Genetically engineered bacterium for producing 9 alpha-hydroxyandrostane-4-ene-3, 17-diketone at high yield as well as construction method and application of genetically engineered bacterium

Abstract

The invention provides a genetically engineered bacterium for high yield of 9 alpha-hydroxyandrostane-4-ene-3, 17-diketone as well as a construction method and application of the genetically engineered bacterium for high yield of 9 alpha-hydroxyandrostane-4-ene-3, 17-diketone. According to the invention, the production of by-products 4-HBC, 4-AD and 9-OH-3-OPCM is eliminated by blocking the expression of Opccr, SalA and TeB in mycobacterium neoaurum and overexpressing KshA1, the production limitation of 9-OHAD is liberated by overexpressing rate-limiting enzyme ChsH1-2, and finally, under the condition that the concentration of phytosterol is 20g / L, the purposes that the yield of 9-OHAD is 13.4 g / L, the molar yield is 91.1% and almost no by-product exists are achieved. When amplification is carried out at the concentration of 45g / L of phytosterol in a 5L bioreactor, soybean oil and (2-hydroxypropyl)-beta-cyclodextrin are used as auxiliaries, and a 9-OHAD conversion result at the concentration of 20g / L in a shake flask is basically reproduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for constructing a genetically engineered bacterium capable of producing high yields of 9α-hydroxyandrost-4-ene-3,17-dione, the constructed genetically engineered bacterium, and application of the genetically engineered bacterium in preparing 9α-hydroxyandrost-4-ene-3,17-dione by microbial fermentation. Background Art

[0002] Steroidal compounds have become a well-known important natural organic compound due to their special physiological functions and disease efficacy. In the global pharmaceutical market, the annual output value of steroidal compounds is as high as hundreds of billions of dollars, the output is as high as millions of tons, and the annual growth rate is maintained at more than 15%. Steroidal compounds can easily introduce functional groups of different configurations on their parent nucleus to have different unique therapeutic effects, including anti-inflammatory, anti-allergic, anticonvulsant and anti-fever. Among them, 9α-hydroxyandrost-4-ene-3,17-dione is an important steroidal intermediate.

[0003] 9α-Hydroxyandrost-4-ene-3,17-dione (9-OHAD) is widely used in the production of steroidal drugs because its structure is easy to modify into various glucocorticoids. 9-OHAD can form enhanced-effect corticosteroids such as beclomethasone and other corticosteroid drugs by introducing halogens and other functional groups at positions 11 and 16. Mycobacterium neogenes is widely used in the biosynthesis of steroidal drug intermediate 9-OHAD because of its comprehensive plant sterol degradation gene cluster and mature fermentation method. Therefore, it is of great significance to enhance the biosynthesis efficiency of 9-OHAD in Mycobacterium neogenes, reduce synthetic by-products, and simplify the biosynthesis process of 9-OHAD in the field of steroidal drug synthesis.

[0004] In recent years, there have been reports at home and abroad that the yield and purity of 9-OHAD can be increased by eliminating byproducts in the degradation of phytosterols to 9-OHAD. For example, the gene encoding the key enzyme KshA1 in the phytosterol degradation pathway is overexpressed to reduce the production of the byproduct 4-androstene-3,17-dione (4-AD). In addition, the overexpression of four key enzymes, Hsd, Hsd4A, KshA1 and KshB, in the phytosterol degradation pathway can also significantly increase the yield of 9-OHAD. To date, most methods for increasing the yield of 9-OHAD have focused on eliminating the over-complete degradation product 4-AD or balancing the metabolic flux of side chain degradation. However, there is still a lack of research on improving the yield and purity of 9-OHAD by solving byproducts such as 20-hydroxymethylpregn-4-ene-3-one (4-HBC) caused by incomplete degradation of the side chain. Summary of the invention

[0005] In order to solve the problems of low productivity and low product purity due to the metabolic diversion effect of various by-products in the process of producing 9-OHAD with plant sterols in Mycobacterium neoaureum in the prior art, the present invention provides a method for constructing a genetically engineered bacterium with high 9-OHAD production, wherein the method genetically modifies the sterol decomposition metabolism in the chassis bacterium Mycobacterium neoaureum, and increases the production of 9-OHAD by reducing the formation of by-products. The constructed genetically engineered bacterium has a high conversion rate and product purity in the application of microbial fermentation to prepare 9-OHAD.

