Method for improving cell permeability of mycobacterium neoaurum, mycobacterium neoaurum engineering bacteria and application of mycobacterium neoaurum engineering bacteria in preparation of 9 alpha-hydroxyandrostane-4-ene-3, 17-diketone

Abstract

The invention provides a method for improving cell permeability of mycobacterium neoaurum, a mycobacterium neoaurum engineering bacterium and an application of the mycobacterium neoaurum engineering bacterium in preparation of 9 alpha-hydroxyandrostane-4-ene-3, 17-diketone (9-OHAD). Biotransformation of phytosterol into high-value steroid intermediates such as 9-OHAD in mycobacteria is a footstone of steroid drugs. However, the limited permeability of its dense cell wall of Mycobacterium severely hinders the effective intracellular transport of phytosterols and its bioconversion to 9-OHAD. The endogenous synthesis capability and exogenous circulation capability of mycobacterium neoaurum trehalose are destroyed through gene knockout, the permeability of cells is improved, the molar conversion rate of 9-OHAD is increased to 65.7% from 44.7%, the extracellular production of 9-OHAD is also improved, the extracellular yield of 9-OHAD is increased to 32.1% from 12%, and subsequent separation and purification are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for improving the cell permeability of new mycobacterium aureum, a constructed new mycobacterium aureum engineered bacterium with high cell permeability, and an application of the new mycobacterium aureum engineered bacterium in the preparation of 9α-hydroxyandrost-4-ene-3,17-dione. Background Art

[0002] Steroidal compounds are important natural organic compounds widely present in organisms. Steroidal drugs are the second largest drug category in the global pharmaceutical market because they have many unique physiological functions and unique therapeutic effects. Globally, millions of tons of steroids with a cumulative output value of hundreds of billions of dollars are used to treat various diseases, including anti-inflammatory, anti-allergic, anticonvulsant and anti-fever, with an annual growth rate of more than 15%. Steroidal compounds have different physiological and biochemical functions due to the different substituents, double bond positions or stereo configurations on their parent nuclei. Common steroidal compounds include sex hormones, adrenal cortex hormones, progesterone in animal tissues, and diosgenin and sitosterol commonly found in plants. 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. Its hydroxylation site at position 9 can introduce F or Cl substituents through simple chemical halogenation reactions. In addition, 9-OHAD can also be modified at other sites such as positions 16 and 11 to obtain drugs with enhanced efficacy, including dexamethasone, beclomethasone and 16β-methylcorticosteroids. Mycobacterium neogoldenum is used to produce the steroidal intermediate 9-OHAD because it has a comprehensive plant sterol degradation gene cluster, simple tools for genetic manipulation and effective fermentation methods. Therefore, enhancing the biosynthesis efficiency of 9-OHAD in Mycobacterium neogoldenum and optimizing the biosynthesis process of 9-OHAD are of great significance in the field of steroidal synthesis.

[0004] The uptake of phytosterols by mycobacteria depends mainly on the direct contact and transport efficiency between phytosterol particles and the cell wall. However, the limited permeability of the compact mycobacterial cell wall severely hinders the effective intracellular transport of phytosterols and their biotransformation to 9-OHAD. The core of the mycobacterial cell wall is a covalently bound layer composed of mycolic acid methyl ester (MA), arabinogalactan (AG), and peptidoglycan (PG). In addition, the surface of the layer is decorated with various non-covalent capsular lipids, including lipomannan (LM), lipoarabinomannan (LAM), and other glycolipids, all of which are considered to be important elements in cell wall formation and play a leading role in preventing the material exchange of substrate phytosterols and product 9-OHAD in Mycobacterium neogoldenum. It has been reported that the addition of vancomycin, glycine, and ethanolamine can inhibit the formation of the cell wall, thereby increasing cell permeability. However, due to the high cost of adding inhibitors, the above methods are almost unsuitable for industrial applications (Fernandes, P., Cruz, A., Angelova, B., Pinheiro, H., Cabral, J. 2003. Microbial conversion of steroid compounds: recent developments. Enzyme and Microbial Technology, 32 (6), 688-705). In contrast, disrupting cell wall synthesis by genetic manipulation is an effective method to enhance the permeability of mycobacterial cells. Summary of the invention

