Genetically engineered bacterium for producing 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester at high yield as well as construction method and application of genetically engineered bacterium

Abstract

The invention provides a genetically engineered bacterium for producing 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester at high yield as well as a construction method and application of the genetically engineered bacterium. In the process of producing 9-OH-3-OPCM by using phytosterol in mycobacterium neoaurum, the problems of low product yield and low purity caused by the metabolic shunt effect of byproducts exist. Therefore, the catabolism of the phytosterol in the mycobacterium neoaurum is subjected to gene modification, and the production of by-products, namely 9-OH-4-HBC, 9-OHAD, 9-OH-3-OPDCM and 9-OH-3-OPC, is eliminated by regulating and controlling the expression intensity of key genes, so that the yield of the 9-OHAD is increased. The molar conversion rates of the 9-OH-3-OPCM of the finally constructed genetically engineered bacterium respectively reach 85.9% and 81.3% under the condition that the phytosterol concentration is 2g / L and 5g / L.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to a genetic engineering bacterium capable of producing high-yield 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester, a construction method and application thereof. Background Art

[0002] Steroidal compounds are widely known as important natural organic compounds because of their special physiological functions and therapeutic effects on diseases. In the global pharmaceutical market, the annual output value of steroidal compounds is as high as hundreds of billions of dollars, and the output is as high as nearly 10 million tons. Steroidal compounds are rich in various medicinal effects, including anti-inflammatory, anti-allergic, anticonvulsant and treatment of certain cancers. However, the synthesis method of C-22 steroidal compounds has been less studied, among which 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester is an important steroidal intermediate.

[0003] 9-Hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester (9-OH-3-OPCM) is widely used in the production of steroidal drugs because its structure is easy to modify into various progestins. 9-OH-3-OPCM can be converted into pregnatetraenedione through simple chemical reactions, and can also be converted into corticosteroids later. Mycobacterium neogoldenum is widely used in the biotransformation process of producing 9-OH-3-OPCM because of its comprehensive plant sterol degradation gene cluster and mature gene manipulation and fermentation methods. Therefore, enhancing the construction of the biological pathway of 9-OH-3-OPCM in Mycobacterium neogoldenum is of great significance to the field of steroidal biosynthesis.

[0004] In recent years, relevant reports at home and abroad have explored the synthesis of C-22 steroidal compounds, including 9-OH-4-HBC, 9-OH-3-OPDCM, etc., and revealed some key genes of C-22 steroidal compounds, and regulated such genes to make the metabolic flow of phytosterol degradation shift between C-19 compounds and C-22 compounds. However, the synthesis pathway of 9-OH-3-OPCM has never been deeply explored, and there is a lack of research on the high-yield and high-purity synthesis method of 9-OH-3-OPCM. Summary of the invention

[0005] In order to solve the problem of low yield and low product purity of 9-OH-3-OPCM produced by microbial fermentation in the prior art, the purpose of the present invention is to construct a 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester production strain with high conversion rate and high product purity.

[0006] The present invention first analyzes and identifies the product 9-OH-3-OPCM and the byproducts 9,20-hydroxymethylpregnane-4-ene-3-one (9-OH-4-HBC), 9-hydroxy-androstenedione (9-OHAD), 3-oxo-4,17-pregnene-9-OH-20-carboxylic acid methyl ester (9-OH-3-OPDCM) and 9-hydroxy-3-oxopregnenedione (9-OH-3-OPC) respectively in the process of constructing a 9-OH-3-OPCM production strain, and determines the expression level of key genes that may be involved in the degradation process of phytosterols. After analyzing the correlation between the expression level change of the key genes and the generation of byproducts, the enzymes with a strong correlation with the generation of byproducts in the production process of 9-OH-3-OPCM are determined, and then the genes are knocked out by genetic engineering technology, thereby obtaining a genetically engineered bacterium with high yield of 9-OH-3-OPCM, and the product has a high purity.