[0006] The technical scheme adopted by the present invention is: a method for constructing a genetically engineered bacterium with high production of 9α-hydroxyandrost-4-ene-3,17-dione, the method comprising: using Mycobacterium neogoldensis as a base bacterium, knocking out KstD1 / 2 / 3 genes, Opccr genes, SalA genes and TeB genes in the base bacterium genome, and constructing a genetically engineered bacterium with high production of 9α-hydroxyandrost-4-ene-3,17-dione.

[0007] Preferably, the method further comprises: overexpressing the KshA1 gene in the chassis bacteria.

[0008] Preferably, the method further comprises: overexpressing ChsH1-2 genes in the chassis bacteria.

[0009] Preferably, the chassis bacteria is Mycobacterium neoaurum ATCC 25795.

[0010] Please refer to Figure 1 The schematic diagram of the metabolic pathway of phytosterols in microorganisms shown in the figure shows that 9-OHAD is decomposed into water and carbon dioxide under the action of KstD enzyme, so the unmodified Mycobacterium neoaurum does not have the ability to produce 9-OHAD. Therefore, the present invention first uses Mycobacterium neoaurum ATCC 25795 as the initial strain, knocks out the KstD1 / 2 / 3 genes in its genome, and constructs the Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3 strain (denoted as engineered bacteria WL00), which has the initial ability to produce 9-OHAD. On this basis, the products and by-products obtained in the process of phytosterol degradation by the WL00 strain were analyzed and identified, and the results showed that at high concentrations (20g / L) of phytosterols, compared with 2g / L phytosterol concentration, in addition to the by-products 4-HBC and 4-AD, a by-product ( Figure 2 ), which was determined by liquid chromatography-mass spectrometry to be 3-oxo-4-pregnene-9-OH-20-carboxymethyl ester (9-OH-3-OPCM, Figure 5). Therefore, the present invention further detects and evaluates the expression levels of related genes in the process of bioconversion of WL00 strain to produce 9-OHAD, and not only determines that the destruction of key enzymes Opccr and SalA plays a key role in the elimination of byproduct 4-HBC, but also determines the gene TeB involved in the generation of byproduct 9-OH-3-OPCM, and the rate-limiting enzyme ChsH1-2 for the production of product 9-OHAD. Based on this, the phytosterol decomposition metabolism in the WL00 strain is genetically modified, specifically by blocking the expression of Opccr gene, SalA gene, and TeB gene to eliminate the production of byproducts 4-HBC and 9-OH-3-OPCM, thereby increasing the yield of 9-OHAD. In addition, the KshA1 gene is further overexpressed to strengthen the conversion of precursor 4-AD to 9-OHAD, and / or the rate-limiting enzyme ChsH1-2 is overexpressed to release the production restriction of 9-OHAD, thereby increasing the yield of 9-OHAD.

[0011] Preferably, the method comprises the following steps:

[0012] (1) Using Mycobacterium neoaurum ATCC 25795 as the base bacteria, using CRISPR-Cas9 gene editing technology, knocking out KstD1, KstD2, and KstD3 genes in the genome to obtain an engineered bacterium WL00, wherein the engineered bacterium WL00 has the ability to produce 9-OHAD;

[0013] (2) Using the engineered bacterium WL00 as the base bacterium, the CRISPR-Cas9 gene editing technology was used to knock out the Opccr and SalA genes in the genome to obtain the engineered bacterium WL01, wherein the engineered bacterium WL01 did not produce the byproduct 4-HBC and had a high yield of 9-OHAD;

[0014] (3) Using the engineered bacterium WM01 as the base bacterium, the CRISPR-Cas9 gene editing technology was used to knock out the TeB gene in the genome to obtain the engineered bacterium WL02, wherein the engineered bacterium WL02 did not produce byproducts 4-HBC and 9-OH-3-OPCM and produced 9-OHAD in high yield;

[0015] (4) Using the engineered bacterium WM02 as the base bacterium, the expression of the KshA1 gene was enhanced by using an overexpression vector to obtain the engineered bacterium WL03, wherein the engineered bacterium WL03 did not produce byproducts 4-HBC, 4-AD and 9-OH-3-OPCM and produced 9-OHAD in high yield;

[0016] (4) The engineered bacteria WM03 was used as the base bacteria, and the expression of the ChsH1-2 gene was enhanced by using an overexpression vector to obtain the engineered bacteria WL04. The engineered bacteria WL04 did not produce byproducts 4-HBC, 4-AD and 9-OH-3-OPCM and had a higher yield of 9-OHAD.

