A sterol c24-reductase mutant with reduced by-product and a saccharomyces cerevisiae producing 7-dehydrocholesterol

Abstract

The application discloses a sterol C24-reductase mutant for reducing by-products and a Saccharomyces cerevisiae for producing 7-dehydrocholesterol, and belongs to the technical field of biology. In order to solve the problems of by-product production and low catalytic activity caused by the wide substrate spectrum of the key enzyme DHCR24 in the current 7-DHC synthesis process, the leucine at the 162th position and the aspartic acid at the 386th position of DHCR24 are mutated through enzyme engineering modification, and a sterol C24-reductase mutant DHCR24M is obtained. The mutant is introduced into a Saccharomyces cerevisiae starting strain, and it is found through fermentation verification that, compared with the unmutated strain, the by-products cholest-7-en-3-ol and cholesta-5,7-dien-3-ol of the mutant strain are reduced by 31.5% and 46.1% respectively, and the target product is increased by 90.2%, which provides a new idea and method for industrial production of 7-dehydrocholesterol and active vitamin D3, and has a very wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a sterol C24-reductase mutant with reduced by-products and a 7-dehydrocholesterol-producing Saccharomyces cerevisiae, and belongs to the field of biotechnology. BACKGROUND

[0002] 7-dehydrocholesterol (7-DHC) is a steroid compound with high added value in the medical and industrial fields, which can not only be used for liquid crystal manufacturing and electromagnetic detection, but also has a greater demand in biomedical and human health. 7-DHC can be converted into vitamin D3 (VD3) under ultraviolet irradiation, so 7-DHC is a direct precursor of VD3. VD3 can promote the absorption of calcium in the intestine to maintain bone calcium balance, promote bone resorption by increasing the number of osteoclasts, maintain the calcium and phosphorus levels required for bone formation, and can also treat senile osteoporosis, rickets, reduce the incidence of common cancers, anti-inflammatory, immune regulation, hypothyroidism, improve skin quality, prevent infection of new coronavirus, etc. With the gradual improvement of people's health consciousness and the aggravation of population aging, the demand for VD3 is increasing year by year, resulting in a huge market for its direct precursor 7-DHC and a broad application prospect.

[0003] There are mainly two ways to obtain VD3 on the market at present, one is to extract from tuna liver oil, and the other is to synthesize through photochemical reaction of 7-dehydrocholesterol. Direct extraction will produce allergens, and the yield is severely limited, so the method of synthesizing VD3 in industry is to first prepare 7-DHC by chemical synthesis. The mainstream chemical synthesis method is to use lanolin as raw material, and 7-DHC is obtained through esterification, oxidation, reduction and hydrolysis reaction, and then VD3 is obtained through photo reaction. 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 microbial fermentation production of 7-DHC is green, environmentally friendly and sustainable.

[0004] Currently, the synthesis of 7-dehydrocholesterol by microorganisms mainly uses Saccharomyces cerevisiae as a 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), and the yield is 5.5 mg / g of cell dry weight (CN 103275997A). Since the 7-dehydrocholesterol synthesis pathway involves multiple organelles, it may cause difficulty in transferring intermediate metabolites, therefore, the 7-dehydrocholesterol synthesis pathway is restructured and fixed in mitochondria, peroxisome, endoplasmic reticulum, lipid droplet and the like, further improving the yield of 7-dehydrocholesterol (CN 112813129A, CN 113151027A, CN 116751698A). The above patents all introduce heterologous DHCR24, which is the most critical enzyme in the 7-DHC synthesis pathway, and without DHCR24, 7-DHC cannot be synthesized.

