A recombinant bacterium for increasing the production of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol and its application

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a recombinant bacterium for increasing the production of (22R, 23R)-22,23-dihydroxy-7-dehydrorapesterol and its application. The recombinant bacterium of the present invention is a saccharomyces cerevisiae YQC512 expressing a truncated cytochrome P450 reductase, a cytochrome P450 enzyme derived from sesame, and a cytochrome P450 enzyme derived from peanut. The engineered bacterium provided by the present invention has improved the ability to synthesize (22R, 23R)-22,23-dihydroxy-7-dehydrorapesterol compared to existing engineered bacteria. Furthermore, the present invention converts the sucrose-inducible promoter P SUC2 The expression cassette for GAL4 was integrated into the genome of Saccharomyces cerevisiae YQC512, enabling the engineered bacteria to express strongly under culture conditions with sucrose as the sole carbon source, reducing the safety risks of using ethanol as a carbon source in existing fermentation production processes.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant bacterium for increasing the yield of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol and an application thereof. Background Art

[0002] Brassinosteroids, a class of sixth-class plant hormones, play a crucial regulatory role in plant growth processes such as seed germination, root growth, and reproduction. As the most biologically active of the brassinosteroids, brassinolide (also known as brassinolide) has broad application prospects in agriculture. Due to the extremely low content of brassinolide in plants and the lack of scalable chemical synthetic precursors in nature, mass production of brassinolide is difficult. Therefore, the most viable technical route for mass production of brassinolide is to use synthetic biology methods to construct an artificially designed biosynthetic pathway for semi-synthetic precursors of brassinolide in microorganisms and obtain these precursors on a large scale through fermentation.

[0003] In the semi-synthetic reaction process, the most critical step is the double bond dihydroxylation reaction catalyzed by rare platinum group metal (such as osmium) oxide. Among them, the Sharpless asymmetric dihydroxylation reaction assisted by chiral ligands and catalyzed by OsO4 can obtain 22R,23R-dihydroxy brassinolide compounds (such as brassinolide lactone) with higher activity in higher yields. However, the use of highly toxic OsO4 and expensive chiral ligands will lead to high risk in the reaction process and high manufacturing costs, which limits the commercial application of brassinolide. Therefore, with the help of synthetic biology methods, semi-synthetic precursors with 22R,23R-dihydroxy structures can be directly obtained through microbial fermentation, which can avoid the use of OsO4 and its chiral ligands, control the synthesis cost of brassinolide compounds, and promote the green, environmental protection and safety of the production process. In Chinese patent CN116496337A, by introducing Arabidopsis thaliana ( Arabidopsis thaliana ) derived from cytochrome P450 reductase AtCPR1, C-22 sterol hydroxylase AtDWF4 (AtCYP90B1) and C-23 sterol hydroxylase AtCYP90D1, to achieve the synthesis of a semi-synthetic precursor with a 22R,23R-dihydroxy structure, namely (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0004] However, Chinese patent CN116496337A is limited by the weak catalytic activity of Arabidopsis thaliana-derived cytochrome P450 enzymes and the poor substrate selectivity of AtDWF4. Through high-density fermentation, the yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol is 100.60 mg / L, and the synthesis level is low. Summary of the Invention

[0005] To address the above issues, the present invention provides a recombinant bacterium for increasing the production of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol and its application. The recombinant bacterium expressed by the present invention exhibits higher cytochrome P450 enzyme activity and better substrate selectivity, thereby increasing the production of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant bacterium for increasing the yield of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, wherein the recombinant bacterium expresses truncated cytochrome P450 reductase, flax ( Cannabis sativa ) derived cytochrome P450 enzymes and peanuts ( Arachis stenosperma ) cytochrome P450 enzymes from Saccharomyces cerevisiae YQC512;

[0008] The amino acid sequence of the truncated cytochrome P450 reductase is shown in SEQ ID NO.1;

[0009] The amino acid sequence of the cytochrome P450 enzyme derived from sesame is shown in SEQ ID NO.2;

[0010] The peanut-derived cytochrome P450 enzyme includes a wild-type P450 enzyme and / or a mutant of the wild-type P450 enzyme; the amino acid sequence of the wild-type P450 enzyme is shown in SEQ ID NO.3;

[0011] The mutants of the wild-type P450 enzyme include one or more of a first mutant, a second mutant, a third mutant, a fourth mutant and a fifth mutant; the amino acid sequences of the first mutant, the second mutant, the third mutant, the fourth mutant and the fifth mutant are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0012] Preferably, the genome of the recombinant bacterium is further integrated with a fourth expression cassette; the fourth expression cassette expresses sterol Δ 24(28) reductase and the peanut-derived cytochrome P450 enzyme, the sterol Δ 24(28) The amino acid sequence of the reductase is shown in SEQ ID NO.4.

[0013] Preferably, the genome of the recombinant bacterium is also integrated with P SUC2 The GAL4 expression cassette expressed; the P SUC2The nucleotide sequence of the expressed GAL4 expression cassette is shown in SEQ ID NO.10.

[0014] The present invention provides a method for constructing the recombinant bacteria described in the above technical solution, comprising the following steps:

[0015] The CRISPR-Cas9 gene editing tool is used to integrate an expression cassette for expressing the enzyme into the genome of Saccharomyces cerevisiae YQC512; the enzyme is an enzyme expressed by the recombinant bacteria.

[0016] Preferably, the promoter of the expression cassette comprises P GAL1 、P GAL2 or P GAL7 .

[0017] Preferably, the CRISPR-Cas9 gene editing tool includes gRNA; the expression cassette of the truncated cytochrome P450 reductase is recorded as the first expression cassette; the expression cassette of the sesame-derived cytochrome P450 enzyme is recorded as the second expression cassette; and the expression cassette of the peanut-derived cytochrome P450 enzyme is recorded as the third expression cassette;

[0018] The nucleotide sequence of the gRNA integrated into the first expression cassette is shown in SEQ ID NO.19;

[0019] The nucleotide sequence of the gRNA integrated into the second expression cassette is shown in SEQ ID NO.31;

[0020] The nucleotide sequence of the gRNA integrated into the third expression cassette is shown in SEQ ID NO.39;

[0021] The nucleotide sequence of the gRNA integrated into the fourth expression cassette is shown in SEQ ID NO.62;

[0022] Integrated P SUC2 The nucleotide sequence of the gRNA of the expressed GAL4 expression cassette is shown in SEQ ID NO.71.

[0023] The present invention provides the use of the recombinant bacteria described in the above technical solution or the recombinant bacteria constructed by the method described in the above technical solution in synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0024] The present invention provides a method for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, comprising: fermenting and culturing an engineered bacterium using glucose and / or sucrose as a carbon source to obtain a fermentation broth containing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol; the engineered bacterium is the recombinant bacterium described in the above technical solution or a recombinant bacterium constructed using the method described in the above technical solution.

[0025] Preferably, the fermentation temperature is 25-35° C., and the pH value is 4.5-5.5.

[0026] Preferably, when the engineered bacteria is an integrated P SUC2 When the recombinant bacteria expressing the GAL4 expression cassette are grown, fermentation culture is started until the OD of the engineered bacteria reaches 600 When the value is 150, glucose is used as the carbon source. 600 When the value is ≥150, sucrose is used as the carbon source.

