Design, construction method and application of De novo biosynthesis route of key intermediate of didrogesterone

Abstract

The invention discloses a design and construction method and application of a De novo biosynthesis route of a key intermediate of didrogesterone, and belongs to the technical field of synthesis of key intermediates of didrogesterone. According to the invention, a recombinant S.cerevisiae strain ScBL6 capable of synthesizing 7-DHC from the beginning is constructed, and when the recombinant S.cerevisiae strain ScBL6 is used for fermentation in a 500mL shake flask, the total yield of 7-DHC reaches 214mg / L, and compared with a contrast ScBL2 strain, the total yield of 7-DHC is increased by 15.24 times. In a 5L fermentation tank, the yield of 7-DHC reaches 1.06 g / L, thalli in the tank are crushed, extracted and then concentrated to 20 g / L. Finally, by utilizing the side chain degradation capacity of mycobacterium neoaurum, after microbial conversion is performed for 72 hours, the molar conversion rate of the target product 6-dehydrodidowncohol reaches 78.54%, the accumulated concentration of the product is 15.71 g / L, the molar conversion rate of the 6-dehydrodidowncohol is detected to be 83.49% in 144 hours, and the yield of 7-DHC in the fermentation tank is detected to be 83.49% in 144 hours. And the concentration of the final product reaches 16.72 g / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthesis of key intermediates of dydrogesterone, and relates to a design, construction method and application of a route for synthesizing 6-dehydrodihydro- ... Background Art

[0002] Dydrogesterone, also known as dehydroprogesterone, is a synthetic progestogen that can be used to treat diseases caused by endogenous progesterone deficiency, such as dysmenorrhea, endometriosis, secondary amenorrhea, irregular menstrual cycle, dysfunctional uterine bleeding, premenstrual syndrome, threatened abortion or habitual abortion caused by progesterone deficiency, infertility caused by corpus luteum deficiency, etc. At present, there are two main routes for the synthesis of dydrogesterone. One is to use 9,10-position pregnane compounds as substrates to construct the 20-position carbonyl group. The problems are long steps and complex operations, resulting in low final yields. The second is to construct the 9,10-position configuration through photoreaction and use photocatalytic double bond shift to construct 5,7-diene. However, it is found that 5,7-diene and 9,10-position configurations are not easy to achieve, atom economy is poor, and the photoreaction yield is low.

[0003] As a new type of progestin, the chemical structure of dehydrogenated progesterone has a similar 2-phenylchromone nucleus structure to classic estrogens such as estradiol and progesterone. However, there are double bonds at C6 and C7 of the B ring. This unsaturated bond has two advantages: (1) it increases metabolic stability and is orally active; (2) it does not have androgen, estrogen and corticosteroid properties. Dydrogesterone is rapidly absorbed after oral administration, metabolized to dihydroxydydrogesterone in the liver, and begins to be excreted in the urine after 20 minutes. Its metabolites also have the above advantages. At present, this compound is only marketed by the original developer Abbott, and its technology has been kept secret. Some of the processes that have been disclosed now have problems such as long reaction steps and low product recovery rates. How to obtain an efficient synthesis method has important economic benefits and social value.

[0004] The transformation of upstream raw materials used in steroidal drugs from plant resource extraction to microbial transformation is revolutionary. The combination of biotransformation and chemical synthesis can replace highly polluting and high-cost plant raw materials, which has significant economic and social benefits. At present, Mycobacterium aureum has a unique decomposition pathway. Patent CN202410599722.7 reports that cheap plant sterols are used as substrates, and the side chain degradation pathway of mycobacteria is modified to achieve the accumulation of 22-hydroxy-23,24-dicholest-4-ene-3-one (4-HBC, dicholestol). After that, the synthesis of progesterone can be achieved through simple chemical synthesis steps (Angewandte Chemie, 2021, 60 (10): 5414-5420).

[0005] Compared with the structure of progesterone, how to introduce the unsaturated double bond has become a key step in the synthesis of dydrogesterone. Zhejiang Shenzhou Pharmaceutical Co., Ltd. believes that alcohol microorganisms can be used to synthesize 7-hydroxyprogesterone on the route of synthesizing dehydrogenated progesterone, and then the product dydrogesterone (CN201910479266.1) can be obtained through subsequent acidic hydrolysis. This synthetic route has many reaction steps, and the activity of C-7 hydroxylase and reductase is low, which cannot support industrial production needs.

[0006] As a model edible fungus, Saccharomyces cerevisiae has a natural synthesis pathway for ergosterol. The synthesis involves about 30 enzymes, and the entire pathway includes three modules: mevalonate biosynthesis, farnesyl pyrophosphate biosynthesis, and ergosterol biosynthesis. In addition, Saccharomyces cerevisiae has a short growth cycle, strong fermentation ability, and a complete genetic operating system, making it an ideal chassis for the de novo synthesis of sterol compounds. The current mainstream strategy is to use metabolic engineering transformation strategies to achieve efficient de novo synthesis of sterol compounds. What is particularly important is that based on a clear de novo synthesis pathway for ergosterol, the presence of a C6,7 nucleus structure in the synthesis pathway is an ideal chassis for the synthesis of dydrogesterone precursor 6-dehydrodihydrocholine. Patent CN2023100585156 reports a Saccharomyces cerevisiae cell factory for the de novo synthesis of 7-DHC. In this patent, by further strengthening the synthesis pathway of 7-DHC and coupling it with the Mycobacterium cell factory for the microbial transformation of dydrogesterone, the directed side chain degradation and core modification of 7-DHC from scratch are achieved. Through the metabolic coupling of the two synthetic chassis, the de novo synthesis of 6-dehydro-dydrogesterone, a key synthetic precursor of dydrogesterone, is achieved. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a technical solution for designing and constructing a de novo biosynthetic route of a key intermediate of dydrogesterone and its application.