[0007] The technical scheme adopted by the present invention is: a method for constructing a genetically engineered bacterium with high yield of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester, the method comprising: using mycobacterium as a base bacterium, blocking the expression of genes KstD1, KstD2, KstD3, ChsE1-2, ChsH1-2, Opccr, and SalA in the base bacterium, and constructing a genetically engineered bacterium with high yield of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester.

[0008] Preferably, the method further comprises: blocking the expression of gene ChsE4-5 in the chassis bacteria.

[0009] Preferably, the method further comprises: overexpressing gene MT4b in the chassis bacteria.

[0010] refer to Figure 1 Regarding the metabolic pathway of phytosterol degradation to synthesize 9-OH-3-OPCM, the present invention first uses Mycobacterium neoaurum ATCC 25795 as the base bacteria, knocks out the genes KstD1, KstD2, and KstD3 on its genome, and constructs the WL01 (ΔKstD1 / 2 / 3) strain to eliminate the dehydrogenation ability at positions 1 and 2 on the steroid nucleus. On this basis, CHsE1-2 and ChsH1-2 in strain WL01 were knocked out to preliminarily obtain the 9-OH-3-OPCM-producing strain WL02 (ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2). Based on the WL02 strain, the present invention detected and evaluated the expression levels of related genes in the process of its biotransformation to produce 9-OHAD ( Figure 5), it was preliminarily determined that genes OpccR, SalA, ChsE3 and ChsE4-5 may be directly related to the production of byproducts 9-OH-4-HBC and 9-OH-3-OPDCM and product 9-OH-3-OPCM. Therefore, according to the above test results, the present invention knocked out or overexpressed the key genes involved, and constructed the following strains based on strain WM02:

[0011] WL03, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccR;

[0012] WL04, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔSalA;

[0013] WL05, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccrΔSalA;

[0014] WL06, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccrΔSalAΔChsE4-5;

[0015] WL07, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccrΔSalAΔChsE4-5::pMV261-MT4b.

[0016] According to the above strain construction results, the newly constructed strains were fermented and verified. Among them, strain WL05 did not produce byproduct 9-OH-4-HBC after fermentation and had the ability to produce high 9-OH-3-OPCM. Strain WL06 did not produce byproducts 9-OH-4-HBC and 9-OH-3-OPDCM after fermentation and had a higher 9-OH-3-OPCM production capacity than strain WL05. Finally, strain WL07 was obtained, which did not produce byproducts 9-OH-4-HBC, 9-OH-3-OPC and 9-OH-3-OPDCM and had a higher 9-OH-3-OPCM production capacity than strains WL05 and WL06. Strain WL07 was fermented at 2g / L and 5g / L phytosterol concentrations, and produced 9-OH-3-OPCM at 85.9% and 81.3% molar conversion rates, respectively.

[0017] Preferably, the method comprises: using Mycobacterium as the base bacteria, knocking out the genes KstD1, KstD2, KstD3, ChsE1-2, ChsH1-2, Opccr, SalA, and ChsE4-5 in the base bacteria genome, and introducing a plasmid that overexpresses the gene MT4b to construct a genetically engineered bacterium that produces high-yield 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester.

[0018] Preferably, the chassis bacteria is Mycobacterium neoaurum ATCC 25795.

[0019] Preferably, the nucleotide sequence of gene Opccr is shown as SEQ ID NO.1; the nucleotide sequence of gene SalA is shown as SEQ ID NO.2; the nucleotide sequence of gene ChsH1-2 is shown as SEQ ID NO.3; the nucleotide sequence of gene ChsE1-2 is shown as SEQ ID NO.4; the nucleotide sequence of gene ChsH4-5 is shown as SEQ ID NO.5; and the nucleotide sequence of gene MT4b is shown as SEQ ID NO.6.

[0020] The present invention also provides a genetic engineering bacterium for high-yield 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester constructed by the method.

[0021] The present invention also provides the use of the genetically engineered bacteria in the preparation of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester by microbial fermentation.