[0005] DHCR24 is composed of 516 amino acids and can remove the double bond at position C24 of the sterol side chain, but its substrate spectrum is wide and can recognize lanosterol, 4,4-dimethylcholesta-8,14,24-trien-3β-ol, 4,4-dimethylcholesta-8,24-dien-3β-ol, zymosterol, cholesta-7,24-dien-3β-ol, 7-dehydrodesmosterol and desmosterol, and is converted into different substances, thereby deriving the Kandutsch-Russell pathway, and through the action of other enzymes in the synthesis pathway, causing the generation and accumulation of by-products, affecting the yield and purification of the final product 7-DHC.

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

[0007] To solve the above problems, the present application provides a sterol C24-reductase mutant, which overcomes the defect of wide substrate spectrum of sterol C24-reductase, reduces the synthesis of by-products, and improves the catalytic efficiency of 7-dehydrodesmosterol. Meanwhile, the present application provides a genetically engineered Saccharomyces cerevisiae based on the mutant, which not only reduces the synthesis of by-products, but also efficiently produces 7-DHC, with a yield increase of 90.2% compared with the unmutated strain, and simplifies the subsequent purification process.

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

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

[0010] the leucine at position 162 is mutated to arginine;

[0011] or the aspartic acid at position 386 is mutated to methionine;

[0012] or the leucine at position 162 is mutated to arginine and the aspartic acid at position 386 is mutated to methionine.

[0013] The existing technology is to express chicken-derived / human-derived DHCR24 in a yeast cell chassis to synthesize 7-DHC, which can synthesize 7-DHC, but introducing a heterologous enzyme with a wide substrate spectrum will inevitably produce other by-products in addition to the target product 7-DHC, resulting in a decrease in the catalytic efficiency of DHCR24. The present application uses enzyme engineering methods, not just the integration of gene fragments, but through molecular docking to determine the active center and key residues, mutating the key residues, detecting the yield of 7-DHC to determine the optimal mutant, and further elucidating the mechanism of yield increase through molecular dynamics, providing a reference for the key enzyme modification of 7-DHC synthesis in the later stage.

[0014] Further, the recombinant Saccharomyces cerevisiae uses a 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 coenzyme A reductase (tHMG1),

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

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

[0019] (5) overexpression of sterol 14a-demethylase (ERG11),

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

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

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

[0023] (9) upregulation of expression of any one of the genes of (1) to (8) using a transcription activation system,

[0024] (10) downregulation of expression of any one of the genes of sterol C-24 methyltransferase (ERG6), malate synthase (MLS1), citrate synthase (CIT2) using a transcription inhibition system,

[0025] (11) overexpression of delta 14-sterol reductase (ERG24),

[0026] (12) overexpression of methyl sterol monooxygenase (ERG25),

[0027] (13) overexpression of sterol 4a-carboxylate 3-dehydrogenase (ERG26),

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

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

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

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

[0032] Further, the amino acid sequence of sterol C24-reductase (DHCR24) is shown as SEQ ID NO. 1; the nucleotide sequence of truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase (tHMG1) is shown as SEQ ID NO. 3; the NCBI number of isopentenyl diphosphate delta-isomerase (IDI1) is NM_001183931.1; the NCBI number of squalene epoxidase gene (ERG1) is NM_001181304.1; the NCBI number of sterol 14a-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 transcription factor UPC2 mutant is shown as 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 methyl sterol monooxygenase (ERG25) is NM_001181189.3; the NCBI number of sterol 4a-carboxylate 3-dehydrogenase (ERG26) is NM_001180866.1; the NCBI number of sterol reductase (ERG27) is NM_001181987.1; the NCBI number of lanosterol synthase (ERG7) is NM_001179202.2; the NCBI number of farnesyl pyrophosphate synthetase (ERG20) is NM_001181600.1; the NCBI number of dimethylallyl diphosphate 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] Further, the enzymes are expressed using localization peptides to localize the enzymes to the endoplasmic reticulum; the localization peptides include localization peptide CNE1, the amino acid sequence of which is shown as SEQ ID NO. 5.

[0034] Further, the starting strain includes but is not limited to recombinant strain XG20.