[0027] Beneficial effects:

[0028] The present invention obtained cytochrome P450 enzymes with higher enzyme activity and better substrate selectivity through screening experiments, namely CsCYP724B1 (NCBI ID: XP_030484427.2) and AsCYP90D1 (NCBI ID: XP_057729888.1). The two cytochrome P450 enzymes were co-expressed with a truncated cytochrome P450 reductase from Arabidopsis thaliana in Saccharomyces cerevisiae YQC512 to obtain a recombinant bacterium that can synthesize (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol ( Figure 1 ); and using a rational design approach, the site in the AsCYP90D1 catalytic pocket that binds to the steroid ring was modified to obtain a highly active AsCYP90D1 mutant, strengthening the synthesis pathway and improving the (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol synthesis capacity of Saccharomyces cerevisiae.

[0029] Furthermore, the present invention increases sterol Δ 24(28) The copy number of reductase and peanut-derived cytochrome P450 enzymes increased the supply of hydroxylated precursors (7-dehydrocampesterol) while further improving the ability of Saccharomyces cerevisiae to synthesize (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0030] Furthermore, the present invention utilizes CRISPR-Cas9 gene editing tools to modify the sucrose-inducible promoter P SUC2 The transcriptional regulatory factor GAL4 was integrated into the genome of Saccharomyces cerevisiae YQC512, so that the promoter regulated by GAL4 (P GAL1 、P GAL2 、P GAL7 ) can be strongly expressed under the culture conditions where sucrose is the only carbon source ( Figure 3), so that sucrose can be used as a carbon source in the middle and late stages of high-density fermentation, increasing the yield of (22R,23R)-22,23-dihydroxy-7-dehydrorapesterol while reducing the safety risks of using ethanol as a carbon source in the existing fermentation production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0032] Figure 1 The biosynthetic pathway of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol in Saccharomyces cerevisiae;

[0033] Figure 2 Schematic diagram of the regulatory mechanism of the GAL series promoters under galactose, glucose, and ethanol before and after the knockout of the GAL80 gene in Saccharomyces cerevisiae;

[0034] Figure 3 Schematic diagram of the inducible expression system established in the present invention using sucrose as an inducer;

[0035] Figure 4 The liquid chromatography-ultraviolet detection analysis diagram of the recombinant bacteria YQC443 and the recombinant bacteria introduced with AtCYP90B1 and CsCYP724B1 respectively;

[0036] Figure 5 The liquid chromatography-ultraviolet detection analysis diagram of the recombinant bacteria YQD680 and the recombinant bacteria introduced with AtCYP90D1 and AsCYP90D1 respectively;

[0037] Figure 6 The yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol of AsCYP90D1 single point mutant and combined mutants;

[0038] Figure 7 The yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol of recombinant strains YQD680-AsM4, YQD692 and YQD693;

[0039] Figure 8 The yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol in high-density fermentation of recombinant bacteria YQD693 and recombinant bacteria YQD630. DETAILED DESCRIPTION

[0040] The present invention provides a recombinant bacterium for increasing the yield of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, wherein the recombinant bacterium expresses truncated cytochrome P450 reductase, flax ( Cannabis sativa ) derived cytochrome P450 enzymes and peanuts ( Arachis stenosperma ) cytochrome P450 enzymes from Saccharomyces cerevisiae YQC512;

[0041] The amino acid sequence of the truncated cytochrome P450 reductase is shown in SEQ ID NO.1;

[0042] The amino acid sequence of the cytochrome P450 enzyme derived from sesame is shown in SEQ ID NO.2;

[0043] The peanut-derived cytochrome P450 enzyme includes a wild-type P450 enzyme and / or a mutant of the wild-type P450 enzyme; the amino acid sequence of the wild-type P450 enzyme is shown in SEQ ID NO.3;

[0044] The mutants of the wild-type P450 enzyme include one or more of a first mutant, a second mutant, a third mutant, a fourth mutant and a fifth mutant; the amino acid sequences of the first mutant, the second mutant, the third mutant, the fourth mutant and the fifth mutant are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0045] As an embodiment, the peanut-derived cytochrome P450 enzyme may be a mutant of a wild-type P450 enzyme. As an embodiment, the mutant of the wild-type P450 enzyme may be a fifth mutant. As an embodiment, the promoter of the gene encoding the mutant of the wild-type P450 enzyme may be P GAL1 promoter.

[0046] As an embodiment, the genome of the recombinant bacteria is further integrated with a fourth expression cassette; the fourth expression cassette expresses sterol Δ 24(28) reductase and the peanut-derived cytochrome P450 enzyme, the sterol Δ 24(28) The amino acid sequence of the reductase is shown in SEQ ID NO. 4. As an embodiment, the cytochrome P450 enzyme expressed by the fourth expression cassette can be the fifth mutant. As an embodiment, the sterol Δ 24(28) The promoter of the reductase encoding gene can be P GAL7 The sterol Δ 24(28) The reductase is an enzyme expressed by Saccharomyces cerevisiae YQC512. The present invention further increases the sterol Δ 24(28)The copy numbers of the reductase and the peanut-derived cytochrome P450 enzyme not only increase the supply of the hydroxylated precursor (7-dehydrocampesterol), but also further enhance the (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol synthesis ability of Saccharomyces cerevisiae.

[0047] As an embodiment, the genome of the recombinant bacteria is also integrated with P SUC2 The GAL4 expression cassette expressed; the P SUC2 The nucleotide sequence of the expressed GAL4 expression cassette is shown in SEQ ID NO.10.

[0048] The AsCYP90D1 mutants AsT117G, AsS213G, AsM216V, AsE289H and AsM4 obtained by rational design in the present invention have significantly improved catalytic activities compared with the wild type.

[0049] The present invention obtains cytochrome P450 enzymes with higher enzyme activity and better substrate selectivity by mining, and then uses a rational design method to rationally design the site in the catalytic pocket of AsCYP90D1 that binds to the steroid ring to improve its catalytic ability for non-natural substrates. Secondly, the rate-limiting enzyme is overexpressed to increase the yield of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol. Finally, a sucrose-inducible promoter P is used to SUC2 The transcriptional regulatory factor GAL4 is expressed to achieve the purpose of using sucrose as a carbon source and inducing gene expression at the same time.

[0050] In order to avoid the expression and catalysis of plant-derived cytochrome P450 enzymes affecting the growth of bacteria in the early stage of fermentation, Chinese patent CN116496337A uses a lactose-inducible promoter ( P GAL1 、 P GAL2 ), this type of promoter is strongly induced by ethanol in a Saccharomyces cerevisiae strain with the GAL80 gene knocked out ( Figure 2 ), in the middle and late stages of the high-density fermentation process, ethanol is used as a carbon source to induce the expression of Arabidopsis thaliana-derived cytochrome P450 enzymes and their reductases. However, the amount of ethanol used is close to one-third of the volume of the fermentation tank. In industrial fermentation production (using 5-ton, 10-ton, 20-ton tanks), tons of sterile ethanol need to be reserved as a carbon source. Not only is it difficult to sterilize ethanol, but it also contains huge safety risks. The present invention uses CRISPR-Cas9 gene editing tools to modify the sucrose-inducible promoter P SUC2 The transcriptional regulatory factor GAL4 was integrated into the genome of Saccharomyces cerevisiae YQC512, so that the promoter regulated by GAL4 (P GAL1 、P GAL2 、P GAL7) can be strongly expressed under culture conditions where sucrose is the sole carbon source, so that sucrose can be used as a carbon source in the middle and late stages of high-density fermentation to induce exogenous gene expression, thereby increasing the yield of (22R,23R)-22,23-dihydroxy-7-dehydrorapesterol and reducing the safety risks of using ethanol as a carbon source in existing fermentation production processes.