[0008] This application is implemented using the following technical solutions:

[0009] The invention provides a method for constructing a biological de novo synthesis route of a key intermediate of dydrogesterone, wherein a cerevisiae strain for de novo synthesis of 7-DHC is coupled with mycobacteria, and 7-DHC is used as a substrate for microbial conversion to synthesize 6-dehydrodigestrol.

[0010] Furthermore, the method specifically comprises: collecting the high-density fermented Saccharomyces cerevisiae cells by centrifugation, separating and extracting the sterol precursors, and then putting them into a transformation system of Mycobacterium aureus capable of directing biotransformation of phytosterols to synthesize 4-HBC, culturing with shaking at 30°C-180rpm, and reacting for 72-144h;

[0011] The sterol precursors include ergosterol or 7-DHC.

[0012] Furthermore, in the method, the new golden mycobacterium is transferred to M3 culture medium at a ratio of 1 to 10%, and the M3 culture medium contains 7-DHC emulsified with hydroxypropyl cyclodextrin and Tween 80, with a working concentration of 20 g / L.

[0013] Furthermore, the 7-DHC de novo synthesizing yeast strain in the method is constructed by the following steps: using high-ergosterol-producing yeast as a base strain to enhance the expression of squalene epoxidase ERG1, lanosterol demethylase ERG11, C-8 sterol isomerase ERG2, C-5 sterol desaturase ERG3, and NADH kinase POS5, and heterologously expressing Gallus Gallus-derived 24-sterol reductase DHCR24, knocking out C-22 sterol desaturase ERG5 required for ergosterol synthesis, knocking out pseudogenes YJL064W, YPL062W, YMR206W, YBR197C, and YNR014W, and weakening △(24)-sterol C-methyltransferase ERG6, to obtain the 7-DHC de novo synthesizing yeast strain.

[0014] Furthermore, the bottom plate bacteria is the ergosterol-high-producing Saccharomyces cerevisiae BL3, wherein the construction of Saccharomyces cerevisiae BL3 can refer to the method of CN202410034174.3.

[0015] Furthermore, the enhanced expression specifically refers to the enhanced expression of 1 copy of ERG1, 1 copy of ERG11, 1 copy of POS5, 1 copy of ERG2, and 1 copy of ERG3 on the Saccharomyces cerevisiae genome, and the heterologous expression specifically refers to the expression of three copies of DHCR24 on the Saccharomyces cerevisiae genome.

[0016] Further, the Gene ID of POS5 is 855913, the Gene ID of ERG1 is 853086, the Gene ID of ER11 is 856398, the Gene ID of ERG2 is 855242, the Gene ID of ERG3 is 850745, the Gene ID of ERG5 is 855029, the Gene ID of YJL064W is 853380, the Gene ID of YPL062W is 856043, the Gene ID of YMR206W is 855246, the Gene ID of YBR197C is 852496, the Gene ID of YNR014W is 855748, and the Gene ID of ERG6 is 855003.

[0017] Further, through P TEF1 The promoter enhances the expression of ERG3 through P PGK1The promoter enhances the expression of ERG2 through P TEF1 The promoter enhances the expression of ERG1 through P PGK1 The promoter enhances the expression of ERG11 through P TEF1 Promoter heterologous expression of DHCR24, through P PGK1 The promoter enhances the expression of POS5 through P ERG7 Promoter weakening of ERG6.

[0018] Further, through P TEF The promoter-enhanced DHCR24 was integrated into the YJL064W site on the Saccharomyces cerevisiae genome, and the Gene ID of YJL064W is 853380;

[0019] By P TEF The promoter-enhanced DHCR24 was integrated into the YPL062W site on the Saccharomyces cerevisiae genome, and the Gene ID of YPL062W is 856043;

[0020] By P TEF Promoter-enhanced expression of DHCR24 and P PGK1 The promoter-enhanced expression of POS5 was integrated into the YMR206W site on the Saccharomyces cerevisiae genome, and the Gene ID of YMR206W is 855246;

[0021] By P TEF Promoter-enhanced expression of ERG3 and P PGK1 The promoter-enhanced ERG2 was integrated into the YBR197C site on the Saccharomyces cerevisiae genome, and the Gene ID of YBR197C is 852496;

[0022] By P TEF Promoter-enhanced expression of ERG1 and P PGK1 The promoter-enhanced ERG11 was integrated into the YNR014W site on the Saccharomyces cerevisiae genome, and the Gene ID of YNR014W is 855748.