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

[0036] The recombinant plasmid containing the gene encoding the sterol C24-reductase mutant is introduced into the starting strain or the gene encoding the sterol C24-reductase mutant 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] A third object of the present application is to provide the use of the recombinant Saccharomyces cerevisiae in the preparation of 7-dehydrocholesterol and / or active VD3.

[0039] A fourth object of the present application is to provide a method for producing 7-dehydrocholesterol and reducing the production of by-products, comprising the step of fermentation using the recombinant Saccharomyces cerevisiae.

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

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

[0042] Further, the fermentation includes the following steps: inoculating the recombinant Saccharomyces cerevisiae into a fermentation medium for fermentation, and supplementing ethanol until the end of fermentation at 16-28 h; the final concentration of the ethanol is 1 g / L.

[0043] Further, the culture is carried out at 28-32°C and 180-280 rpm.

[0044] Further, the fermentation medium contains the following components: soybean peptone 30-70 g / L, glucose 10-40 g / L, sucrose 10-40 g / L, glycerol 10-40 g / L, potassium phosphate 0.1-5 g / L.

[0045] A fifth object of the present application is to provide a sterol C24-reductase mutant, which is obtained by mutating the sterol C24-reductase mutant with the amino acid sequence shown in SEQ ID NO. 1, and the mutation includes:

[0046] the leucine at position 162 is mutated to arginine;

[0047] or the aspartic acid at position 386 is mutated to methionine;

[0048] or the leucine at position 162 is mutated to arginine and the aspartic acid at position 386 is mutated to methionine.

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

[0050] A seventh object of the present application is to provide a genetic integration expression frame or a recombinant plasmid carrying the nucleic acid molecule.

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

[0052] Further, the host cell is a microorganism, such as a bacterium or a fungus.

[0053] A ninth object of the present application is to provide the use of the sterol C24-reductase, the nucleic acid molecule, the genetic expression frame or the recombinant plasmid, or the recombinant cell in the preparation of 7-dehydrocholesterol.

[0054] Further, the production is performed with 7-dehydrodesmosterol as the substrate.

[0055] Advantages of the present application:

[0056] The present application provides a sterol C24-reductase mutant (DHCR24M) by performing enzyme engineering on DHCR24 and mutating leucine at position 162 and aspartic acid at position 386, which overcomes the problems of wide substrate spectrum of the key enzyme DHCR24 in 7-DHC production, leading to the generation of by-products and low catalytic activity. After the mutant is introduced into the Saccharomyces cerevisiae starting strain XG20, the by-products 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 DRAWINGS

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

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

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

[0060] Figure 4 Figure 4 is the kinetic results of the original DHCR24 and the mutant DHCR24M.

[0061] Figure 5 Figure 5 is a diagram of the distance of amino acid 162 to the substrate. DETAILED DESCRIPTION

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

[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 mutant sterol C24-reductase DHCR24M is shown in SEQ ID NO. 2. Specifically, as follows, wherein the sequence of amino acid residues key to the active center of sterol C24-reductase is indicated by underlining:

[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 number of the isopentenyl-diphosphate delta-isomerase IDI1 is NM_001183931.1.

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

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

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

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

[0074] The nucleotide sequence of the mutated UPC2 (transcription factor mutant UPC2 G888A ) is shown in SEQ ID NO. 4.

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

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

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

[0078] The NCBI number of the delta 14-sterol reductase ERG24 is NM_001183118.1.

[0079] The NCBI number of the methyl sterol monooxygenase ERG25 is NM_001181189.3.

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

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

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

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

[0084] The NCBI number of diphosphomevalerate decarboxylase MVD1 is NM_001183220.1.

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

[0086] The NCBI 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 number of endoplasmic reticulum regulatory factor INO2 is NM_001180431.1.