[0017] The present invention also provides a genetically engineered bacterium with high yield of 9α-hydroxyandrost-4-ene-3,17-dione obtained by the method. The genetically engineered bacterium is preferably any one of the following strains:

[0018] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB;

[0019] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB::pMV261-KshA1;

[0020] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB::pMV261-ChsH1-2&KshA1;

[0021] More preferably:

[0022] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB::pMV261-ChsH1-2&KshA1.

[0023] The present invention also provides the use of the genetically engineered bacteria in preparing 9-OHAD by microbial fermentation.

[0024] Preferably, the application comprises: inoculating the genetically engineered bacteria into a fermentation medium containing sterols, culturing at 25-40° C. and 100-300 rpm for 48-168 hours, and obtaining the 9α-hydroxyandrost-4-ene-3,17-dione in the fermentation broth.

[0025] Preferably, the concentration of sterols in the fermentation medium is 20-50 g / L.

[0026] Preferably, the sterol is selected from at least one of cholesterol, stigmasterol, sitosterol and phytosterol, preferably phytosterol.

[0027] Preferably, the fermentation medium further comprises an auxiliary agent, which is selected from one or both of (2-hydroxypropyl)-β-cyclodextrin and soybean oil. When the plant sterols are amplified at a high concentration (45-50 g / L) in a fermentation tank, it is preferred to use a mixture of one or both of soybean oil and (2-hydroxypropyl)-β-cyclodextrin as a cosolvent for the plant sterols.

[0028] Beneficial effects of the present invention: The present invention systematically explores the correlation between changes in the expression levels of key genes involved in the degradation of phytosterols in Mycobacterium neogoldensis and the production of by-products, and selectively regulates the expression of key enzymes according to their changing rules, and finally achieves a 9-OHAD yield of 13.4 g / L, a molar yield of 91.1%, and almost no by-products when the phytosterol concentration is 20 g / L. When the phytosterol concentration is amplified in a 5L bioreactor at 45 g / L, soybean oil and (2-hydroxypropyl)-β-cyclodextrin are used as adjuvants to basically reproduce the 9-OHAD conversion results at 20 g / L in the shake flask. Therefore, the present invention provides an effective strategy to increase the yield of 9-OHAD by reducing the formation of three by-products, and provides a research idea for improving the ability of Mycobacterium neogoldensis to biodegrade sterols and synthesize 9-OHAD. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the metabolic pathway of phytosterols in Mycobacterium neoaureum in the present invention.

[0030] Figure 2 The figure is a comparison of the production of by-products of the engineered bacteria WL00 of the present invention at different phytosterol concentrations.

[0031] Figure 3 This is the electrophoresis diagram of gene knockout in the example of the present invention.

[0032] Figure 4 This is the electrophoresis diagram of gene overexpression in the embodiment of the present invention.

[0033] Figure 5 This is a mass spectrum detection diagram of the by-product 9-OH-3-OPCM in the example of the present invention.

[0034] Figure 6 The changes in gene expression levels under different phytosterol concentrations in the examples of the present invention.

[0035] Figure 7 This is a liquid phase detection diagram of 9-OHAD biotransformation of different mutant strains in the examples of the present invention. DETAILED DESCRIPTION

[0036] The following is an explanation of the embodiments of the present invention by specific specific examples, 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 by 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, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0037] Preparation of culture medium and solution required:

[0038] (1) LB medium: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L.

[0039] (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.

[0040] (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 syrup 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.

[0041] (4) LB solid plate medium: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, agar 20 g / L.

[0042] (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.