[0023] Further, the construction of Saccharomyces cerevisiae specifically includes the following steps:

[0024] (1) Knockout of ERG5, a C-22 sterol desaturase required for ergosterol synthesis, and the resulting Saccharomyces cerevisiae strain was named ScBL1;

[0025] (2) P TEF1 -DHCR24-T PDH3 The fragment was integrated into the genome of the ScBL1 strain at the YJL064W site, and the resulting Saccharomyces cerevisiae strain was named ScBL2;

[0026] (3) P TEF1 -DHCR24-T PDH3 The fragment was integrated into the genome of the ScBL2 strain at the YPL062W site, and the resulting Saccharomyces cerevisiae strain was named ScBL3;

[0027] (4) Using P on the genome of ScBL3 strain ERG7 The promoter was weakened ERG6, and the resulting Saccharomyces cerevisiae strain was named ScBL3-1;

[0028] (5)P TEF -DHCR24-T PDH3 -P PGK1 -POS5-T CYC1 The fragment was integrated into the YMR206W site on the genome of the ScBL3-1 strain, and the resulting Saccharomyces cerevisiae strain was named ScBL4;

[0029] (6) P TEF -ERG3-T PDH3 -P PGK1 -ERG2-T CYC1 The fragment was integrated into the YBR197C site on the genome of the ScBL4 strain, and the resulting Saccharomyces cerevisiae strain was named ScBL5;

[0030] (7)P TEF -ERG1-T PDH3 -P PGK1 -ERG11-T CYC1 The fragment was integrated into the YNR014W site on the genome of the ScBL5 strain, and the resulting Saccharomyces cerevisiae strain was named ScBL6.

[0031] The present invention provides the use of any of the above-mentioned constructed 7-DHC de novo synthesizing yeast strains and mycobacterium coupled microorganisms for transforming and synthesizing 6-dehydrodihydrocarbendazim.

[0032] The new golden mycobacterium used in the present invention is a genetically engineered bacterium for producing and preparing biredoethanol 4-HBC mycobacterium constructed in Chinese patent CN202410599722.7.

[0033] The beneficial effects of the present invention are mainly reflected in: the present invention constructs a recombinant S.cerevisiae strain ScBL6 that can synthesize 7-DHC from scratch. When using it for 500mL shake flask fermentation, the total yield of 7-DHC reaches 214mg / L, which is 15.24 times higher than that of the control ScBL2 strain. In a 5L fermentation tank, the yield of 7-DHC reaches 1.06g / L, and the bacteria in the tank are crushed and extracted and concentrated to 20g / L. Finally, the side chain degradation ability of the new gold mycobacterium is used. After 72h of microbial transformation, the molar conversion rate of the target product 6-dehydrodihydrodihydrogen alcohol reaches 78.54%, and the cumulative concentration of the product is 15.71g / L. The molar conversion rate of 6-dehydrodihydrodihydrogen alcohol detected at 144h is 83.49%, and the final product concentration reaches 16.72g / L.

[0034] The recombinant strain constructed by the present invention is combined with Mycobacterium neogoldenum to effectively produce 6-dehydrodihydroprogesterone, which provides a guiding idea for the subsequent production of dehydroprogesterone and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Comparison of intracellular 7-DHC production in starting strains ScBL2 to ScBL6.

[0036] Figure 2 This is the fermentation process of the ScBL6 strain in a 5L tank. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0038] The culture medium involved in the embodiment is as follows:

[0039] SOB medium: Each liter contains 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, and 1g MgCl2.

[0040] SD-Ura3 medium: each liter contains 20 mg leucine, 20 mg tryptophan, 20 mg histidine, 6.7 g amino-free yeast nitrogen source, and 20 g anhydrous glucose.

[0041] Fermentation medium: each liter contains 20g tryptone, 40g anhydrous glucose, and 10g yeast extract.

[0042] SOB+Amp plate: Each liter contains 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, 1g MgCl2, and 20g agar powder.

[0043] M3TW medium: 0.5g / L KH2PO4, 0.5g / L K2HPO4, 2g / L (NH4)2HPO4, 0.005g / L FeSO4·7H2O, 0.002g / L ZnSO4·7H2O, 0.2g / L MgSO4·7H2O, 5g / L yeast extract powder, 5g / L glycerol, pH 7.2, 0.05% (v / v) Tween 80.

[0044] Detection of 7-DHC content:

[0045] The fermented bacterial liquid was centrifuged, the remaining bacterial cells were broken with 3M hydrochloric acid, and the cell fragments were collected by centrifugation, and the intracellular sterols were extracted by saponification with 1.5mol / L KOH methanol solution in a 60℃ water bath. After the reaction, the above liquid was extracted with n-hexane, and the obtained n-hexane was evaporated to dryness in a 75℃ water bath, and a certain amount of ethyl acetate was added to dissolve it, and it was filtered into the liquid phase bottle with a filter membrane.

[0046] The gas phase column used was Thermo Fisher's HP-5 (30m*0.25mm*0.25μm); the gas phase program was set as follows: inlet temperature: 300℃; program temperature: 190℃ (1min) 10℃ / min 300℃ (10min); injection volume: 1μL, split ratio 20:1; carrier gas: N2, flow rate 1.0mL / min; transfer rod temperature: 250℃.