[0089] (2) Strain:

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

[0091] Table 1 XG20 gene modification site and gene operation

[0092]

[0093]

[0094] (3) Medium composition:

[0095] Soybean peptone fermentation medium: soybean peptone 50 g / L, glucose 25 g / L, sucrose 25 g / L, glycerol 25 g / L, potassium phosphate dibasic 0.6 g / 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 for Saccharomyces cerevisiae as the expression host) as the template, using primers Tu162-F1 and Tu162-R1 to amplify the DHCR24-162P1 gene fragment, using primers Tu162-F2 and Tu162-R2 to amplify the DHCR24-162P2 gene fragment, using primers Tu386-F1 and Tu386-R1 to amplify the DHCR24-386P1 gene fragment, using primers Tu386-F2 and Tu386-R2 to amplify the DHCR24-386P2 gene fragment, using the pY14-TEF1 plasmid as the template, the map of which is shown in Figure 1

[0099] (b) using the plasmid pY14-TEF1-DHCR24-162 as the template, using primers Tu386-F1 and Tu386-R1 to amplify the DHCR24-386P1 gene fragment, using primers Tu386-F2 and Tu386-R2 to amplify the DHCR24-386P2 gene fragment, using the pY14-TEF1 plasmid as the template, using primers P14D24-F and P14D24-R to amplify the pY14-TEF1 gene fragment, and using the DHCR24-386P1 fragment, the DHCR24-386P2 fragment, and the pY14-TEF1 fragment to perform ligation by means of a seamless ligase, thereby obtaining the plasmid pY14-TEF1-DHCR24-162-386.

[0100] ​(c) using plasmid pY14-TEF1-DHCR24-162-386 as a template, primer D24Tu-F and D24Tu-R to amplify DHCR24M gene fragment, using plasmid pY14-TEF1-DHCR24-162 as a template, primer D24Tu-F and D24Tu-R to amplify DHCR24-162 gene fragment, using plasmid pY14-TEF1-DHCR24-386 as a template, primer D24Tu-F and D24Tu-R to amplify DHCR24-386 gene fragment, using pMHyLp-trp plasmid as a template, primer loxTrp-F and loxTrp-R to amplify gene fragment lox-trp, using S. cerevisiae S228C genome as a template, primer mot3UP-F and mot3UP-R to amplify gene fragment MOT3UP fragment, primer mot3Down-F and mot3Down-R to amplify 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 fragments were recovered by gel cutting 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, mot3-lox-trp-DHCR24-162 obtained in step (c) were transformed into the competent cells of S. cerevisiae XG20 strain (the genetic modification sites and contents involved in the engineering bacteria are listed in Table 1), and then coated on SD-Trp plates and cultured at 30°C for 3 days; using primers YZ-D24Tu-F and YZ-D24Tu-R, the colony PCR fragments were subjected to 1% agarose gel electrophoresis verification, and then the fragments were recovered by gel cutting, sequenced, and verified to obtain strains XG21, XG22 and XG23.

[0102] (e) The strains XG21, XG22 and XG23 obtained in step (d) are made competent, and the plasmid PY26-Cre is transformed into the Saccharomyces cerevisiae, after a single colony is grown on the SD-Ura screening solid plate, it is inoculated into YPD medium and cultured for 12 h, then it is inoculated on YPD solid plate containing 5-FOA and cultured at 30°C for 3 d, after that, the single colonies grown are respectively transferred to YPD solid plate and SD Ura screening solid plate for comparison and verification, the single colony which grows normally on YPD plate but cannot grow on SD Ura screening plate is the correct genetically engineered bacteria, and is named as 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] Fermentation of the mutant strains of Example 2 and detection of 7-DHC and its by-products