[0022] Preferably, the application comprises: inoculating the genetically engineered bacteria into a fermentation medium containing sterols, and culturing at 25-40° C. and 100-300 rpm with shaking for 48-168 hours to obtain 9α-hydroxyandrost-4-ene-3,17-dione.

[0023] Preferably, the sterol is selected from at least one of cholesterol, stigmasterol, sitosterol and phytosterol.

[0024] Preferably, the fermentation medium further comprises a phytosterol cosolvent, and the phytosterol cosolvent is selected from (2-hydroxypropyl)-β-cyclodextrin and / or soybean oil.

[0025] The beneficial effects of the present invention are as follows: in the process of producing 9-OH-3-OPCM with phytosterols in Mycobacterium neogoldensis, the metabolic shunting effect of various byproducts leads to the problems of low yield and low purity in the current production process. Therefore, the present invention genetically modifies the phytosterol decomposition metabolism in Mycobacterium neogoldensis, and eliminates the production of byproducts 9-OH-4-HBC, 9-OHAD, 9-OH-3-OPDCM and 9-OH-3-OPC by blocking the expression of KstD1, KstD2, KstD3, ChsE1-2, ChsH1-2, Opccr, SalA, ChsE4-5 and overexpressing MT4b to increase the yield of 9-OHAD. The molar conversion rate of 9-OH-3-OPCM in the genetically engineered bacteria finally obtained when the phytosterol concentration was 2g / L and 5g / L reached 85.9% and 81.3%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Metabolic pathway of phytosterols in microorganisms.

[0027] Figure 2 This is the degradation of phytosterols into 9-OH-3-OPCM in strain WL02 after preliminary metabolic pathway modification in the example of the present invention.

[0028] Figure 3 This is a mass spectrometry detection diagram of 9-OH-3-OPCM, the product of fermentation culture of strain WL02 in the embodiment of the present invention.

[0029] Figure 4 This is the nuclear magnetic resonance detection image of 9-OH-3-OPCM product after fermentation culture of strain WL02 in the embodiment of the present invention.

[0030] Figure 5 The differences in gene transcription levels in the production of 9-OH-3-OPCM by strain WL02 in the examples of the present invention.

[0031] Figure 6 This is the effect of knocking out genes ChsE4-5, SalA and OpccR in strains WL03 to WL06 on the synthesis of 9-OH-3-OPCM in the examples of the present invention.

[0032] Figure 7 This is the effect of overexpressing gene MT4b in strain WL07 on the synthesis of 9-OH-3-OPCM in the example of the present invention.

[0033] in, Figure 2 In / 6 / 7, peak a represents 9-OHAD, peak b represents 9-OH-3-OPC, peak c represents 9-OH-4-HBC, peak d represents 9-OH-3-OPDCM, and peak e represents 9-OH-3-OPCM. DETAILED DESCRIPTION

[0034] The following describes the embodiments of the present invention through specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0035] Unless otherwise specified, the methods used in the examples of the present invention are conventional methods, and the reagents used can be obtained from commercial sources. The substrate phytosterol was purchased from COFCO Corporation, and the product 9-OH-3-OPCM standard was purchased from Nanjing Tengyi Biotechnology Co., Ltd.

[0036] Preparation of culture medium and solution required:

[0037] (1) LB medium: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L.

[0038] (2) M1 culture medium: KH2PO4 1.3 g / L, K2HPO4 1.3 g / L, NaNO3 2 g / L, FeSO4·7H2O 0.1 g / L, ZnSO4·7H2O 0.1 g / L, MgSO4 0.4 g / L, corn steep liquor 10 g / L.

[0039] (3) M2 medium: KH2PO4 1.3 g / L, K2HPO4 1.3 g / L, NaNO3 2 g / L, FeSO4·7H2O 0.1 g / L, ZnSO4·7H2O 0.1 g / L, MgSO4 0.4 g / L, corn steep liquor 10 g / L, glucose 20 g / L, (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) 40 g / L. In the bioconversion process, 20 g / L phytosterol f was added as a raw material. When the conversion was scaled up in a 5 L fermenter, the added phytosterol concentration was 45 g / L.