[0047] Recombinant Saccharomyces cerevisiae OD 600 Detection method:

[0048] The yeast seed solution cultured for 16-24 hours was inoculated into a 500 mL shake flask containing 100 mL fermentation medium at a 1% inoculation rate and cultured at 30°C and 220 rpm. When sampling, the OD was measured using a UV spectrophotometer after dilution in an appropriate ratio. 600 .

[0049] The construction of the plasmid involved in the embodiment was carried out in E. coli Dh5α, and after the plasmid construction was completed, it was used as a template to amplify the expression cassette.

[0050] The plasmids involving PAM site mutations in the embodiments all need to be sequenced at the corresponding positions after construction to ensure that the pdc5 plasmid with the correct mutation at the PAM site is obtained.

[0051] The primer sequences involved in the examples are shown in Table 1:

[0052] Table 1: Primer list

[0053]

[0054]

[0055]

[0056] The strain genotypes involved in the examples are shown in Table 2:

[0057] Table 2: Detailed information of strains

[0058]

[0059] Example 1: Construction of tool plasmid

[0060] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PG2 F and PG2 R were used to obtain the gene fragment P GAL2 ;

[0061] Using pYES2 plasmid as template, primers LpYES2 F and LpYES2 R were used to obtain linearized pYES2 plasmid;

[0062] The gene fragment P GAL2 The linearized pYES2 plasmid was connected and introduced into competent Dh5α, and primers VG2F and VG2R were used to verify the P GAL1 The promoter was replaced by P GAL2 P GAL2 -pYES2 plasmid.

[0063] Using pYES2 plasmid as template, primers emp F and emp R were used to obtain linearized plasmid, which was transformed into Dh5α competent cells to obtain P GAL1 -T CYC1 Plasmid emp with empty expression cassette.

[0064] Example 2: Construction of high-yield 7-DHC Saccharomyces cerevisiae chassis ScBL1

[0065] The specific steps are as follows:

[0066] (1) Synthesis of fragments:

[0067] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment ERG5-HOMO1 was obtained using primers rERG5-HOMO1F and rERG5-HOMO1R;

[0068] The gene fragment ERG5-HOMO2 was obtained using primers rERG5-HOMO2F and rERG5-HOMO2R;

[0069] (2) The two fragments ERG5-HOMO1 and ERG5-HOMO2 in step (1) are fused by PCR, and the correct bands obtained by running the gel are excised and recovered.

[0070] (3) The fusion gene fragment in step (2) was transformed into the competent strain of Saccharomyces cerevisiae BL3, cultured on Sc-ura plates at 30°C for 2-3 days, and verified by single colony PCR using primers VER H1 F and VER H2R described in Table 1. Single colonies with correct bands were selected to obtain strain BL3-ΔERG5, which was named Saccharomyces cerevisiae ScBL1.

[0071] Example 3: Construction of high-yield 7-DHC Saccharomyces cerevisiae chassis ScBL2

[0072] (1) Construction of sgRNA:

[0073] Using plasmid pdc5 as a template, using the primers described in Table 1, using primers NCYJL R and NCYJL F; linearized pdc5 plasmid was obtained, and introduced into competent Dh5α, spread on SOB+Amp plates, and cultured at 37°C to obtain pdc5-NCYJL plasmid.

[0074] (2) Construction of donor fragment

[0075] The artificial gene fragment DHCR24 (existing fragment) was ligated to pYES2 and the gene fragment DHCR24 was obtained using primers D24 F and D24R;

[0076] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment YJL-HOMO1 was obtained using primers rYJL-HOMO1F and rYJL-HOMO1R;

[0077] The gene fragment YJL-HOMO2 was obtained using primers rYJL-HOMO2F and rYJL-HOMO2R;

[0078] The three fragments YJL-HOMO1, YJL-HOMO2, and DHCR24 were fused by PCR, and the correct bands obtained by running the gel were cut and recovered to obtain donor H1-P TEF1 -DHCR24-T PDH3 -H2.

[0079] (3) The sgRNA dc5-NCYJL and donor H1-P constructed in (1) and (2) were TEF1 -DHCR24-T PDH3 -H2 was simultaneously introduced into the competent cell of Saccharomyces cerevisiae ScBL1, cultured on Sc-ura plates at 30°C for 2-3 days, and verified by PCR using P-YJL F and P-YPLR to obtain the Saccharomyces cerevisiae chassis ScBL2.

[0080] Example 4: Construction of high-yield 7-DHC Saccharomyces cerevisiae chassis ScBL3

[0081] (1) Construction of sgRNA:

[0082] Using plasmid pdc5 as a template, using the primers described in Table 1, using primers NCYPL R and NCYPL F; linearized pdc5 plasmid was obtained, and introduced into competent Dh5α, spread on SOB+Amp plates, and cultured at 37°C to obtain pdc5-NCYPL plasmid.

[0083] (2) Construction of donor fragment

[0084] The gene fragment DHCR24 was artificially synthesized and connected to pYES2, and the gene fragment DHCR24 was obtained using primers D24 F (YPL) and D24R (YPL);

[0085] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1 , the gene fragment YPL-HOMO1 was obtained using primers rYPL-HOMO1F and rYPL-HOMO1R;

[0086] The gene fragment YPL-HOMO2 was obtained using primers rYPL-HOMO2F and rYPL-HOMO2R;

[0087] The three fragments YPL-HOMO1, YPL-HOMO2, and DHCR24 were fused by PCR, and the correct bands obtained by running the gel were cut and recovered to obtain donor H1-P TEF1 -DHCR24-T PDH3 -H2.