[0125] Inoculate the single colonies of Saccharomyces cerevisiae XG21M, XG22M and XG23M on solid YPD plates into 5 ml YPD medium, respectively, and incubate at 30°C, 220 rpm for 16-20 h. Then, inoculate 10% of the culture into 25 ml YPD liquid medium in a 250 ml round-bottom flask, and incubate at 30°C, 220 rpm for 60 h. Add anhydrous ethanol to a final concentration of 1 g / L at 16-28 h of fermentation. After 120 h of fermentation, centrifuge at 13,000 rpm for 30 s to remove the supernatant, resuspend the precipitate with distilled water, centrifuge at 13,000 rpm for 30 s, remove the supernatant, and add a certain amount of 1.5 mol / L potassium hydroxide methanol solution, 0.5 mm glass beads, 1 g ascorbic acid and 0.5 g HBT to the precipitate. Mix well, shake in a high-speed homogenizer at 8 m / s for 12 cycles, and then saponify the broken liquid in a water bath at 80°C for 2 h. After saponification, perform shock extraction with petroleum ether, remove the lower impurities using a separatory funnel, and then distill the petroleum ether layer under reduced pressure. Further resuspend the precipitate with methanol or acetonitrile or ethanol or a mixture of methanol and acetonitrile, filter at 0.55 μm, and then perform high-performance liquid chromatography analysis. Use methanol and water as the mobile phase, use a UV detector, use a C18 chromatographic column, set the column temperature to 40°C, and set the detection wavelength to 265 nm.

[0126] Gas chromatography-mass spectrometry is used to analyze the byproducts. First, extract the products with ethyl acetate, perform rotary evaporation on the extracted liquid, then add 50 μL of derivatization reagent N, O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), heat at 60°C for 30 min, cool to room temperature, and perform gas chromatography-mass spectrometry detection. The detection chromatographic column is DB-5, the flow rate is 1.0 mL / min, the injection port temperature is 250°C, the initial temperature is 180°C, and the initial temperature is maintained for 2 min, then increased to 280°C at a rate of 10°C / min, and maintained for 5 min.

[0127] After liquid and gas chromatography-mass spectrometry analysis, the 7-DHC yield of the successfully constructed DHCR24M mutant strain XG21M is 867 mg / L, the peak area of the byproduct cholesta-5,7-dien-3-ol is 2460, and the peak area of the byproduct cholest-7-en-3-ol is 2355; the 7-DHC yield of the successfully constructed DHCR24-386 mutant strain XG22M is 540 mg / L, the peak area of the byproduct cholesta-5,7-dien-3-ol is 3750, and the peak area of the byproduct cholest-7-en-3-ol is 3100; and the 7-DHC yield of the successfully constructed DHCR24-162 mutant strain XG23M is 670 mg / L, the peak area of the byproduct cholesta-5,7-dien-3-ol is 3510, and the peak area of the byproduct cholest-7-en-3-ol is 2891 (as shown in Table 1).Figure 2 Figure 6 shows the docking results of the substrate 7-dehydrodesmosterol in the active site of the mutant DHCR24M.

[0128] Example 3 Molecular docking and analysis of kinetics analysis of mutant DHCR24M

[0129] Firstly, the structure of DHCR24M was modeled using Alphafold, then the protein structure was dehydrated and hydrogenated, and the structure of the substrate 7-dehydrodesmosterol was optimized and energy minimized, and the molecular docking was performed using the semi-flexible docking module LibDock in Discovery Studio, and the best docking result was determined according to the scoring function of LibDock scoring, as shown in Figure 3 The amino acid residues in the substrate pocket range were selected. The molecular dynamics used the Desmond module in Mastro, the temperature was set to 303.15 K, the others were kept default, the total duration was 100 ns, and the dynamics results were as shown in Figure 4 It can be seen that the energy of the whole protein binding with the substrate is lower, more stable, and the interaction force between the residues in the active center and the substrate is more, and the binding is more closely, and at the same time, as shown in Figure 5 The distance between the key amino acids and the substrate has changed from to which is more conducive to the reaction.