[0040] (4) LB solid plate medium: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, agar 20 g / L.

[0041] (5) Kanamycin (Kan) stock solution: Weigh 0.5 g of kanamycin and dissolve it in 10 mL of sterile water. Sterilize it through a 0.22 μm filter membrane to obtain a stock solution with a final concentration of 50 g / L.

[0042] (6) 10% glycerol: Take 10 mL of glycerol and add sterile water to make up to 100 mL.

[0043] (7) 0.1 M CaCl2 solution: Weigh 11.1 g of CaCl2 powder and add water to make up to 1 L.

[0044] (8) 50×TAE buffer: Weigh 242 g of Tris and 37.2 g of disodium ethylenediaminetetraacetate dihydrate and dissolve in 800 mL of sterile water. Stir to dissolve. Add 57.1 mL of acetic acid and continue stirring. Add the solution to a volumetric flask and adjust the volume to 1 L.

[0045] (9) 50% sucrose solution: Weigh 50 g of sucrose, dissolve it in sterile water and make up to 100 mL.

[0046] Example 1: Construction of strains WL02 to WL07

[0047] 1. Construction of gene knockout plasmid and screening of knockout strains

[0048] (1) Construction of knockout plasmid

[0049] The construction of knockout plasmids includes pNS-ΔChsE1-2, pNS-ΔChsH1-2, pNS-ΔOpccr, pNS-ΔSalA, and pNS-ΔChsE4-5. Taking the knockout plasmid pNS-ΔChsE4-5 as an example, the target gene ChsE4-5 is first located, and a fragment of about 1000 bp in length is selected from the upstream and downstream sequences of the gene as the arms of homologous exchange during gene knockout. When knocking out genes by homologous exchange, a certain overlapping region must be retained between the homologous arms and the target gene, and the preset homology length is about 1000 bp. Then, two pairs of knockout primers D ChsE4-5U f / r and D ChsE4-5D f / r and a pair of vector primers Pvector f / r are designed for the construction of knockout plasmids. D ChsE4-5U f / r and D ChsE4-5D f / r are forward primers / reverse primers for the upstream homology arm of the knockout target gene ChsE4-5 and forward primers and reverse primers for the downstream homology arm of the knockout target gene ChsE4-5, respectively. There is a homology portion of about 25 bp between the upstream and downstream homology arm primers, and both have a homology portion of about 25 bp with the vector connection position, and are convenient for subsequent one-step cloning and connection. The relevant sequence is cloned from the template and matched with the corresponding target fragment after comparison. After sequencing analysis, the results show that the amplified sequence is the same as the ideal sequence. The primer sequences are shown in Table 1.

[0050] The target fragment and vector fragment obtained by PCR were cleaned up and collected for later use, and connected using the Vazyme One Step Cloning Kit to obtain a recombinant plasmid vector named pNS-ΔChsE4-5. The ligation product was transformed into the pre-made DH5α large intestine competent medium. Using Kan resistance screening, transformants were selected for verification. The plasmid was then extracted using a plasmid extraction kit. The construction of other knockout plasmids is shown in pNS-ChsE4-5.

[0051] Table 1. Primers for construction of gene knockout and overexpression plasmids

[0052]

[0053]

[0054] (2) Screening of knockout strains

[0055] First, prepare a fresh competent state of Mycobacterium niger. The preparation method is as follows: culture the Mycobacterium in M1 medium to the mid-logarithmic phase, collect the bacterial solution and wash it with sterile water and 10% glycerol (three times) to obtain the competent state of Mycobacterium niger. After the constructed knockout plasmid is treated with alkali, take an appropriate amount and add it to the competent state of Mycobacterium niger, and let it stand on ice for 10 minutes; set the voltage of the electroporator to 2.5kV, select the aperture of the electric shock cup to 2mm, confirm that the electric shock frequency is within the range of 3.5-5ms, and then continuously shock twice, then add 800μL of pre-cooled LB medium, fully suspend the bottom bacteria, and transfer to a sterile centrifuge tube. Culture at a constant temperature of 37℃ / 180rpm for 3-4h, take an appropriate amount of supernatant and apply it to Kan solid medium, and culture it upside down at 30℃ for 3-5 days.