[0051] The engineered bacteria constructed by the present invention can produce up to 32.52 mg / L of protein after being cultured in YPD medium for 72 hours, which is 5.42 times that of the existing patented technology under the same culture conditions.

[0052] The engineered bacteria constructed in the present invention can produce up to 388.91 mg / L of ethanol after culturing for 144 hours in a fermentation medium using glucose-sucrose as a carbon source, which is 3.85 times that of the existing technology (glucose-ethanol).

[0053] Based on the above advantages, the present invention provides a method for constructing the recombinant bacteria described in the above technical solution, comprising the following steps:

[0054] The CRISPR-Cas9 gene editing tool is used to integrate an expression cassette for expressing the enzyme into the genome of Saccharomyces cerevisiae YQC512; the enzyme is an enzyme expressed by the recombinant bacteria.

[0055] As an embodiment, the promoter of the expression cassette includes P GAL1 、P GAL2 or P GAL7 .

[0056] As an embodiment, the CRISPR-Cas9 gene editing tool includes gRNA; the expression cassette of the truncated cytochrome P450 reductase is recorded as the first expression cassette; the expression cassette of the sesame-derived cytochrome P450 enzyme is recorded as the second expression cassette; the expression cassette of the peanut-derived cytochrome P450 enzyme is recorded as the third expression cassette;

[0057] The nucleotide sequence of the gRNA integrated into the first expression cassette is shown in SEQ ID NO.19;

[0058] The nucleotide sequence of the gRNA integrated into the second expression cassette is shown in SEQ ID NO.31;

[0059] The nucleotide sequence of the gRNA integrated into the third expression cassette is shown in SEQ ID NO.39;

[0060] The nucleotide sequence of the gRNA integrated into the fourth expression cassette is shown in SEQ ID NO.62;

[0061] Integrated P SUC2The nucleotide sequence of the gRNA of the expressed GAL4 expression cassette is shown in SEQ ID NO.71.

[0062] Based on the above advantages, the present invention provides the use of the recombinant bacteria described in the above technical solution or the recombinant bacteria constructed using the method described in the above technical solution in the synthesis of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0063] Based on the above advantages, the present invention provides a method for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, comprising: fermenting and culturing an engineered bacterium using glucose and / or sucrose as a carbon source to obtain a fermentation broth containing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol; the engineered bacterium is the recombinant bacterium described in the above technical solution or a recombinant bacterium constructed using the method described in the above technical solution.

[0064] In one embodiment, the fermentation temperature is 25-35° C., and the pH value is 4.5-5.5. In another embodiment, the fermentation temperature is 28-32° C., and the pH value is 5.0.

[0065] As an embodiment, when the engineered bacteria is an integrated P SUC2 When the recombinant bacteria expressing the GAL4 expression cassette are grown, fermentation culture is started until the OD of the engineered bacteria reaches 600 When the value is 150, glucose is used as the carbon source. 600 When the value is ≥150, sucrose is used as the carbon source.

[0066] To further illustrate the present invention, the following detailed description of a recombinant bacterium for increasing the production of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol and its application is provided by the present invention in conjunction with the accompanying drawings and examples. However, these should not be construed as limiting the scope of protection of the present invention.

[0067] Preparation Example

[0068] The culture medium used in the present invention is as follows:

[0069] (1) YPD medium: 10 g / L yeast powder, 20 g / L peptone and 20 g / L glucose; solid medium with 20 g / L agar powder added, sterilized at 115°C for activation and pre-culture of Saccharomyces cerevisiae.

[0070] (2) YPD (HygB / G418) medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 100 μg / mL hygromycin B (HygB), and 200 μg / mL geneticin (G418); solid medium with 20 g / L agar powder added and sterilized at 115°C for screening of KanMX markers.

[0071] (3) LB medium: 10 g / L sodium chloride, 10 g / L peptone and 5 g / L yeast powder; add 20 g / L agar powder to the solid medium and sterilize at 121°C for activation and pre-culture of E. coli.

[0072] (4) LB (Amp) medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, and 100 μg / mL ampicillin. For solid culture medium, add 20 g / L agar powder and sterilize at 121°C for culturing Escherichia coli containing plasmids.

[0073] The detection methods involved in the following examples are as follows:

[0074] 1. Detection of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol and its precursors:

[0075] A single clone was picked from the YPD plate streaked with recombinant bacteria and inoculated into 5 mL of YPD liquid medium. The culture was carried out at 30°C and 220 rpm for 96 h. After the culture was completed, 0.5 mL of the fermentation broth was collected and centrifuged to remove the supernatant. 600 μL of ethanol-KOH solution (250 g / L KOH was added to a 50% volume concentration of ethanol solution) was added and vortexed thoroughly. The solution was saponified in a boiling water bath for 1 h, cooled to room temperature, and 400 μL of water and 800 μL of petroleum ether were added. After vortexing thoroughly, 500 μL of the upper petroleum ether extract was vacuum-evacuated and redissolved in 500 μL of anhydrous ethanol. The extract was filtered through a 0.22 μm PVDF filter membrane into the cannula in the liquid phase detection bottle as the test sample.

[0076] High-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2050 with a Thermo C-18 column (ODS Hypersil, 4.6×250 mm, 5 μm). The detection wavelength was 280 nm, the column temperature was 30°C, the flow rate was 1 mL / min, the injection volume was 10 μL, and the mobile phase was the following gradient: 90% methanol for 15 min, 100% methanol for 9 min, and 90% methanol for 6 min.

[0077] The Saccharomyces cerevisiae YQC512 involved in the following examples was constructed by the construction method disclosed in Chinese patent CN116496337A.

[0078] The amino acid sequences or nucleotide sequences involved in the following examples are as follows:

[0079] trCPR1 (SEQ ID NO.1):

[0080] MAWKKTTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYGDGEPTDNAARFYKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPRFTTQKSMESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSRVHVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFIYEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW;

[0081] CsCYP724B1 (SEQ ID NO.2):

[0082] MMMSSSYSSTILFITMIFGVMGSLLLLLILMKYFLPTKPQNLPAGTMGWLPFFGETLAFLKPHNSNSLGSFLQDRCLRYGKVFKSHLFGSGAIVSCDVELNMFILQNEEKLFEAHYPKSMHGILGKYSLLVVSGDVHKKLRNIIVRFINSSKSQPKFLACVDNLVISMLKSWNDRTQVSFFKEAKRMTLSVMVKSVLNIEPDDPLTVQILEEFETYMKGFVSLPILFPGTSYYKAIKARERLSSIVKGIIKERVLGKTTRNEVGEEELFLDMIIEKEENLSEEEKLSIVLDILLGGYETTATLMSLLLYFISNQPHVFQQLKEEHQAIRENKKDGEPLNWEDYKKMDFTSHVTCEAMRCGNVVKFVHRKALQDVKYKDIVIPSGWKVFPIFTGVHFDESLHDKPHDFNPWRWTSTDNNKEMRKKVTPFGGGPRLCPGSELAKVVIGFTLHHILLNYRWKIKADESPLAYPYVEFRRGLILEIEPAENTYINITN;

[0083] AsCYP90D1 (SEQ ID NO.3):