[0005] In order to solve the problem of low biosynthesis efficiency of 9-OHAD in the prior art due to low cell wall permeability of Mycobacterium neoaureum, the present invention targets genes related to trehalose synthesis and external recycling pathways in Mycobacterium neoaureum (such as Figure 1 A systematic investigation was conducted on the cell permeability of Mycobacterium aureum (M. aureum), and the cell wall formation, cell growth, cell wall permeability and 9-OHAD biotransformation involved were studied respectively. This provided a method for improving the cell permeability of Mycobacterium aureum, and by destroying the endogenous trehalose synthesis ability and the exogenous trehalose circulation ability to reduce the trehalose content in the cell wall, a new Mycobacterium aureum engineered bacterium with high cell permeability was constructed, and it was applied to the preparation of 9α-hydroxyandrost-4-ene-3,17-dione, thereby achieving the enhancement of the cell permeability of Mycobacterium aureum and the improvement of 9-OHAD yield.

[0006] The technical solution adopted by the present invention is: a method for improving the cell permeability of new Mycobacterium aureum, comprising: taking new Mycobacterium aureum as the base bacteria, knocking out the KstD1 / 2 / 3 genes in the base bacteria genome, destroying the endogenous synthesis pathway of trehalose in the base bacteria, destroying the exogenous circulation ability of trehalose in the base bacteria, and constructing a new Mycobacterium aureum engineered bacterium with high cell permeability.

[0007] Specifically, the method for destroying the endogenous synthesis pathway of trehalose in the chassis bacteria includes: knocking out one or more of the OtsA gene, the TreS gene, and the TrsY gene in the chassis bacteria genome.

[0008] Specifically, the method for destroying the exogenous circulation ability of trehalose in the chassis bacteria includes: knocking out one or more of the LpqY gene, the SugA gene, the SugB gene, and the SugC gene in the chassis bacteria genome.

[0009] The present invention uses Mycobacterium ATCC 25795 as the base bacteria, first knocks out KstD1, KstD2, and KstD3 genes in sequence, and constructs a strain WM00 with 9-OHAD synthesis ability. Then, using WM00 as the base bacteria, endogenous synthesis trehalose-deficient strains WM01 (ΔOtsA), WM02 (ΔTreS), and WM03 (ΔTreY) are constructed, and the effects of the individual destruction of the three endogenous trehalose synthesis pathways on cell wall formation are tested, and the three endogenous trehalose synthesis pathways are further destroyed to obtain the endogenous trehalose synthesis-deficient strain WM04 (ΔOtsA / TreS / TreY). The cell growth, cell wall permeability, and 9-OHAD bioconversion of the above-mentioned trehalose-deficient strains are tested and studied, and the cell permeability and 9-OHAD production of the strain WM04 are greatly improved. Therefore, the exogenous trehalose recycling ability was destroyed on the basis of WM04, and the exogenous trehalose recycling-deficient strains WM05 (ΔOtsA / TreS / TreY / LpqY), WM06 (ΔOtsA / TreS / TreY / LpqY / SugC) and WM07 (ΔOtsA / TreS / TreY / LpqYSugA / B / C) were constructed. The cell growth, cell wall permeability and 9-OHAD biotransformation of strains WM05, WM06 and WM07 were tested and studied. The cell permeability of strain WM06 was further improved compared with WM04, and its 9-OHAD production was increased from 44.7% (WM00) to 65.7%, reaching 9.6 g / L when the phytosterol concentration was 20 g / L in the shake flask. In addition, the present invention also analyzes and tests the cell wall components of WM00, WM04 and WM06 and the transport of 9-OHAD inside and outside the cell. The components of lipid trehalose monomycolate (TMM) and trehalose dimycolate (TDM) in the cell wall of the obtained mutant strains change significantly, and verifies that the increase of TMM content in the cell wall will directly affect the distribution of 9-OHAD inside and outside the cell, and finally leads to an increase in the extracellular production of 9-OHAD from 12% to 32.1%, which is convenient for subsequent separation and purification.

[0010] Therefore, the method preferably includes: using the new Mycobacterium aureum as the base bacteria, knocking out the KstD1 / 2 / 3 gene, OtsA gene, TreS gene, TrsY gene, and LpqY gene in the base bacteria genome, and constructing a new Mycobacterium aureum engineered bacterium with high cell permeability. Further, the method also includes: knocking out one or more of the SugA gene, SugB gene, and SugC gene in the base bacteria genome, preferably the SugC gene.