[0056] Select appropriate transformants on the plate and culture them at 37°C-180rpm for 12h. When OD600 is between 0.4-0.5, take 100μL of bacterial solution and transfer it to sucrose plate (8% sucrose LB solid medium). After inverted culture at 30°C for 3-5 days, screen the transformants. First, select the transformants with good colony status and copy them on Kan resistance and non-resistance plates at the same time. Perform PCR screening on the transformants that do not grow in Kan plates and grow in non-resistance plates to finally obtain the knockout strain.

[0057] 2. Construction of gene overexpression plasmid and screening of strains

[0058] (1) Construction of overexpression plasmid

[0059] The overexpression plasmid was constructed as pMV261-MT4b. Taking the overexpression plasmid pMV261-MT4b as an example, the target gene MT4b was first located, and primers O MT4bf / r and O pMV261f / r were used to amplify the ATCC 25795 genome and pMV261 plasmid respectively. A homologous part of about 25bp was designed between the amplified products of the two, and it was convenient for subsequent one-step cloning and connection. The relevant sequence was cloned from the template and matched with the corresponding target fragment. After sequencing analysis, the results showed that the amplified sequence was the same as the ideal sequence. The primer sequences are shown in Table 1.

[0060] The target fragment and vector fragment obtained by PCR were cleaned up and collected for later use. They were connected using the Vazyme One Step Cloning Kit to obtain a recombinant plasmid vector named pMV261-MT4b. The ligation product was transformed into the pre-made DH5α large intestine competent medium. The transformants were selected for verification using Kan resistance screening. The plasmid was then extracted using a plasmid extraction kit.

[0061] (2) Screening of overexpression strains

[0062] Take an appropriate amount of the constructed overexpression plasmid and add it to the mycobacterium competent state, and let it stand on ice for 10 minutes; set the voltage of the electroporator to 2.5kV, select the aperture of the electric shock cup to 2mm, confirm that the electric shock frequency is within the range of 3.5-5ms, and then continuously shock twice, then add 800μL of pre-cooled LB medium, fully suspend the bottom bacteria, and transfer to a sterile centrifuge tube. Culture at a constant temperature of 37℃ / 180rpm for 3-4 hours, take an appropriate amount of supernatant and apply it to Kan solid medium, and culture it inverted at 30℃ for 3-5 days. Pick the transformants with better colony status for PCR screening and culture, and finally determine the correct overexpression strain.

[0063] The starting host strain WL01 was derived from the original strain ATCC 25795 by destroying three endogenous KstD enzymes (3-sterol-Δ1-dehydrogenase). Subsequent mutant strains WL02-WL07 were all modified on the basis of WM01.

[0064] The following strains were constructed:

[0065] WL02, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2;

[0066] WL03, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccR;

[0067] WL04, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔSalA;

[0068] WL05, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccrΔSalA;

[0069] WL06, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccrΔSalAΔChsE4-5;

[0070] WL07, ΔKstD1 / 2 / 3ΔChsE1-2&ChsH1-2ΔOpccrΔSalAΔChsE4-5::pMV261-MT4b.

[0071] Example 2: 9-OH-3-OPCM transformation study of strain WL02

[0072] 1. Production and verification of 9-OH-3-OPCM of WL02 strain

[0073] Take a loop of bacterial liquid from the glycerol tube of strain WL02, streak it on LB solid medium, and culture it at 30℃ for 48 hours; pick a single clone in 5mL LB liquid, shake and culture it at 30℃ for 36 hours; transfer it to 90mL M2 medium at a ratio of 2%, shake and culture it at 30℃ for 12 hours, add appropriate amount of hydroxypropyl cyclodextrin emulsified sterol to the final concentration of 2g / L, 5g / L and 10g / L, shake and culture the reaction at 32℃-180rpm for 168 hours, and then take a sample. The cell transformation suspension is extracted with an equal amount of ethyl acetate, and the biotransformation mixed product is obtained after drying.