[0084] MDKVVLIVVTPLLLFFILFYRRITWLRLIKSKHMKNQLPLGTLGWPFVGETIEFISCAYSDHPESFMDKRRSMYGKVFKSHIFGSPTIVSTDAEVNKFILQSDAKAFVPSYPKSLTELMGKSSILLINGSLQRRIHGLVGAFFKSQQLKVQITREMQNYVQQSMTNWKEDQPVYIQDETKNIAFQVLVKALISLDPGEEMELLKKHFQEFMSGLMSLPINLPGTKLHQSLQAKKKMVKLMLRTIESRRECGISKVPKDVVDVLLNDENDRLTDELIADNMIDMMIPGEDSVPVLMTLAVKYLSECPTALQQLREENVKLKKLKDQLGEPLNWSDYLSLPFTQTVITETLRIGNVIIGVMRKAMKDVEIKGYFIPKGWCVLAYFRSIHLDDKNYDKPYQFNPWRWQDKDTSTCNFTPFGGGQRLCPGLDLARLEASIFLHHLVTQFRWYAEEDTIVNFPTVRMKRRMPILVRRVKS;

[0085] ArDWF1 (SEQ ID NO.4):

[0086] MSDLEVPLRPKRKKIWVDYFVQFRWIIVIFLVLPISFTLYFLTYLGDVRSESKSFKQRQREHDENVEKVVKRLKERNPKKDGLVCTARKPWISVGMRNVDYKRARHFEVDLSAFRNVLDIDKERMIARVEPLVNMGQISRVTGPMNLSLAVVAELDDLTVGGLINGYGIEGSSHLYGLFSDTVVSYEIVLADGRLVRATKDNQYSDLFYAIPWSQGTLGLLVAAEVKLIPVKEYMKVTYKPIVGNLKELAQGYIDSFAPRDGDQDNPEKVPDFVETMIYSPTEGVCMTGRYASKEEAKKKGNKINPVGWWFKPWFYQHAQTALKKGEFVEYIPTRDYHHRHTRSLYWEGELILPFADQWWFRFLLGWLMPPKVSLLKATQGEAIRNYYHEMHVIQDMLVPLYKVGDALEFVHREMELYPVWLCPHRLFKLPVKPMISPEPGFELQRRQGDTHYAQMYTDILLCYAPGPVLRGEQFDGAEAVHRMENWLIENHGYEPQYTVSELSEKNFWRMFDGGLYEQCRRKYGAVGTFMSVYYKSKKGRKTEKEVQEAEQAILESPDAEVA;

[0087] AsT117G(SEQ ID NO.5):

[0088] MDKVVLIVVTPLLLFFILFYRRITWLRLIKSKHMKNQLPLGTLGWPFVGETIEFISCAYSDHPESFMDKRRSMYGKVFKSHIFGSPTIVSTDAEVNKFILQSDAKAFVPSYPKSLGELMGKSILLINGSSLQRRIHGLVGAFFKSQQLKVQITREMQNYVQQSMTNWKEDQPVYIQDETKNIAFQVLVKALISLDPGEEMELLKKHFQEFMSGLMSLPINLPGTKLHQSLQAKKKMVKLMLRTIESRRECGISKVPKDVVDVLLNDENDRLTDELIADNMIDMMIPGEDSVPVLMTLAVKYLSECPTALQQLREENVKLKKLKDQLGEPLNWSDYLSLPFTQTVITETLRIGNVIIGVMRKAMKDVEIKGYFIPKGWCVLAYFRSIHLDDKNYDKPYQFNPWRWQDKDTSTCNFTPFGGGQRLCPGLDLARLEASIFLHHLVTQFRWYAEEDTIVNFPTVRMKRRMPILVRRVKS;

[0089] AsS213G (SEQ ID NO. 6):

[0090] MDKVVLIVFVTPLLLFFILFYRRITWLRLIKSKHMKNQLPLGTLGWPFVGETIEFISCAYSDHPESFMDKRRSMYGKVFKSHIFGSPTIVSTDAEVNKFILQSDAKAFVPSYPKSLTELMGKSSILLINGSLQRRIHGLVGAFFKSQQLKVQITREMQNYVQQSMTNWKEDQPVYIQDETKNIAFQVLVKALISLDPGEEMELLKKHFQEFMGGLMSLPINLPGTKLHQSLQAKKKMVKLMLRTIESRRECGISKVPKDVVDVLLNDENDRLTDELIADNMIDMMIPGEDSVPVLMTLAVKYLSECPTALQQLREENVKLKKLKDQLGEPLNWSDYLSLPFTQTVITETLRIGNVIIGVMRKAMKDVEIKGYFIPKGWCVLAYFRSIHLDDKNYDKPYQFNPWRWQDKDTSTCNFTPFGGGQRLCPGLDLARLEASIFLHHLVTQFRWYAEEDTIVNFPTVRMKRRMPILVRRVKS;

[0091] AsM216V(SEQ ID NO.7):

[0092] MDKVVLIVFVTPLLLFFILFYRRITWLRLIKSKHMKNQLPLGTLGWPFVGETIEFISCAYSDHPESFMDKRRSMYGKVFKSHIFGSPTIVSTDAEVNKFILQSDAKAFVPSYPKSLTELMGKSSILLINGSLQRRIHGLVGAFFKSQQLKVQITREMQNYVQQSMTNWKEDQPVYIQDETKNIAFQVLVKALISLDPGEEMELLKKHFQEFMSGLVSLPINLPGTKLHQSLQAKKKMVKLMLRTIESRRECGISKVPKDVVDVLLNDENDRLTDELIADNMIDMMIPGEDSVPVLMTLAVKYLSECPTALQQLREENVKLKKLKDQLGEPLNWSDYLSLPFTQTVITETLRIGNVIIGVMRKAMKDVEIKGYFIPKGWCVLAYFRSIHLDDKNYDKPYQFNPWRWQDKDTSTCNFTPFGGGQRLCPGLDLARLEASIFLHHLVTQFRWYAEEDTIVNFPTVRMKRRMPILVRRVKS;

[0093] AsE289H(SEQ ID NO.8):

[0094] MDKVVLIVVTPLLLFFILFYRRITWLRLIKSKHMKNQLPLGTLGWPFVGETIEFISCAYSDHPESFMDKRRSMYGKVFKSHIFGSPTIVSTDAEVNKFILQSDAKAFVPSYPKSLTELMGKSSILLINGSLQRRIHGLVGAFFKSQQLKVQITREMQNYVQQSMTNWKEDQPVYIQDETKNIAFQVLVKALISLDPGEEMELLKKHFQEFMSGLMSLPINLPGTKLHQSLQAKKKMVKLMLRTIESRRECGISKVPKDVVDVLLNDENDRLTDELIADNMIDMMIPGHDSVPVLMTLAVKYLSECPTALQQLREENVKLKKLKDQLGEPLNWSDYLSLPFTQTVITETLRIGNVIIGVMRKAMKDVEIKGYFIPKGWCVLAYFRSIHLDDKNYDKPYQFNPWRWQDKDTSTCNFTPFGGGQRLCPGLDLARLEASIFLHHLVTQFRWYAEEDTIVNFPTVRMKRRMPILVRRVKS;

[0095] AsM4 (SEQ ID NO.9):