[0130] Comparative Example 1

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

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

[0133] (b) The gene fragment mot3-lox-trp-DHCR24 obtained in step (a) is transformed into the competent cells of the Saccharomyces cerevisiae XG20 strain, spread on a SD-Trp plate, and cultured at 30°C for 3 days. The colonies are subjected to PCR using primers YZ-D24-F and YZ-D24-R, and the fragments obtained by colony PCR are subjected to 1% agarose gel electrophoresis to verify the correctness. The fragments are recovered by gel cutting and subjected to sequencing. After verification, the strain XG24 is obtained.

[0134] (c) The strain XG24 obtained in step (b) is made competent, and the plasmid PY26-Cre is transformed into Saccharomyces cerevisiae. After single colonies grow on a SD-Ura solid plate, they are inoculated into YPD medium and cultured for 12 h. Then, they are inoculated on a YPD solid plate containing 5-FOA and cultured at 30°C for 3 d. After that, the single colonies grown are transferred to a YPD solid plate and a SD Ura solid plate for comparison and verification. The single colonies that grow normally on the YPD plate but cannot grow on the SD Ura plate are the correct genetically engineered bacteria, and are 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 byproducts

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

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

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

[0142] As can be seen, the 7-DHC yield of the mutant strains with D386 and / or L162 mutation screened is improved relative to the wild strain constructed in the present comparative example, and the byproduct yield is obviously decreased, and the DHCR24M mutant is the best.

[0143] Obviously, the above examples are only examples for clearly illustrating, and are not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A recombinant Saccharomyces cerevisiae, characterized in that, The recombinant Saccharomyces cerevisiae overexpresses a sterol C24-reductase mutant; The sterol C24-reductase mutant is obtained by mutating the sterol C24-reductase with the amino acid sequence shown in SEQ ID NO. 1, and the mutation is: the leucine at position 162 is mutated into arginine; or the aspartic acid at position 386 is mutated into methionine; or the leucine at position 162 is mutated into arginine and the aspartic acid at position 386 is mutated into methionine.

2. The recombinant Saccharomyces cerevisiae of claim 1, wherein, The recombinant Saccharomyces cerevisiae uses the Saccharomyces cerevisiae with or without modification as the starting strain; the modification includes one or more of the following: (1) overexpressing sterol C24-reductase DHCR24, (2) overexpressing truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase, the nucleotide sequence of which is shown in SEQ ID NO. 3, (3) overexpressing isopentenyl diphosphate delta-isomerase IDI1, (4) overexpressing squalene epoxidase ERG1, (5) overexpressing sterol 14α-demethylase ERG11, (6) overexpressing ethanol dehydrogenase 2 ADH2, (7) overexpressing lanosterol synthase ERG7, (8) overexpressing transcription factor UPC2 mutant, the nucleotide sequence of which is shown in SEQ ID NO. 4, (9) overexpressing △14-sterol reductase ERG24, (10) overexpressing methyl sterol monooxygenase ERG25, (11) overexpressing sterol 4α-carboxylate 3-dehydrogenase ERG26, (12) overexpressing sterol reductase ERG27, (13) overexpressing one or more enzymes of lanosterol synthase ERG7, farnesyl pyrophosphate synthetase ERG20, isopentenyl diphosphate delta-isomerase IDI1, dimethylallyl pyrophosphate decarboxylase MVD1, phosphomevalonate kinase ERG8, and mevalonate kinase ERG12, and positioning one or more enzymes to express in the endoplasmic reticulum, (14) overexpressing endoplasmic reticulum regulatory factor INO2.