[0074] The biotransformation mixture was extracted with an equal volume of ethyl acetate, and the solid residue was dried and redissolved in methanol. The resulting solution was then detected by high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). HPLC detection was performed on a Waters system equipped with a C18 column (250 mm × 4.6 mm × 5 μm) and a diode array detector (DAD). The conditions were as follows: the detection wavelength was 210 nm, the mobile phase consisted of a mixture of methanol and water (80:20, v / v), and the flow rate was 1 mL / min. The retention times of the standard samples 9-OHAD, 9-OH-3-OPC, 9-OH-3-OPCM, 9-OH-3-OPDCM, and 9-OH-4-HBC were compared. For the structural confirmation test of 9-OH-3-OPCM, its retention time was first compared with the literature records, and it was analyzed by LC-MS working in positive ion mode in the range of 50-1000 m / z, and further confirmed by nuclear magnetic resonance spectroscopy. The structure of the product was confirmed by LC-MS and nuclear magnetic resonance to be 3-oxo-4-pregnene-9-OH-20-carboxymethyl ester (9-OH-3-OPCM) (see Figure 3 and Figure 4 ). Combined Figure 2 It can be seen that strain WL02 has preliminary 9-OH-3-OPCM production capacity.

[0075] 2. Different gene expression levels of WL02 strain

[0076] The WL02 strain was inoculated in M2 medium containing phytosterols for culture at 37°C / 200rpm / 24 hours. After centrifugation to obtain cells, qRT-PCR was performed as follows: First, total RNA was extracted using the Vazyme HiScriptII RT Supermix Kit and Fast Prep instrument. Double distilled water (ddH2O) 4XgDNA was added to the total RNA obtained to 16μL and incubated at 42°C for 2 minutes. 4μL of 5X HiScript II qRT Supermix was added and incubated as follows: 50°C for 15 minutes and 85°C for 5 minutes. Further, cDNA analysis was performed on the Real Time PCR system. 10μL of 2X Chem QTM containing SYBR, 0.4μL of forward primer and reverse primer (as shown in Table 2), 50ng cDNA were added and diluted to a volume of 20μL with ddH2O. Amplification was performed by the following program: 10 min of initial denaturation, followed by 40 cycles of a second step: denaturation at 95 °C for 15 s, annealing at 58 °C for 15 s, and extension at 72 °C for 30 s. Melting curve analysis was performed after each cycle, and the expression of 16S rRNA was used as an internal standard for normalization calculations of qRT-PCR data. The detection results are shown in Figure 5 shown.

[0077] According to the results, it was preliminarily determined that genes OpccR, SalA, ChsE3 and ChsE4-5 may be directly related to the production of byproducts 9-OH-4-HBC and 9-OH-3-OPDCM and product 9-OH-3-OPCM. Therefore, the key genes involved in Example 1 were knocked out or overexpressed, and strains WL03 to WL07 were constructed based on strain WM02.

[0078] Table 2. Gene qRT-PCR primer design

[0079]

[0080] Example 3: 9-OH-3-OPCM transformation study of strains WL03 to WL07

[0081] Take a loop of bacterial liquid from the glycerol tubes of strains WL03 to WL07, streak on LB solid culture medium, and culture at 30°C for 48 hours; pick a single clone in 5mL LB liquid, and shake culture at 30°C for 36 hours; transfer to 90mL M2 culture medium at a ratio of 2%, shake culture at 30°C for 12 hours, add 10mL of 200g / L sterol solution emulsified with hydroxypropyl cyclodextrin, and shake culture at 32°C-180rpm; sample the reaction every 24 hours, and the reaction lasts for 168 hours. The cell transformation suspension is extracted with an equal amount of ethyl acetate, and the crude product is obtained after drying for subsequent analysis and detection. Among them, WL03-06 are strains that attempt to eliminate the production of 9-OH-4-HBC and 9-OH-3-OPDCM byproducts, and WL07 is a strain constructed to eliminate the byproduct 9-OH-3-OPC.