[0096] MDKVVLIVFVTPLLLFFILFYRRITWLRLIKSKHMKNQLPLGTLGWPFVGETIEFISCAYSDHPESFMDKRRSMYGKVFKSHIFGSPTIVSTDAEVNKFILQSDAKAFVPSYPKSLGELMGKSSILLINGSLQRRIHGLVGAFFKSQQLKVQITREMQNYVQQSMTNWKEDQPVYIQDETKNIAFQVLVKALISLDPGEEMELLKKHFQEFMGGLVSLPINLPGTKLHQSLQAKKKMVKLMLRTIESRRECGISKVPKDVVDVLLNDENDRLTDELIADNMIDMMIPGHDSVPVLMTLAVKYLSECPTALQQLREENVKLKKLKDQLGEPLNWSDYLSLPFTQTVITETLRIGNVIIGVMRKAMKDVEIKGYFIPKGWCVLAYFRSIHLDDKNYDKPYQFNPWRWQDKDTSTCNFTPFGGGQRLCPGLDLARLEASIFLHHLVTQFRWYAEEDTIVNFPTVRMKRRMPILVRRVKS;

[0097] GAL4 expression cassette (SEQ ID NO.10) containing P: SUC2

[0098] ​

[0099] AtCYP90B1(SEQ ID NO.24):

[0100] MFETEHHTLLPLLLLPSLLSLLLFLILLKRRNRKTRFNLPPGKSGWPFLGETIGYLKPYTATTLGDFMQQHVSKYGKIYRSNLFGEPTIVSADAGLNRFILQNEGRLFECSYPRSIGGILGKWSMLVLVGDMHRDMRSISLNFLSHARLRTILLKDVERHTLFVLDSWQQNSIFSAQDEAKKFTFNLMAKHIMSMDPGEEETEQLKKEYVTFMKGVVSAPLNLPGTAYHKALQSRATILKFIERKMEERKLDIKERKQRTDDDLLGWVLKHSNLSTEQILDLILSLLFAGHETSSVAIALAIFFLQACPKAVEELREEHLEIARAKKELGESELNWDDYKKMDFTQCVINETLRLGNVVRFLHRKALKDVRYKGYDIPSGWKVLPVISAVHLDNSRYDQPNLFNPWRWQQQNTWGNNYMPFGGGPRLCAGSELAKLEMAVFIHHLVLKFNWELAEDDKPFAFPFVDFPNGLPIRVSRIL;

[0101] AtCYP90D1(SEQ ID NO.32):

[0102] MDTSSSLLFFSFFFFIIIVIFNKINGLRSSPASKKKLNDHHVTSQSHGPKFPHGSLGWPVIGETIEFVSSAYSDRPESFMDKRRLMYGRVFKSHIFGTATIVSTDAEVNRAVLQSDSTAFVPF YPKTVRELMGKSSILLINGSLHRRFHGLVGSFLKSPLLKAQIVRDMHKFLSESMDLWSEDQPVLLQDVSKTVAFKVLAKALISVEKGEDLEELKREFENFISGLMSLPINFPGTQLHRSLQAK KNMVKQVERIIEGKIRKTKNKEEDDVIAKDVVDVLLKDSSEHLTHNLIANNMIDMMIPGHDSVPVLITLAVKFLSDSPAALNLLTEENMKLKSLKELTGEPLYWNDYLSLPFTQKVITETLRM GNVIIGVMRKAMKDVEIKGYVIPKGWCFLAYLRSVHLDKLYYESPYKFNPWRWQERDMNTSSSFSPFGGGQRLCPGLDLARLETSVFLHHLVTRFRWIAEEDTIINFPTVHMKNKLPIWIKRI;

[0103] Table 1 Nucleotide sequences of different primers or gRNAs

[0104]

[0105]

[0106]

[0107] Example 1 Construction of Saccharomyces cerevisiae strain YQC443 expressing the truncated cytochrome P450 reductase trCPR1 gene from Arabidopsis thaliana

[0108] The genome of Saccharomyces cerevisiae YQC512 was used as a template and primers PP GAL2 -F (SEQ ID NO. 11) and PP GAL2 -R (SEQ ID NO.12) was amplified by PCR to obtain P GAL2 fragment; using the genome of Saccharomyces cerevisiae YQC512 as a template, the primers PT ADH2 -F (SEQ ID NO. 13) and PT ADH2 -R (SEQ ID NO.14) was amplified by PCR to obtain T ADH2 fragment.

[0109] The product synthesized by Shanghai Sangon Biotechnology Co., Ltd. trCPR1 The plasmid of the gene was used as a template and primers PP GAL2 -trCPR1 (SEQ ID NO. 15) and PT ADH2 -trCPR1 (SEQ ID NO.16) was obtained by PCR amplification trCPR1 fragment; trCPR1 The protein encoded by the gene is a truncated cytochrome P450 reductase derived from Arabidopsis thaliana with 47 amino acids at the N-terminus replaced by methionine-methionine. The amino acid sequence is shown in SEQ ID NO.1.

[0110] Three fragments (P GAL2 Fragment, T ADH2 Fragments and trCPR1 The fragments were fused by overlap extension PCR and PCR using pEASY ® -Blunt Zero Cloning Kit (containing pEASY®-Blunt Zero plasmid, purchased from Beijing Quanshijin Biotechnology Co., Ltd., Cat. No. CB501), constructed pEB-P GAL2 -trCPR1; plasmid pEB-P GAL2 -trCPR1 was used as template and primer Dor.-V07-P with 50 bp homology arm was used. GAL2 (SEQ ID NO. 17) and Dor.-V07-T ADH2 (SEQ ID NO.18) was amplified by PCR to obtain trCPR1 expression cassette; using lithium acetate / PEG3350 chemical conversion method, the trCPR1 The expression cassette was transformed into Saccharomyces cerevisiae YQC512; using the CRISPR-Cas9 gene editing tool, double-strand breaks were made at the V07 site under the guidance of V07-gRNA (SEQ ID NO.19), and the Saccharomyces cerevisiae strain YQC443 expressing the cytochrome P450 reductase gene was constructed.

[0111] Example 2 Construction of recombinant bacteria YQD679 with the introduction of AtCYP90B1 and recombinant bacteria YQD680 with the introduction of CsCYP724B1

[0112] The genome of Saccharomyces cerevisiae YQC512 was used as a template and primers PP GAL7 -F (SEQ ID NO. 20) and PP GAL7 -R (SEQ ID NO.21) was amplified by PCR to obtain P GAL7fragment; using the genome of Saccharomyces cerevisiae YQC512 as a template, the primers PT HIS5 -F (SEQ ID NO. 22) and PT HIS5 -R (SEQ ID NO.23) was amplified by PCR to obtain T HIS5 fragment.

[0113] The product synthesized by Shanghai Sangon Biotechnology Co., Ltd. is derived from Arabidopsis thaliana. AtCYP90B1 The plasmid of the gene was used as a template and primers PP GAL7 -At90B1 (SEQ ID NO. 25) and PT HIS5 -At90B1 (SEQ ID NO. 26) was amplified by PCR to obtain the AtCYP90B1 fragment, AtCYP90B1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.24.

[0114] The product synthesized by Shanghai Shenggong Biotechnology Co., Ltd. is derived from sesame ( Cannabis sativa )of CsCYP724B1 The plasmid of the gene was used as a template and primers PP GAL7 -Cs724B1 (SEQ ID NO. 27) and PT HIS5 -Cs724B1 (SEQ ID NO. 28) was amplified by PCR to obtain the CsCYP724B1 fragment, CsCYP724B1 The amino acids encoded by the gene are shown in SEQ ID NO.2.