[0043] (6) 10% glycerol: Take 10 mL of glycerol and add sterile water to make up to 100 mL.

[0044] (7) 0.1 M CaCl2 solution: Weigh 11.1 g of CaCl2 powder and add water to make up to 1 L.

[0045] (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.

[0046] (9) 50% sucrose solution: Weigh 50 g of sucrose, dissolve it in sterile water and make up to 100 mL.

[0047] The substrate phytosterols were purchased from COFCO Corporation, and the product 9-OHAD standard was purchased from Beijing Bailingwei Technology Co., Ltd.

[0048] Example 1: Genetically engineered bacteria with high 9-OHAD production and construction method thereof

[0049] Construction of gene knockout plasmids and screening of knockout strains

[0050] (1) Construction of knockout plasmid

[0051] The construction of knockout plasmids includes pNS-ΔKstD1, pNS-ΔKstD2, pNS-ΔKstD3, pNS-ΔOpccr, pNS-ΔSalA, and pNS-ΔTeB. Taking the knockout plasmid pNS-ΔOpccr as an example, the target gene Opccr 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 100 bp. Then, two pairs of knockout primers D OpccrU f / r and D OpccrD f / r and a pair of vector primers Pvector f / r are designed for the construction of knockout plasmids. D Opccr U f / r and D Opccr D f / r are respectively the forward primer / reverse primer for the upstream homology arm of the knockout target gene Opccr and the forward primer and reverse primer for the downstream homology arm of the knockout target gene Opccr. There is a homology portion of about 25 bp between the upstream and downstream homology arm primers, and both carry a homology portion of about 25 bp with the vector connection position, and are for subsequent one-step cloning and connection. The relevant sequence is cloned from the template and matched after comparison with the corresponding target fragment. After sequencing analysis, the results show that the amplified sequence is the same as the ideal sequence. The nucleotide sequences of the three knockout genes are shown in SEQ ID NO.1-NO.3. The primer sequences are shown in Table 1.

[0052] 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 pNS-ΔOpccr. The ligation product was transformed into the pre-made DH5α colon competent medium. The transformants were selected for verification using Kan resistance screening. The verification results were as follows: Figure 3 As shown. Then the plasmid was extracted using a plasmid extraction kit. The construction of other knockout plasmids is shown in pNS-ΔOpccr.

[0053] Table 1. Primers for construction of knockout plasmids and overexpression plasmids

[0054]

[0055] (2) Screening of knockout strains

[0056] 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 shock twice continuously, 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.

[0057] Select appropriate transformants from the plate and culture them at 37°C / 180 rpm for 12 h. 600 When the p-value is between 0.4 and 0.5, 100 μL of bacterial solution is transferred to a sucrose plate (8% sucrose LB solid medium), and then inverted and cultured at 30°C for 3 to 5 days before screening for transformants. First, select transformants with good colony status and copy them on Kan resistance and non-resistance plates at the same time. Perform PCR screening on transformants that do not grow on Kan plates and grow on non-resistance plates, and finally obtain knockout strains. The following engineering strains are obtained:

[0058] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3, denoted as engineered strain WL00;

[0059] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalA, denoted as engineered strain WL01;

[0060] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB, denoted as engineered strain WL02.

[0061] Construction of gene overexpression plasmid and screening of strains

[0062] (1) Construction of overexpression plasmid

[0063] The construction of the overexpression plasmid includes pMV261-KshA1, pMV261-ChsH1-2&KshA1, wherein ChsH1 and ChsH2 are adjacent on the genome and have complementary functions, so the overexpression plasmid is amplified and constructed in the form of a group of genes. Taking the overexpression plasmid pMV261-KshA1 as an example, the target gene KshA1 is first located, and the primers OKshA1 f / r and OpMV261f / r are used to amplify the ATCC 25795 genome and the pMV261 plasmid respectively. A homologous part of about 25bp is designed between the amplified products of the two, and the subsequent one-step cloning and connection is carried out. The relevant sequence is cloned from the template and matched after comparison with the corresponding target fragment. After sequencing analysis, the results show that the amplified sequence is the same as the ideal sequence. The nucleotide sequences of the three overexpressed genes are shown in SEQ ID NO.4-NO.6. The primer sequences are shown in Table 1.