[0088] (3) The sgRNA dc5-NCYPL and donor H1-P constructed in (1) and (2) were TEF1 -DHCR24-T PDH3 -H2 was simultaneously introduced into the competent cell of Saccharomyces cerevisiae ScBL2, cultured on Sc-ura plates at 30°C for 2-3 days, and verified by PCR using P-YPL F and P-YPL R to obtain the Saccharomyces cerevisiae chassis ScBL3.

[0089] Example 5: Construction of high-yield 7-DHC Saccharomyces cerevisiae chassis ScBL3-1

[0090] (1) Construction of sgRNA:

[0091] Using plasmid pdc5 as a template, using the primers described in Table 1, and using primers NCERG6 R and NCERG6F; linearized pdc5 plasmid was obtained, and introduced into competent Dh5α, spread on SOB+Amp plates, and cultured at 37°C to obtain pdc5-NCERG6 plasmid.

[0092] (2) Construction of donor fragment

[0093] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment P was obtained using primers pERG7-F and pERG7-R. ERG7 ;

[0094] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment ERG6-HOMO1 was obtained using primers rERG6-HOMO1F and rERG6-HOMO1R;

[0095] The gene fragment ERG6-HOMO2 was obtained using primers rERG6-HOMO2F and rERG6-HOMO2R;

[0096] The above three fragments ERG6-HOMO1, ERG6-HOMO2, P ERG7 Fusion PCR was performed using PCR, and the correct band obtained by running the gel was cut and recovered to obtain donor H1-P ERG7 -H2.

[0097] (3) The sgRNA dc5-NCERG6 and donor H1-P constructed in (1) and (2) were ERG7 -H2 was simultaneously introduced into the competent cell of Saccharomyces cerevisiae ScBL3, cultured on Sc-ura plates at 30°C for 2-3 days, and verified by PCR using P-ERG6-F and P-ERG6-R to obtain the Saccharomyces cerevisiae chassis ScBL3-1.

[0098] Example 6: Construction of high-yield 7-DHC Saccharomyces cerevisiae chassis ScBL4

[0099] (1) Construction of sgRNA:

[0100] Using plasmid pdc5 as a template, using the primers described in Table 1, using primers NCYMR R and NCYMRF; linearized pdc5 plasmid was obtained, and introduced into competent Dh5α, spread on SOB+Amp plates, and cultured at 37°C to obtain pdc5-NCYMR plasmid.

[0101] (2) Construction of donor fragment

[0102] The gene fragment DHCR24 was artificially synthesized and connected to pYES2, and the gene fragment DHCR24 was obtained using primers D24 F (YMR) and D24R (YMR);

[0103] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1 , the gene fragment POS5 was obtained using primers POS5-F and POS5-R;

[0104] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1 , the gene fragment YMR-HOMO1 was obtained using primers rYMR-HOMO1F and rYMR-HOMO1R;

[0105] The gene fragment YMR-HOMO2 was obtained using primers rYMR-HOMO2F and rYMR-HOMO2R;

[0106] The above four fragments YMR-HOMO1, YMR-HOMO2, DHCR24, and POS5 were fused by PCR, and the correct bands obtained by running the gel were cut and recovered to obtain donor H1-P TEF -DHCR24-T PDH3 -P PGK1 -POS5-T CYC1 -H2.

[0107] (3) The sgRNA dc5-NCYMR and donor H1-P constructed in (1) and (2) were TEF -DHCR24-T PDH3 -P PGK1 -POS5-T CYC1 -H2 was simultaneously introduced into the competent cell of Saccharomyces cerevisiae ScBL3-1, cultured on Sc-ura plates at 30°C for 2-3 days, and verified by PCR using P-YMR-F and P-YMR-R to obtain the Saccharomyces cerevisiae chassis ScBL4.

[0108] Example 7: Construction of high-yield 7-DHC Saccharomyces cerevisiae chassis ScBL5

[0109] (1) Construction of sgRNA:

[0110] Using plasmid pdc5 as a template, using the primers described in Table 1, using primers NCYBR R and NCYBRF; linearized pdc5 plasmid was obtained, and introduced into competent Dh5α, spread on SOB+Amp plates, and cultured at 37°C to obtain pdc5-NCYBR plasmid.

[0111] (2) Construction of donor fragment

[0112] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment ERG2 was obtained using primers ERG2-F and ERG2-R;

[0113] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment ERG3 was obtained using primers ERG3-F and ERG3-R;

[0114] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment YBR-HOMO1 was obtained using primers rYBR-HOMO1F and rYBR-HOMO1R;

[0115] Gene fragment YBR-HOMO2 was obtained using primers rYBR-HOMO2F and rYBR-HOMO2R;

[0116] The above four fragments YBR-HOMO1, YBR-HOMO2, ERG2, and ERG3 were fused by PCR, and the correct bands obtained by running the gel were cut and recovered to obtain donor H1-P TEF -ERG3-T PDH3 -P PGK1 -ERG2-T CYC1 -H2.