[0082] An equal amount of ethyl acetate was added to the obtained 1 mL sample for extraction, and the mixture was shaken for 30 minutes. 200 μL of the upper organic phase was taken and heated in an EP tube for volatilization, and 0.8 mL of methanol was added for re-dissolution. The sample was subjected to liquid phase detection, the detection wavelength was 210 nm, and the mobile phase was a mixture of methanol and water (8:2, v / v). The transformation results of strain WL07 are shown in Table 3, and the liquid phase results of strains WL03 to 07 products are shown in Table 3. Figure 6 and Figure 7 shown.

[0083] Table 3. WL07 strain 9-OHAD biotransformation results

[0084]

[0085] According to the fermentation verification results, strain WL05 produced no byproduct 9-OH-4-HBC after fermentation and had the ability to produce high 9-OH-3-OPCM. Strain WL06 produced no byproduct 9-OH-4-HBC and 9-OH-3-OPDCM after fermentation and had a higher 9-OH-3-OPCM production capacity than strain WL05. Finally, strain WL07 was obtained, which had no byproduct 9-OH-4-HBC, 9-OH-3-OPC and 9-OH-3-OPDCM and had a higher 9-OH-3-OPCM production capacity than strains WL05 and WL06. Strain WL07 was fermented at 2g / L and 5g / L phytosterol concentrations, and produced 9-OH-3-OPCM at 85.9% and 81.3% molar conversion rates, respectively.

[0086] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A method for constructing a genetically engineered bacterium that produces high yield of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester, characterized in that: The method comprises: using mycobacteria as the base bacteria, blocking the expression of genes KstD1, KstD2, KstD3, ChsE1-2, ChsH1-2, Opccr and SalA in the base bacteria, and constructing a genetically engineered bacterium with high production of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester.

2. The method according to claim 1, characterized in that The method also includes: blocking the expression of gene ChsE4-5 in the chassis bacteria.

3. The method according to claim 1 or 2, characterized in that The method further comprises: overexpressing gene MT4b in the chassis bacteria.

4. The method according to claim 1, characterized in that The method comprises: Using Mycobacterium as the base bacteria, the genes KstD1, KstD2, KstD3, ChsE1-2, ChsH1-2, Opccr, SalA, and ChsE4-5 in the base bacteria genome were knocked out, and a plasmid overexpressing the gene MT4b was introduced to construct a genetically engineered bacterium that produces high levels of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester.

5. The method according to claim 1, characterized in that The bottom plate bacteria is Mycobacterium neoaurumATCC 25795.

6. The method according to claim 3, characterized in that The nucleotide sequence of gene Opccr is shown in SEQ ID NO.1; The nucleotide sequence of gene SalA is shown in SEQ ID NO.2; The nucleotide sequence of gene ChsH1-2 is shown in SEQ ID NO.3; The nucleotide sequence of gene ChsE1-2 is shown in SEQ ID NO.4; The nucleotide sequence of gene ChsH4-5 is shown in SEQ ID NO.5; The nucleotide sequence of gene MT4b is shown in SEQ ID NO.

6.

7. A genetically engineered bacterium for high production of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester constructed according to any one of claims 1 to 6.

8. Use of the genetically engineered bacteria as claimed in claim 7 in the preparation of 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester by microbial fermentation.

9. The use according to claim 8, characterized in that The application comprises: inoculating the genetically engineered bacteria into a fermentation medium containing sterols, and culturing at 25-40° C. and 100-300 rpm for 48-168 hours to obtain 9α-hydroxyandrost-4-ene-3,17-dione.

10. The use according to claim 9, characterized in that The sterol is selected from at least one of cholesterol, stigmasterol, sitosterol and phytosterol; and / or, The fermentation medium further comprises a phytosterol cosolvent, wherein the phytosterol cosolvent is selected from (2-hydroxypropyl)-β-cyclodextrin and / or soybean oil.

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