[0064] 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-KshA1. The ligation product was transformed into the pre-made DH5α colon competent medium. The transformants were selected for verification using Kan resistance screening. See the schematic diagram for details. Figure 4 The plasmid was then extracted using a plasmid extraction kit. The construction of other overexpression plasmids is shown in pMV261-KshA1.

[0065] (2) Screening of overexpression strains

[0066] Take an appropriate amount of the constructed overexpression plasmid and add it to the competent state of mycobacteria, 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 spread it on Kan solid medium, and invert and culture 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. Based on the engineered bacteria WL02, the following engineered strains were obtained:

[0067] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB::pMV261-KshA1, denoted as engineered strain WL03;

[0068] Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB::pMV261-ChsH1-2&KshA1, denoted as engineered strain WL04.

[0069] Embodiment 2:

[0070] Production and verification of byproducts of engineered bacteria WL00 at different phytosterol concentrations

[0071] Take a loop of bacterial liquid from the glycerol tube, streak it on LB solid medium, and culture it at 30℃ for 48 hours; pick a single clone in 5mL LB liquid, shake culture at 30℃ for 36 hours; transfer it to 90mL M2 medium at a ratio of 2%, shake culture at 30℃ for 12 hours, add appropriate amount of hydroxypropyl cyclodextrin emulsified sterol to the final concentration of 2g / L, 10g / L and 20g / L, shake culture 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.

[0072] The biotransformation mixed product was extracted with an equal volume of ethyl acetate, dried to obtain a solid residue, 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 carried out on a Waters system equipped with a C18 column (250 mm × 4.6 mm × 5 μm) and a diode array detector (DAD), and the conditions were set as follows: the detection wavelength was 210 nm, the mobile phase consisted of a mixture of methanol and water (70:30, v / v), and the flow rate was 1 mL / min. The retention times were compared with those of the standards 4-AD, 9-OHAD, and 4-HBC, respectively. For the detection of byproducts of 9-OH-3-OPCM, its retention time was first compared with that recorded in the literature, and then analyzed by LC-MS operating in the positive ion mode in the range of 50-1000 m / z. The results are shown in the figure. Figure 5 shown.

[0073] Gene expression levels of engineered bacteria WL00 under different phytosterol concentrations

[0074] Fresh new gold mycobacterium WL00 strains were inoculated in M2 medium composed of different concentrations of phytosterols (2g / L, 20g / L) to culture new gold mycobacterium cells at 37℃ / 200rpm / 24 hours. After centrifugation to obtain cells, qRT-PCR was performed as follows: First, total RNA was extracted using the Vazyme HiScript II RT Supermix Kit and FastPrep instrument. Double distilled water (ddH2O) 4X gDNA was added to the total RNA obtained to 16μL and incubated at 42℃ for 2 minutes. 5X HiScript II qRT Supermix 4μL was added and incubated as follows: 50℃ for 15 minutes and 85℃ for 5 minutes. Further, cDNA analysis was performed on the Real Time PCR system. Add 10 μL of 2X ChemQTM containing SYBR, 0.4 μL of forward primer and reverse primer (as shown in Table 2), 50 ng of cDNA, and dilute to 20 μL volume with ddH2O. Amplification was performed by the following program: 10 minutes of initial denaturation, followed by 40 cycles of the second step: denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and then extension at 72°C for 30 seconds. Melting curve analysis was performed after each cycle, and the expression of 16S rRNA was used as an internal standard for normalization calculation of qRT-PCR data. The detection results are shown in Figure 6 shown.

[0075] Table 2. qPCR primers

[0076]

[0077]

[0078] Example 3: Application of genetically engineered bacteria in the preparation of 9-OHAD by microbial fermentation.