[0117] (3) The sgRNA dc5-NCYBR and donor H1-P constructed in (1) and (2) were TEF -ERG3-T PDH3 -P PGK1 -ERG2-T CYC1 -H2 was simultaneously introduced into the competent cell of Saccharomyces cerevisiae ScBL4, cultured on Sc-ura plates at 30°C for 2-3 days, and verified by PCR using P-YBR-F and P-YBR-R to obtain the Saccharomyces cerevisiae chassis ScBL5.

[0118] Example 8: Construction of high-yield 7-DHC Saccharomyces cerevisiae chassis ScBL6

[0119] (1) Construction of sgRNA:

[0120] Using plasmid pdc5 as a template, using the primers described in Table 1, using primers NCYNRR and NCYNRF; obtaining a linearized pdc5 plasmid, and introducing it into competent Dh5α, spreading it on a SOB+Amp plate, and culturing it at 37°C to obtain a pdc5-NCYNR plasmid.

[0121] (2) Construction of donor fragment

[0122] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment ERG1 was obtained using primers ERG1-F and ERG1-R;

[0123] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1 , the gene fragment ERG11 was obtained using primers ERG11-F and ERG11-R;

[0124] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and the primers described in Table 1, the gene fragment YNR-HOMO1 was obtained using primers rYNR-HOMO1F and rYNR-HOMO1R;

[0125] The gene fragment YNR-HOMO2 was obtained using primers rYNR-HOMO2F and rYNR-HOMO2R;

[0126] The above four fragments YNR-HOMO1, YNR-HOMO2, ERG1, and ERG11 were fused by PCR, and the correct bands obtained by running the gel were cut and recovered to obtain donor H1-P TEF -ERG1-T PDH3 -P PGK1 -ERG11-T CYC1 -H2.

[0127] (3) The sgRNA dc5-NCYNR and donor H1-P constructed in (1) and (2) were TEF -ERG1-T PDH3 -P PGK1 -ERG11-T CYC1 -H2 was simultaneously introduced into the competent cell of Saccharomyces cerevisiae ScBL5, cultured on Sc-ura plates at 30°C for 2-3 days, and verified by PCR using P-YNR-F and P-YNR-R to obtain the Saccharomyces cerevisiae chassis ScBL6.

[0128] Example 9: Production of 7-DHC by high-density fermentation of Saccharomyces cerevisiae

[0129] (I) Shake flask fermentation

[0130] (1) First, recombinant cerevisiae ScBL6 is inoculated into a seed culture medium to prepare a seed solution, and the prepared seed solution is inoculated into a fermentation medium at an inoculation rate of 1 to 10% (v / v). The seed culture medium is usually a YPD culture medium. In the present invention, the YPD culture medium used has the following composition: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L anhydrous glucose.

[0131] (2) 7-DHC extraction and yield analysis:

[0132] Drain the fermentation liquid and dilute it 10 times, then use a UV spectrophotometer to measure the OD 600 , draw 8mL of fermentation liquid, wash with deionized water and resuspend, then put it into 10mL EP tube with 3mL 3M hydrochloric acid, and break it in boiling water for 5min. After centrifugation, remove the supernatant and wash with deionized water. Use 1.5mol / L KOH methanol solution to saponify and extract intracellular sterols in a 60℃ water bath. After the reaction, extract the above liquid with n-hexane, evaporate the obtained n-hexane in a 75℃ water bath, add a certain amount of ethyl acetate to dissolve it, and filter it into the liquid phase bottle with a filter membrane. Gas chromatography detection was performed, and the fermentation yield of the engineering strain was obtained by converting it with the peak area of ​​the 7-DHC standard. The final yield of the ScBL6 strain shake flask reached 214mg / L.

[0133] (ii) 5L tank fermentation

[0134] (1) Under sterile conditions, a single colony of strain ScBL6 was inoculated into YPD medium and cultured at 30°C and 220 rpm for 24 h to prepare an activated bacterial solution for fermentation;

[0135] (2) Under sterile conditions, the activated bacterial solution of ScBL6 was inoculated into a 500 ml YPD shake flask at a 2% inoculation rate and cultured at 30°C and 220 rpm for 24 h for subsequent tank fermentation;

[0136] (3) Batch fed-batch fermentation was carried out in a 5 L fermenter with a liquid volume of 3 L. The initial ventilation rate was 3 L / min, the initial stirring was 300 rpm, the culture temperature was 30°C, and the pH was controlled at 5.5 with 50% ammonia water. After the initial dissolved oxygen dropped to 30%, the dissolved oxygen was controlled at about 30% by controlling the stirring speed and air flow. Figure 2 The fermentation process is shown, including residual sugar, biomass (OD 600 ), ethanol content and 7-DHC production changes over time. The initial 20% of glucose in the fermentation broth was basically consumed in 24 hours. When the glucose consumption reached 1g / L and the ethanol content was below 10g / L, feeding began. The pH value of the fermentation broth will decrease during the fermentation process, so it is necessary to turn on automatic alkali replenishment to maintain the pH at around 5.5. At the same time, glucose should be controlled below 1g / L to avoid the inhibitory effect of high concentration of glucose on the growth of strain cells. At the end of fermentation, the maximum intracellular production of 7-DHC reached 1.06g / L.