3. The recombinant Saccharomyces cerevisiae of claim 2, wherein, The amino acid sequence of the sterol C24-reductase is shown as SEQ ID NO. 1; the NCBI number of the isopentenyl diphosphate delta-isomerase is NM_001183931.1; the NCBI number of the squalene epoxidase gene is NM_001181304.1; the NCBI number of the sterol 14alpha-demethylase is NM_001179137.1; the NCBI number of the alcohol dehydrogenase 2 is NM_001182812.1; the NCBI number of the lanosterol synthase is NM_001179202.2; the NCBI number of the sterol C-24 methyltransferase is NM_001180521.1; the NCBI number of the malate synthase is NM_001182955.1; the NCBI number of the citrate synthase is NM_001178718.1; the NCBI number of the delta 14-sterol reductase is NM_001183118.1; the NCBI number of the methyl sterol monooxygenase is NM_001181189.3; the NCBI number of the sterol 4alpha-carboxylate 3-dehydrogenase is NM_001180866.1; the NCBI number of the sterol reductase is NM_001181987.1; the NCBI number of the lanosterol synthase is NM_001179202.2; the NCBI number of the farnesyl pyrophosphate synthetase is NM_001181600.1; the NCBI number of the dimethylallyl phosphate decarboxylase is NM_001183220.1; the NCBI number of the phosphomevalonate kinase is NM_001182727.1; the NCBI number of the mevalonate kinase is XM_033912620.1; the NCBI number or nucleotide sequence of the endoplasmic reticulum modulator is NM_001180431.

1.

4. The recombinant Saccharomyces cerevisiae of claim 2, wherein, The enzyme is expressed using a localization peptide; the localization peptide includes a localization peptide CNE1, and the amino acid sequence of the localization peptide CNE1 is shown as SEQ ID NO.

5.

5. The recombinant Saccharomyces cerevisiae of claim 2, wherein, The starting strain is a recombinant strain XG20.

6. The method of constructing a recombinant Saccharomyces cerevisiae of any of claims 1-5, characterized in that, The method comprises the following steps: The gene containing the gene encoding the sterol C24-reductase mutant is introduced into the expression frame or recombinant plasmid of the Saccharomyces cerevisiae starting strain to obtain the recombinant Saccharomyces cerevisiae.

7. Use of the recombinant Saccharomyces cerevisiae according to claim 1 for the preparation of 7-dehydrocholesterol, characterized in that, The recombinant Saccharomyces cerevisiae takes the recombinant strain XG20 as the starting strain.

8. A method for producing 7-dehydrocholesterol with reduced production of by-products, characterized in that, The method comprises the step of fermenting using the recombinant Saccharomyces cerevisiae of claim 1, wherein the recombinant Saccharomyces cerevisiae takes the recombinant strain XG20 as the starting strain.

9. The method of claim 8, wherein, The by-product includes cholesta-5,7-dien-3-ol or cholest-7-en-3-ol.

10. The method of claim 8, wherein, The fermentation comprises the following steps: inoculating the recombinant Saccharomyces cerevisiae into a fermentation medium for fermentation, and supplementing ethanol until the end of fermentation when the fermentation is performed for 16-28 h; and the final concentration of the ethanol is 1 g / L.

11. A sterol C24-reductase mutant, characterized in that, The sterol C24-reductase mutant is obtained by mutating the sterol C24-reductase with the amino acid sequence shown as SEQ ID NO. 1, and the mutation is: the leucine at the 162nd position is mutated into arginine; or the aspartic acid at position 386 is mutated to methionine; or the leucine at position 162 is mutated to arginine and the aspartic acid at position 386 is mutated to methionine.

12. A nucleic acid molecule encoding the sterol C24-reductase mutant of claim 11.

13. A genetic integration expression cassette or recombinant plasmid carrying the nucleic acid molecule of claim 12.

14. A recombinant cell expressing the sterol C24-reductase mutant of claim 11.

15. Use of the sterol C24-reductase mutant according to claim 11, the nucleic acid molecule according to claim 12, the genetic integration expression cassette or recombinant plasmid according to claim 13, or the recombinant cell according to claim 14 for the preparation of 7-dehydrocholesterol, characterized in that, with 7-dehydrodesmosterol as substrate. with 7-dehydrodesmosterol as substrate.

Images