[0115] Overlap extension PCR fusion GAL7 fragments, AtCYP90B1 fragments and T HIS5 fragments and using pEASY ® -BluntZero Cloning Vectors Kit, constructed plasmid pEB-P GAL7 -AtCYP90B1; using overlap extension PCR fusion P GAL7 fragment, CsCYP724B1 fragment and T HIS5 fragments and using pEASY ® -Blunt Zero Cloning Vectors Kit, constructed plasmid pEB-P GAL7 -CsCYP724B1.

[0116] plasmid pEB-P GAL7 -AtCYP90B1 and pEB-P GAL7 -CsCYP724B1 was used as template and primer Dor.-V18-P with 50 bp homology arm was used.GAL7 (SEQ ID NO. 29) and Dor.-V18-T HIS5 (SEQ ID NO. 30) was amplified by PCR to obtain the AtCYP90B1 expression cassette and the CsCYP724B1 expression cassette; the AtCYP90B1 expression cassette and the CsCYP724B1 expression cassette were respectively transformed into the strain YQC443 constructed in Example 1 using the lithium acetate / PEG3350 chemical transformation method; using the CRISPR-Cas9 gene editing tool, double-strand breaks were performed on the V18 site under the guidance of V18-gRNA (SEQ ID NO. 31), and the DNA was integrated into the Saccharomyces cerevisiae genome. HygB / G418 plates were coated, and positive clones were screened to construct the recombinant bacteria YQD679 with the introduction of the AtCYP90B1 expression cassette and the recombinant bacteria YQD680 with the introduction of the CsCYP724B1 expression cassette.

[0117] The precursors of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol were detected by fermentation in 50 mL YPD shake flasks at 30°C and 220 rpm for 96 h. The liquid phase diagrams of YQC433 (None), YQD679 (AtCYP90B1) and YQD680 (CsCYP724B1) were analyzed. Figure 4 ), it can be found that the recombinant bacteria YQD680 introduced with CsCYP724B1 does not have a precursor peak around 14 min, indicating that CsCYP724B1 has better substrate selectivity and higher catalytic efficiency.

[0118] Example 3 Construction of recombinant bacteria YQD680-At introduced with AtCYP90D1 and recombinant bacteria YQD680-AsWT introduced with AsCYP90D1

[0119] The product synthesized by Shanghai Sangon Biotechnology Co., Ltd. is derived from Arabidopsis thaliana. AtCYP90D1 The plasmid of the gene was used as a template and primers PP GAL7 -AtCYP90D1 (SEQ ID NO. 33) and PT HIS5 -AtCYP90D1 (SEQ ID NO. 34) was amplified by PCR to obtain the AtCYP90D1 fragment, AtCYP90D1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.32.

[0120] The product synthesized by Shanghai Sangon Biotechnology Co., Ltd. is derived from peanut ( Arachis stenosperma )of AsCYP90D1 The plasmid of the gene was used as a template and primers PP GAL7-AsCYP90D1 (SEQ IDNO.35) and PT HIS5 -AsCYP90D1 (SEQ ID NO. 36) was amplified by PCR to obtain the AsCYP90D1 fragment, AsCYP90D1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

[0121] The method of Example 2 was used to amplify P GAL7 Fragment and T HIS5 Fragments were fused using overlap extension PCR. GAL7 fragments, AtCYP90D1 fragments and T HIS5 The fragment was constructed using the pEASY®-Blunt Zero Cloning Vectors kit to obtain the plasmid pEB-P GAL7 -AtCYP90D1; using overlap extension PCR fusion P GAL7 fragments, AtCYP90B1 fragments and T HIS5 The fragment was constructed using the pEASY®-Blunt Zero Cloning Vectors kit to obtain the plasmid pEB-P GAL7 -AsCYP90D1.

[0122] plasmid pEB-P GAL7 -AtCYP90D1 and pEB-P GAL7 -AsCYP90D1 was used as template and primer Dor.-V35-P with 40 bp homology arm was used. GAL7 (SEQ ID NO. 37) and Dor.-V35-T HIS5(SEQ ID NO.38) was amplified by PCR to obtain the AtCYP90D1 expression cassette and the AsCYP90D1 expression cassette; the AtCYP90D1 expression cassette and the AsCYP90D1 expression cassette were respectively transformed into the recombinant bacteria YQD680 constructed in Example 2 using the lithium acetate / PEG3350 chemical transformation method; using the CRISPR-Cas9 gene editing tool, double-strand breaks were performed on the V35 site under the guidance of V35-gRNA (SEQ ID NO.39), and the DNA was integrated into the Saccharomyces cerevisiae genome. HygB / G418 plates were coated and positive clones were screened to construct the recombinant bacteria YQD680-At introduced with the AtCYP90D1 expression cassette and the recombinant bacteria YQD680-AsWT introduced with the AsCYP90D1 expression cassette. (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol and its precursors were detected by 50mL YPD shake flask fermentation at 30℃ and 220rpm for 96h. The liquid phase diagrams of YQD680 (None), YQD680-At (AtCYP90D1) and YQD680-AsWT (AsCYP90D1) were compared ( Figure 5 ), the (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol yield of YQD680-AsWT was 7.96 mg / L, which was higher than that of YQD680-At (4.26 mg / L).

[0123] Example 4 Rational Design of the Catalytic Pocket of AsCYP90D1 to Enhance Its Catalytic Activity for 22S-22-Hydroxy-7-dehydrocampesterol

[0124] Since the optimal substrate of plant-derived CYP90D1 differs from the 22S-22-hydroxy-7-dehydrocampesterol catalyzed in the present invention mainly in the structure of the sterol ring, and CsCYP724B1 has significantly better catalytic activity for 7-dehydrocampesterol, by comparing the amino acid sequences, protein structures, and substrate binding to the catalytic pocket of AsCYP90D1 and CsCYP724B1, the amino acid residues in the catalytic pockets of the two that are related to the sterol ring binding were analyzed to obtain potential amino acid residue sites that can improve the catalytic ability of AsCYP90D1 to 22S-22-hydroxy-7-dehydrocampesterol. GAL7-AsCYP90D1 was used as a template, and primer pairs As-T117G-F (SEQ ID NO.40) and As-T117G-R (SEQ ID NO.41), As-S213G-F (SEQ ID NO.42) and As-S213G-R (SEQ ID NO.43), As-M216V-F (SEQ ID NO.44) and As-M216V-R (SEQ ID NO.45), As-E289H-F (SEQ ID NO.46) and As-E289H-R (SEQ ID NO.47) were used to perform site-directed mutagenesis on residues 117, 213, 216, and 289 of AsCYP90D1, respectively, in combination with the method for constructing mutants disclosed in Chinese patent CN119242599A (the amino acid sequences after mutation are shown in SEQ ID NOs.5 to 5). IDNO.8), and obtained plasmids carrying the corresponding AsCYP90D1 mutants, which were successively recorded as pEB-P GAL7 -AsT117G、pEB-P GAL7 -AsS213G、pEB-P GAL7 -AsM216V and pEB-P GAL7 -AsE289H.