[0079] Shake flask fermentation: Take a loop of bacterial liquid from the glycerol tubes of engineered bacteria WL00-WL04, streak on LB solid medium, and culture at 30℃ for 48 hours; pick a single clone in 5mL LB liquid, shake and culture at 30℃ for 36 hours; transfer to 90mL M2 medium at a ratio of 2%, shake and culture at 30℃ for 12 hours, add 10mL of 20g / L sterol solution emulsified with hydroxypropyl cyclodextrin, shake and culture at 32℃ / 180rpm; sample 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 by drying for subsequent analysis and detection. Add 5mL ethyl acetate to the obtained 1mL sample for extraction, shake and mix for 30 minutes, take 200μL of the upper organic phase in an EP tube, heat and volatilize, and add 0.8mL of methanol to dissolve. The samples were subjected to liquid phase detection, the detection wavelength was 210 nm, and the mobile phase was a mixture of methanol and water (7:3, v / v). The conversion results are shown in Table 3. The liquid phase detection results are shown in Figure 7 shown.

[0080] Table 3. 9-OHAD biotransformation molar ratios of different mutants

[0081]

[0082] Fermentation in a 5L fermenter: Take an appropriate amount of bacterial liquid from the cryopreserved tube of engineered bacteria WL04 and streak it on LB solid medium for culture. Select a monoclonal colony and transfer it to LB liquid medium. When it grows to the late logarithmic stage, transfer it to M1 medium at a ratio of 2%; then transfer the bacterial liquid in M1 medium after expansion to a sterilized and pre-cooled 5L fermenter at a ratio of 5%. The fermentation conditions are set as follows: the speed is 800rpm, the ventilation volume is 0.5vvm, the temperature is 32℃, and the pH is maintained at 7.2 during the fermentation process by adding alkali; samples are taken every 24 hours during the fermentation process, and the whole process lasts for 168 hours. The cell suspension obtained during the sampling process is extracted and dried by ethyl acetate, and subsequent analysis and detection are performed. The detection steps are the same as above.

[0083] After genetic modification of phytosterol catabolism in Mycobacterium neoaurum, the resulting engineered strain Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB::pMV261-ChsH1-2&KshA1 (engineered strain WL04) achieved a 9-OHAD yield of 13.4 g / L and a molar yield of 91.1% with almost no byproducts at a phytosterol concentration of 20 g / L. When scaled up in a 5L bioreactor at a phytosterol concentration of 45 g / L, soybean oil and (2-hydroxypropyl)-β-cyclodextrin were used as adjuvants, which basically reproduced the 9-OHAD conversion results at 20 g / L in the shake flask. Therefore, this study provides an effective strategy to increase the yield of 9-OHAD by reducing the formation of three byproducts.

[0084] 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 9α-hydroxyandrost-4-ene-3,17-dione, characterized in that: The method comprises: using Mycobacterium neogoldensis as a base bacterium, knocking out KstD1 / 2 / 3 genes, Opccr genes, SalA genes and TeB genes in the base bacterium genome, and constructing a genetically engineered bacterium with high production of 9α-hydroxyandrost-4-ene-3,17-dione.

2. The method according to claim 1, characterized in that The method also includes: overexpressing ChsH1-2 genes in the chassis bacteria.

3. The method according to claim 1, characterized in that The method further comprises: overexpressing the KshA1 gene in the bottom plate bacteria.

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

5. A genetically engineered bacterium that produces high levels of 9α-hydroxyandrost-4-ene-3,17-dione constructed according to any one of claims 1 to 4.

6. The genetically engineered bacterium according to claim 5, characterized in that The genetically engineered bacteria is Mycobacteriumneoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOpccrΔSalAΔTeB::pMV261-ChsH1-2&KshA1.

7. Use of the genetically engineered bacteria as claimed in claim 6 in the preparation of 9α-hydroxyandrost-4-ene-3,17-dione by microbial fermentation.

8. The use according to claim 7, characterized in that The application includes: inoculating the genetically engineered bacteria into a fermentation medium containing sterols, shaking and culturing at 25-40° C. and 100-300 rpm for 48-168 hours, and obtaining the 9α-hydroxyandrost-4-ene-3,17-dione in the fermentation liquid.

9. The use according to claim 8, characterized in that The sterol is selected from at least one of cholesterol, stigmasterol, sitosterol and phytosterol.

10. The use according to claim 8, characterized in that The fermentation medium also includes an auxiliary agent, which is selected from one or both of (2-hydroxypropyl)-β-cyclodextrin and soybean oil.

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