improved compared with the wild strain constructed in this comparative example, and the by-product production was significantly reduced, among which the DHCR24M mutant was the best.

[0143] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant Saccharomyces cerevisiae, characterized in that A sterol C24-reductase mutant is overexpressed in the recombinant Saccharomyces cerevisiae; The sterol C24-reductase mutant is obtained by mutating the sterol C24-reductase whose amino acid sequence is shown in SEQ ID NO.1, and the mutation includes: Mutate leucine at position 162 to arginine; or mutating aspartic acid at position 386 to methionine; Or mutate the leucine at position 162 to arginine and mutate the aspartic acid at position 386 to methionine.

2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that The recombinant Saccharomyces cerevisiae is based on modified or unmodified Saccharomyces cerevisiae as a starting strain; the modification includes one or more of the following: (1) Overexpression of sterol C24-reductase, (2) Overexpression of truncated 3-hydroxy-3-methylglutaryl-CoA reductase, (3) Overexpression of isopentenyl diphosphate δ-isomerase, (4) Overexpression of squalene epoxidase, (5) Overexpression of sterol 14α-demethylase, (6) Overexpression of alcohol dehydrogenase 2, (7) Overexpression of lanosterol synthase, (8) Overexpression of transcription factor UPC2 mutant, (9) using a transcriptional activation system to upregulate the expression of any gene encoding any of (1) to (8), (10) using a transcriptional repression system to down-regulate the expression of any gene encoding sterol C-24 methyltransferase, malate synthase, or citrate synthase, (11) Overexpression of Δ14-sterol reductase, (12) Overexpression of methylsterol monooxygenase, (13) Overexpression of sterol 4α-carboxylate 3-dehydrogenase, (14) Overexpression of sterol reductase, (15) overexpressing one or more of lanosterol synthase, farnesyl pyrophosphate synthase, isopentenyl diphosphate delta-isomerase, diphosphomevalonate decarboxylase, phosphomevalonate kinase, and mevalonate kinase, and localizing one or more of the enzymes to the endoplasmic reticulum for expression, (16) Overexpression of endoplasmic reticulum regulatory factors.

3. The recombinant Saccharomyces cerevisiae according to claim 2, characterized in that The transcription activation system includes a CRISPR / dCpf1-mediated transcription activation system; the transcription inhibition system includes a CRISPR / dCas9-mediated transcription inhibition system.

4. The recombinant Saccharomyces cerevisiae according to claim 2, characterized in that The amino acid sequence of sterol C24-reductase is shown in SEQ ID NO.1; the nucleotide sequence of truncated 3-hydroxy-3-methylglutaryl-CoA reductase is shown in SEQ ID NO.3; the NCBI number of isopentenyl diphosphate δ-isomerase is NM_001183931.1; the NCBI number of squalene epoxidase gene is NM_001181304.1; the NCBI number of sterol 14α-demethylase is NM_001179137.1; the NCBI number of alcohol dehydrogenase 2 is NM_001182812.1; the NCBI number of lanosterol synthase is NM_001179202.2; the nucleotide sequence of transcription factor UPC2 mutant is shown in SEQ ID NO.4; the NCBI number of sterol C-24 methyltransferase is NM_001180521.1; the NCBI number of malate synthase is NM_001182955.1; the NCBI number of citrate synthase is NM_001178718.1; the NCBI number of Δ14-sterol reductase is NM_001183118.1; the NCBI number of methylsterol monooxygenase is NM_001181189.3; the NCBI number of sterol 4α-carboxylate 3-dehydrogenase is NM_001180866.1; the NCBI number of sterol reductase is NM_001183118.1; the NCBI number of sterol 4α-carboxylate 3-dehydrogen ... The NCBI number is NM_001181987.1; the NCBI number of lanosterol synthase is NM_001179202.2; the NCBI number of farnesyl pyrophosphate synthase is NM_001181600.1; the NCBI number of diphosphomevalonate decarboxylase is NM_001183220.1; the NCBI number of phosphomevalonate kinase is NM_001182727.1; the NCBI number of mevalonate kinase is XM_033912620.1; the NCBI number or nucleotide sequence of endoplasmic reticulum regulatory factor is NM_001180431.

1.

5. The recombinant Saccharomyces cerevisiae according to claim 2, characterized in that The enzyme is localized to the endoplasmic reticulum for expression using a localization peptide; the localization peptide includes a localization peptide CNE1, and the amino acid sequence of the localization peptide CNE1 is shown in SEQ ID NO.

5.

6. The recombinant Saccharomyces cerevisiae according to claim 2, characterized in that At least one of the following is included: the host of the starting strain includes Saccharomyces cerevisiae S288C; the starting strain includes the recombinant strain XG20.

7. The method for constructing the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 6, characterized in that: The following steps are involved: The gene integration expression frame or recombinant plasmid containing the sterol C24-reductase mutant encoding gene is introduced into the starting strain of Saccharomyces cerevisiae to obtain the recombinant Saccharomyces cerevisiae.

8. Use of the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 6 in the preparation of 7-dehydrocholesterol and / or active vitamin D3.

9. A method for producing 7-dehydrocholesterol and reducing the production of by-products, characterized in that: The method comprises the step of fermenting with the recombinant brewer's yeast according to any one of claims 1 to 6.

10. The method according to claim 9, characterized in that The by-products include cholesta-5,7-dien-3-ol, cholest-7-en-3-ol or the like.

11. The method according to claim 9, characterized in that The fermentation comprises the following steps: inoculating the recombinant brewer's yeast into a fermentation medium for fermentation, and supplementing ethanol when the fermentation reaches 16-28 hours until the fermentation ends; the final concentration of the ethanol is 1 g / L.

12. A sterol C24-reductase mutant, characterized in that The sterol C24-reductase mutant is obtained by mutating the sterol C24-reductase whose amino acid sequence is shown in SEQ ID NO.1, and the mutation includes: Mutate leucine at position 162 to arginine; or mutating aspartic acid at position 386 to methionine; Or mutate the leucine at position 162 to arginine and mutate the aspartic acid at position 386 to methionine.

13. A nucleic acid molecule encoding the sterol C24-reductase mutant according to claim 12.

14. A gene integration expression cassette or recombinant plasmid carrying the nucleic acid molecule of claim 13.

15. A recombinant cell expressing the sterol C24-reductase mutant according to claim 12.

16. Use of the sterol C24-reductase mutant according to claim 12, the nucleic acid molecule according to claim 13, the gene integration expression cassette or recombinant plasmid according to claim 14, and the recombinant cell according to claim 15 in the preparation of 7-dehydrocholesterol.

17. The use according to claim 16, characterized in that It is produced using 7-dehydrosterol as substrate.

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