[0125] Plasmids carrying the corresponding AsCYP90D1 mutants were used as templates and primers Dor.-V35-P with 40 bp homology arms were used. GAL7 (SEQ ID NO. 37) and Dor.-V35-T HIS5 (SEQ ID NO. 38) was amplified by PCR to obtain four AsCYP90D1 mutant expression cassettes; the four AsCYP90D1 mutant expression cassettes were respectively transformed into the recombinant bacteria YQD680 constructed in Example 2 using the lithium acetate / PEG3350 chemical transformation method; using the CRISPR-Cas9 gene editing tool, double-strand breaks were performed at the V35 site under the guidance of V35-gRNA (SEQ ID NO. 39), and the DNA was integrated into the Saccharomyces cerevisiae genome. HygB / G418 plates were coated, and positive clones were screened to construct recombinant bacteria with the AsCYP90D1 mutant expression cassette introduced.

[0126] Through fermentation culture and liquid phase analysis, positive AsCYP90D1 mutants with improved production were identified. Figure 6As shown, four mutants with significantly improved AsCYP90D1 enzyme activity compared with the wild type were obtained, namely AsT117G, AsS213G, AsM216V, and AsE289H. The production of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol was 11.86 mg / L, 9.87 mg / L, 11.82 mg / L, and 11.8 mg / L, respectively, which was increased to 1.49, 1.24, 1.48, and 1.48 times compared with the wild type, respectively.

[0127] pEB-P GAL7 -AsT117G was used as a template, and primers As-S213G-F (SEQ ID NO.42) and As-S213G-R (SEQ ID NO.43) were used for site-directed mutagenesis to obtain pEB-P containing two point mutations of AsCYP90D1 mutant. GAL7 -AsT117G / S213G; pEB-P GAL7 -AsT117G / S213G was used as a template, and primers As-E289H-F (SEQ ID NO.46) and As-E289H-R (SEQ ID NO.47) were used for site-directed mutagenesis to obtain pEB-P containing the triple-point mutation AsCYP90D1 mutant. GAL7 -AsT117G / S213G / E289H; pEB-P GAL7 -AsT117G / S213G / E289H was used as a template, and primers As-M216V-F2 (SEQ ID NO. 72) and As-M216V-R2 (SEQ ID NO. 73) were used for site-directed mutagenesis to obtain the plasmid pEB-P containing the four-point mutant AsM4 (the encoded amino acid sequence is shown in SEQ ID NO. 9). GAL7 -AsM4, the recombinant strain YQD680-AsM4 was constructed through gene editing. The (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol production of the strain was 17.51 ​​mg / L, which was 2.2 times higher than that of the wild type ( Figure 6 ).

[0128] Example 5 A method for constructing ArDWF1 and AsM4 overexpression strains

[0129] The plasmid pEB-19-ArDWF1-AsM4 used to construct the ArDWF1 and AsM4 co-expression strain was constructed as follows:

[0130] The product synthesized by Shanghai Sangon Biotechnology Co., Ltd. ArDWF1The plasmid of the gene was used as a template, and the ArDWF1 fragment was obtained by PCR amplification using primers PG7-ArDWF1-F (SEQ ID NO.48) and TM1-ArDWF1-R (SEQ ID NO.49). ArDWF1 The gene is derived from Ajuga procumbens ( Ajuga reptans ) of sterol Δ 24(28) The coding gene of reductase, the amino acid sequence of which is shown in SEQ ID NO.4; the plasmid pEB-P constructed in Example 4 GAL7 -AsM4 as a template, primers PG1-AsM4-F (SEQ ID NO.50) and TH5-AsM4-R (SEQ ID NO.51) were used to amplify the AsM4 fragment; the genome of Saccharomyces cerevisiae YQC512 was used as a template, primers P-pEB-P GAL7 (SEQ ID NO.52) and P-ArDWF1-P GAL7 (SEQ ID NO.53) was amplified to obtain P GAL7 fragment; using the genome of Saccharomyces cerevisiae YQC512 as a template, primers P-ArDWF1-T MFA1 (SEQ ID NO. 54) and PP GAL1 -T MFA1 (SEQ ID NO.55) was amplified to obtain T MFA1 fragment; using the genome of Saccharomyces cerevisiae YQC512 as a template, the primers PT MFA1 -P GAL1 (SEQ ID NO.56) and P-AsM4-P GAL1 (SEQID NO.57) was amplified to obtain P GAL1 fragment; using the genome of Saccharomyces cerevisiae YQC512 as a template, the primers P-AsM4-T HIS5 (SEQ ID NO. 58) and P-pEB-T HIS5 (SEQ ID NO.59) was amplified to obtain T HIS5 fragments, fragments (ArDWF1 fragment, AsM4 fragment, P GAL7 Fragment, T MFA1 Fragment, P GAL1 Fragment and T HIS5 The fragments were fused by overlap extension PCR and the pEASY®-Blunt Zero Cloning Vectors kit was used to construct pEB-19-ArDWF1-AsM4.

[0131] The recombinant strain YQD692 overexpressing ArDWF1 and AsM4 was constructed as follows:

[0132] Plasmid pEB-19-ArDWF1-AsM4 was used as template and primer Dor.-V32-P with 50 bp homology arm was used. GAL7 (SEQ ID NO. 60) and Dor.-V32-T HIS5 (SEQ ID NO.61) was amplified by PCR to obtain the ArDWF1-AsM4 expression cassette; the above ArDWF1-AsM4 expression cassette was transformed into the recombinant strain YQD680-AsM4 constructed in Example 4 using the lithium acetate / PEG3350 chemical transformation method; the CRISPR-Cas9 gene editing tool was used to perform double-strand break cleavage of the V32 site under the guidance of V32-gRNA (SEQ ID NO.62), and the DNA was integrated into the Saccharomyces cerevisiae genome. HygB / G418 plates were coated and positive clones were screened to obtain the recombinant strain YQD692. After fermentation in a 50mL YPD shake flask for 96h, the yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol was 29.67mg / L ( Figure 7 ).

[0133] Example 6 A method for constructing a strain expressing a GAL series promoter using sucrose induction

[0134] The genome of Saccharomyces cerevisiae YQC512 was used as a template and primers P-pEB-P SUC2 (SEQ ID NO.63) and P-GAL4-P SUC2 (SEQ ID NO.64) was amplified to obtain P SUC2 fragment; using the genome of Saccharomyces cerevisiae YQC512 as a template, primers PP SUC2 -GAL4 (SEQ ID NO. 65) and PT RPL3 -GAL4 (SEQ ID NO.66) was amplified to obtain the GAL4 fragment; the genome of Saccharomyces cerevisiae YQC512 was used as a template and primers P-GAL4-T RPL3 (SEQ ID NO. 67) and P-pEB-T RPL3 (SEQ ID NO.68) was amplified to obtain T RPL3 Fragment, fragment (P SUC2 fragment, GAL4 fragment and T RPL3 The fragments were fused by overlap extension PCR and the pEASY®-Blunt Zero Cloning Vectors kit was used to construct pEB-P SUC2 -GAL4 plasmid.

[0135] The recombinant strain YQD693 expressing the GAL series promoter induced by a sucrose-inducible promoter was constructed. The construction steps are as follows:

[0136] Plasmid pEB-P SUC2 -GAL4 was used as the template and primer Dor.-V11-P with a 50 bp homology arm was used. SUC2 (SEQ ID NO. 69) and Dor.-V11-T RPL3 (SEQ ID NO.70) was amplified by PCR to obtain a SUC2 The GAL4 expression cassette (SEQ ID NO.10) was converted into the above-mentioned SUC2 The GAL4 expression cassette was transformed into the Saccharomyces cerevisiae strain YQD692. Using the CRISPR-Cas9 gene editing tool, double-strand breaks were made at the V11 locus under the guidance of V11-gRNA (SEQ ID NO. 71), and the DNA was integrated into the Saccharomyces cerevisiae genome. HygB / G418 plates were plated and positive clones were screened to obtain the recombinant strain YQD693. After 96 h of fermentation in 50 mL YPD shake flasks, the yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol was 32.52 mg / L ( Figure 7 ).