[0137] Example 10: Synthesis of 6-dehydrodihydroquinolone by Mycobacterium microbial transformation

[0138] The cells in the fermentation tank in Example 10 were collected by centrifugation, and the cells were broken with 3M hydrochloric acid. The broken cells were washed with deionized water, and then saponified with 1.5mol / L KOH methanol solution in a 60°C water bath to extract intracellular sterols. After the reaction was completed, the liquid was extracted with n-hexane, and the obtained n-hexane was evaporated to dryness in a 75°C water bath. Considering that oils and fats have a good solubility for sterols and can be used as defoamers, it is beneficial to promote the conversion of sterols. Therefore, in addition, cyclodextrin, as a cyclic oligosaccharide with a hydrophobic cavity, can bind hydrophobic molecules through van der Waals forces and hydrogen bonds. Combined with steroid compounds to form an embedding complex, enhance the solubility of sterols in aqueous solution and promote the transfer of substrates into the cell, accelerating the microbial conversion reaction. Therefore, soybean oil and HPDC are preferred to construct an emulsified system for sterols. Take 2g 7-DHC, 0.5% soybean oil and 10g HPDC, dissolve them in water, perform shear emulsification, make the volume to 10mL, prepare 200g / L 7-DHC substrate solution, and sterilize it under pressure at 115°C for 30min.

[0139] According to patent CN202410599722.7, a ring of new gold mycobacterium liquid that can transform more than 90% of 4-HBC was taken from a glycerol tube, streaked on LB solid medium, and cultured at 30°C for 48h; a single clone was picked in 5mL LB liquid and cultured at 30°C for 36h; transferred to 60mL M3 medium (containing soybean oil and HPDC emulsified 6-dehydrocholesterol, working concentration 20g / L) at a ratio of 5%, and cultured at 30oC-180rpm; reacted for 72h, and 1mL sample was taken for product analysis after 144h. The specific operation process is: 1mL of conversion liquid is added to 5mL ethyl acetate for extraction, shaken and mixed for 30min, 200μL of the upper organic phase is volatilized in an EP tube, 0.8mL methanol is added for re-dissolution, and liquid phase detection is performed. The results showed that in this conversion system, after 72 hours of reaction, the molar conversion rate of 6-dehydrodihydrogenated alcohol was 55.4%, and the cumulative concentration of the product was 11.08 g / L. The molar conversion rate of 6-dehydrodihydrogenated alcohol detected at 144 hours was 60.3%, and the final product concentration reached 12.32 g / L.

[0140] Example 11: Optimization of sterol emulsification system using mycobacterium microorganisms to synthesize 6-dehydrodihydrocholine

[0141] The cells in the fermentation tank in Example 10 were collected by centrifugation, and the cells were broken with 3M hydrochloric acid. The broken cells were washed with deionized water, and then saponified with 1.5 mol / L KOH methanol solution in a 60°C water bath to extract intracellular sterols. After the reaction, the liquid was extracted with n-hexane, and the obtained n-hexane was evaporated to dryness in a 75°C water bath. The substrate conversion rate of 7-DHC in Example 11 was not ideal. It is speculated that soybean oil may have a certain effect on the growth of the strain. At the same time, a large amount of organic matter such as soybean oil will burden product extraction and wastewater treatment. Further research on relevant literature found that the addition of surfactant Tween 80 can enhance the conversion and dispersibility of plant sterols, so HPDC and Tween 80 were selected to construct a sterol conversion system. Take 2 g 7-DHC, 40 g HPDC and 2 g Tween 80, dissolve them in water, perform shear emulsification, make the volume to 10 mL, prepare 200 g / L 7-DHC substrate solution, and sterilize it under pressure at 115°C for 30 min.

[0142] According to the patent CN202410599722.7, a ring of new gold mycobacterium liquid that can transform more than 90% of 4-HBC was taken from the glycerol tube, streaked on the LB solid medium, and cultured at 30°C for 48h; a single clone was picked in 5mL LB liquid and cultured at 30°C for 36h; it was transferred to 60mL M3 medium (containing 7-DHC emulsified with hydroxypropyl cyclodextrin and Tween 80, working concentration 20g / L) at a ratio of 5%, and cultured at 30°C-180rpm; reacted for 72h, and 1mL sample was taken after 144h for product analysis. The specific operation process is: 1mL of conversion liquid is added to 5mL ethyl acetate for extraction, shaken and mixed for 30min, 200μL of the upper organic phase is volatilized in an EP tube, 0.8mL of methanol is added for redissolution, and liquid phase detection is performed. The results showed that in this conversion system, after 72 hours of reaction, the molar conversion rate of 6-dehydrodihydrogenated alcohol was 78.54%, and the cumulative concentration of the product was 15.71 g / L. The molar conversion rate of 6-dehydrodihydrogenated alcohol detected at 144 hours was 83.49%, and the final product concentration reached 16.72 g / L.

Claims

1. A method for constructing a de novo biosynthesis route for a key intermediate of dydrogesterone, characterized in that: A Saccharomyces cerevisiae strain that synthesizes 7-DHC de novo was coupled with mycobacteria to synthesize 6-dehydrodihydrocarbamide using 7-DHC as a substrate through microbial conversion.

2. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 1, characterized in that: Specifically include: The high-density fermented Saccharomyces cerevisiae cells were collected by centrifugation, and the sterol precursors were separated and extracted, and then put into the transformation system of the new golden mycobacterium that can biotransform phytosterols to synthesize 4-HBC, and cultured with shaking at 30°C-180rpm for 72-144h; The sterol precursors include ergosterol or 7-DHC.

3. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 2, characterized in that: The new golden mycobacterium is transferred into M3 culture medium at a ratio of 1 to 10%. The M3 culture medium contains 7-DHC emulsified with hydroxypropyl cyclodextrin and Tween 80, and the working concentration is 20 g / L.

4. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 1, characterized in that: The 7-DHC de novo synthesizing yeast strain is constructed by the following steps: using saccharomyces cerevisiae as a base bacterium to enhance the expression of squalene epoxidase ERG1, lanosterol demethylase ERG11, C-8 sterol isomerase ERG2, C-5 sterol desaturase ERG3, and NADH kinase POS5, and heterologously expressing 24-sterol reductase DHCR24 from Gallus Gallus, knocking out C-22 sterol desaturase ERG5 required for ergosterol synthesis, knocking out pseudogenes YJL064W, YPL062W, YMR206W, YBR197C, and YNR014W, and weakening △(24)-sterol C-methyltransferase ERG6, so as to obtain the 7-DHC de novo synthesizing yeast strain.

5. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 4, characterized in that: The bottom plate bacteria is the ergosterol-producing Saccharomyces cerevisiae BL3.

6. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 4, characterized in that: The enhanced expression specifically refers to the enhanced expression of 1 copy of ERG1, 1 copy of ERG11, 1 copy of POS5, 1 copy of ERG2, and 1 copy of ERG3 on the Saccharomyces cerevisiae genome, and the heterologous expression specifically refers to the expression of three copies of DHCR24 on the Saccharomyces cerevisiae genome.

7. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 4, characterized in that: By P TEF1 The promoter enhances the expression of ERG3 through P PGK1 The promoter enhances the expression of ERG2 through P TEF1 The promoter enhances the expression of ERG1 through P PGK1 The promoter enhances the expression of ERG11 through P TEF1 Promoter heterologous expression of DHCR24, through P PGK1 The promoter enhances the expression of POS5 through P ERG7 Promoter weakening of ERG6.

8. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 4, characterized in that: By P TEF The promoter-enhanced DHCR24 was integrated into the YJL064W site on the Saccharomyces cerevisiae genome, and the Gene ID of YJL064W is 853380; By P TEF The promoter-enhanced DHCR24 was integrated into the YPL062W site on the Saccharomyces cerevisiae genome, and the Gene ID of YPL062W is 856043; By P TEF Promoter-enhanced expression of DHCR24 and P PGK1 The promoter-enhanced expression of POS5 was integrated into the YMR206W site on the Saccharomyces cerevisiae genome, and the Gene ID of YMR206W is 855246; By P TEF Promoter-enhanced expression of ERG3 and P PGK1 The promoter-enhanced ERG2 was integrated into the YBR197C site on the Saccharomyces cerevisiae genome, and the Gene ID of YBR197C is 852496; By P TEF Promoter-enhanced expression of ERG1 and P PGK1 The promoter-enhanced ERG11 was integrated into the YNR014W site on the Saccharomyces cerevisiae genome, and the Gene ID of YNR014W is 855748.

9. The method for constructing a de novo biosynthesis route of a key intermediate of dydrogesterone according to claim 4, characterized in that: The construction of the 7-DHC de novo synthesis yeast strain specifically comprises the following steps: (1) Knockout of ERG5, a C-22 sterol desaturase required for ergosterol synthesis, and the resulting Saccharomyces cerevisiae strain was named ScBL1; (2) P TEF1 -DHCR24-T PDH3 The fragment was integrated into the genome of the ScBL1 strain at the YJL064W site, and the resulting Saccharomyces cerevisiae strain was named ScBL2; (3) P TEF1 -DHCR24-T PDH3 The fragment was integrated into the genome of the ScBL2 strain at the YPL062W site, and the resulting Saccharomyces cerevisiae strain was named ScBL3; (4) Using P on the genome of ScBL3 strain ERG7 The promoter was weakened ERG6, and the resulting Saccharomyces cerevisiae strain was named ScBL3-1; (5)P TEF -DHCR24-T PDH3 -P PGK1 -POS5-T CYC1 The fragment was integrated into the YMR206W site on the genome of the ScBL3-1 strain, and the resulting Saccharomyces cerevisiae strain was named ScBL4; (6) P TEF -ERG3-T PDH3 -P PGK1 -ERG2-T CYC1 The fragment was integrated into the YBR197C site on the genome of the ScBL4 strain, and the resulting Saccharomyces cerevisiae strain was named ScBL5; (7)P TEF -ERG1-T PDH3 -P PGK1 -ERG11-T CYC1 The fragment was integrated into the YNR014W site on the genome of the ScBL5 strain, and the resulting Saccharomyces cerevisiae strain was named ScBL6.

10. Use of a cerevisiae strain that synthesizes 7-DHC de novo according to any one of claims 4 to 9 and a mycobacterium-coupled microbial transformation to synthesize 6-dehydrodihydrocarbendazim.

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