[0137] Example 7 Fermentation Verification of Recombinant Strain YQD693

[0138] A single colony was inoculated into 5 mL of YPD medium, cultured at 30°C and 220 rpm for 24 h, and then inoculated into two 50 mL YPD shake flasks at a 2% (v / v) inoculum. After culturing for 20 h, the culture medium in two shake flasks was inoculated into a 2-L fermentor; the initial culture medium formula in the fermentor was 10 g / L D-glucose, 10 g / L (NH4)2SO4, 8 g / L KH2PO4, 3 g / L MgSO4, 0.72 g / L ZnSO4·7H2O, 4.84 mg / L NaMoO4·2H2O, 40 mg / L MnSO4·H2O, 8.68 mg / L CoCl2·6H2O, 2.5 mg / L CuSO4·5H2O, 55.6 mg / L FeSO4·7H2O, 0.3 mg / L biotin, 3.6 mg / L calcium pantothenate, 3.6 mg / L vitamin B1, 3.6 mg / L vitamin B6 and 72 mg / L inositol. The initial fermentation volume after inoculation was 1 L.

[0139] Fermentation tank parameter setting: the temperature was set to 30°C, ammonia was added to control the pH to maintain at 5.0, and the dissolved oxygen was maintained at >25% saturation by adjusting the stirring rate (300 rpm to 950 rpm) and the airflow rate (1 vvm to 3 vvm). After the carbon source in the initial culture medium was used up, a feed solution containing 500 g / L glucose and 12 mL / L vitamin solution was added to the fermentation tank according to the pseudo-exponential feeding model; when the bacterial OD in the tank reached 0.05, the fermentation tank was heated to 0.05. 600 When the fermentation temperature reaches 150, 700 g / L sucrose is added at a rate of 8 mL / h until the end of the fermentation. F S Determined by formula I:

[0140] Formula I;

[0141] In formula I, X0, V0, and S are the initial biomass density (gDCW / L), initial culture volume (L), and glucose concentration in the culture medium (g / L), respectively; Y X / S is the yield of cell biomass to glucose (gDCW / g glucose); μ is the specific growth rate (h -1 m is the maintenance factor (g glucose / gDCW / h), and t is the time after the start of feeding (h). The specific growth rate was set to 0.1h -1 , Y X / S Set to 0.5 and m to 0.05.

[0142] After the fermentation, the yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol in YQD693 strain was detected by HPLC-UV. 5,7 Since the side chain groups of sterols have little effect on the response value, a standard curve was prepared using a solution prepared with ergosterol standard for quantitative analysis. The test results showed that the yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol was 388.91 mg / L, which is 3.85 times that of the YQD630 strain (glucose-ethanol) disclosed in Chinese patent CN116496337A ( Figure 8 ).

[0143] In summary, the cytochrome P450 enzyme expressed by the recombinant bacteria provided by the present invention has higher activity and better substrate selectivity, and can increase the yield of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0144] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A recombinant bacterium for increasing the production of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol, characterized in that: The recombinant bacteria express truncated cytochrome P450 reductase, flax ( Cannabis sativa ) derived cytochrome P450 enzymes and peanuts ( Arachis stenosperma ) cytochrome P450 enzymes from Saccharomyces cerevisiae YQC512; The amino acid sequence of the truncated cytochrome P450 reductase is shown in SEQ ID NO.1; The amino acid sequence of the cytochrome P450 enzyme derived from sesame is shown in SEQ ID NO.2; The peanut-derived cytochrome P450 enzyme includes a wild-type P450 enzyme and / or a mutant of the wild-type P450 enzyme; the amino acid sequence of the wild-type P450 enzyme is shown in SEQ ID NO.3; The mutants of the wild-type P450 enzyme include one or more of a first mutant, a second mutant, a third mutant, a fourth mutant and a fifth mutant; the amino acid sequences of the first mutant, the second mutant, the third mutant, the fourth mutant and the fifth mutant are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

2. The recombinant bacterium according to claim 1, characterized in that The genome of the recombinant bacterium is further integrated with a fourth expression cassette; the fourth expression cassette expresses sterol Δ 24(28) reductase and the peanut-derived cytochrome P450 enzyme, the sterol Δ 24(28) The amino acid sequence of the reductase is shown in SEQ ID NO.

4.

3. The recombinant bacterium according to claim 1 or 2, characterized in that The genome of the recombinant bacterium also integrates P SUC2 The GAL4 expression cassette expressed; the P SUC2 The nucleotide sequence of the expressed GAL4 expression cassette is shown in SEQ ID NO.

10.

4. The method for constructing the recombinant bacterium according to any one of claims 1 to 3, characterized in that: The following steps are involved: The CRISPR-Cas9 gene editing tool is used to integrate an expression cassette for expressing the enzyme into the genome of Saccharomyces cerevisiae YQC512; the enzyme is an enzyme expressed by the recombinant bacteria.

5. The construction method according to claim 4, characterized in that The promoter of the expression cassette includes P GAL1 、P GAL2 or P GAL7 .

6. The construction method according to claim 4 or 5, characterized in that: The CRISPR-Cas9 gene editing tool includes gRNA; the expression cassette of the truncated cytochrome P450 reductase is recorded as the first expression cassette; the expression cassette of the sesame-derived cytochrome P450 enzyme is recorded as the second expression cassette; and the expression cassette of the peanut-derived cytochrome P450 enzyme is recorded as the third expression cassette; The nucleotide sequence of the gRNA integrated into the first expression cassette is shown in SEQ ID NO.19; The nucleotide sequence of the gRNA integrated into the second expression cassette is shown in SEQ ID NO.31; The nucleotide sequence of the gRNA integrated into the third expression cassette is shown in SEQ ID NO.39; The nucleotide sequence of the gRNA integrated into the fourth expression cassette is shown in SEQ ID NO.62; Integrated P SUC2 The nucleotide sequence of the gRNA of the expressed GAL4 expression cassette is shown in SEQ ID NO.

71.

7. Use of the recombinant bacterium according to any one of claims 1 to 3 or the recombinant bacterium constructed by the method according to any one of claims 4 to 6 in the synthesis of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol.

8. A method for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, characterized in that: include: The engineered bacteria are fermented and cultured using glucose and / or sucrose as carbon sources to obtain a fermentation broth containing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol; the engineered bacteria are the recombinant bacteria according to any one of claims 1 to 3 or the recombinant bacteria constructed using the method according to any one of claims 4 to 6.

9. The synthesis method according to claim 8, characterized in that The fermentation culture temperature is 25-35° C., and the pH value is 4.5-5.

5.

10. The synthesis method according to claim 8 or 9, characterized in that When the engineered bacteria are integrated with P SUC2 When the recombinant bacteria expressing the GAL4 expression cassette are grown, fermentation culture is started until the OD of the engineered bacteria reaches 600 When the value is 150, glucose is used as the carbon source. 600 When the value is ≥150, sucrose is used as the carbon source.

Images