[0011] Preferably, the Mycobacterium neoaurum is Mycobacterium neoaurum ATCC 25795.

[0012] The present invention also provides a new engineered Mycobacterium neoaurum constructed by the method. Preferably, the engineered Mycobacterium neoaurum is Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOtsA / TreS / TreY / LpqY / SugC.

[0013] The present invention also provides the use of the new Mycobacterium aureum engineered bacteria in the preparation of 9α-hydroxyandrost-4-ene-3,17-dione by microbial fermentation. Preferably, the use comprises: inoculating the new Mycobacterium aureum engineered bacteria into a fermentation medium containing sterols, and shaking and culturing at 25 to 40° C. and 100 to 300 rpm for 48 to 168 hours to obtain the 9α-hydroxyandrost-4-ene-3,17-dione.

[0014] The beneficial effects of the present invention are as follows: the present invention destroys the endogenous synthesis ability and exogenous circulation ability of trehalose of Mycobacterium neogoldensis through gene knockout, which not only improves the permeability of the cell and increases the molar conversion rate of 9-OHAD from 44.7% to 65.7%, but also increases the extracellular production of 9-OHAD by changing the components in the cell wall, and finally increases the extracellular production of 9-OHAD from 12% to 32.1%, which is convenient for subsequent separation and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure is a schematic diagram of the endogenous synthesis and exogenous circulation of trehalose in the new Mycobacterium aureum of the present invention.

[0016] Figure 2 Schematic diagram of the construction of the knockout plasmid of the present invention.

[0017] Figure 3 It is the electrophoresis diagram of gene knockout in the embodiment of the present invention; Lane M is a marker, Lanes 1 to 7 are OtsA / TreS / TreY / LpqYSugA / SugB / SugC gene knockout lanes, and Lane 8 is a control lane for genes not being knocked out.

[0018] Figure 4 The figures are the results of the change of cell permeability when the endogenous trehalose synthesis capacity is damaged to different degrees in the examples of the present invention; A is the FDA labeling method, and B is the 4-cholesten-3-one labeling method.

[0019] Figure 5 This is a graph showing the yield of 9-OHAD when the endogenous trehalose synthesis capacity is destroyed to different degrees in the examples of the present invention.

[0020] Figure 6 The figures are the results of the changes in cell permeability when the circulating capacity of exogenous trehalose is damaged to different degrees in the examples of the present invention; A is the FDA labeling method, and B is the 4-cholesten-3-one labeling method.

[0021] Figure 7 This is a graph showing the yield of 9-OHAD when the recycling capacity of exogenous trehalose is damaged to varying degrees in the examples of the present invention.

[0022] Figure 8 This is a graph analyzing the changes in the acid layer components of Mycobacterium neogoldenum in an embodiment of the present invention.

[0023] Fig. 9 This is a diagram showing the intracellular and extracellular distribution of 9-OHAD generated by biotransformation of Mycobacterium neogold in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] 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.

[0025] Preparation of culture medium and solution required:

[0026] (1) LB medium: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L.

[0027] (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.

[0028] (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, hydroxypropyl-β-cyclodextrin (HP-β-CD) 40 g / L. In the bioconversion process, 20 g / L phytosterol f was added as a raw material.

[0029] (4) MM medium: NH4NO3 2.0 g / L, K2HPO4 0.5 g / L, MgSO4 0.5 g / L and ammonium ferric citrate 0.05 g / L. For cell growth and cell permeability phenotype identification, add appropriate concentrations of fluorescein diacetate (FDA) or 4-cholesten-3-one.

[0030] (5) LB solid plate medium: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, agar 20 g / L.

[0031] (6) 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.

[0032] (7) 10% glycerol: Take 10 mL of glycerol and add sterile water to make up to 100 mL.

[0033] (8) 0.1 M CaCl2 solution: Weigh 11.1 g of CaCl2 powder and add water to make up to 1 L.

[0034] (9) 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 mixture to a volumetric flask and adjust the volume to 1 L.

[0035] (10) 50% sucrose solution: Weigh 50 g of sucrose, dissolve it in sterile water and make up to 100 mL.

[0036] The substrate phytosterols were purchased from COFCO Corporation, and the product 9-OHAD standard was purchased from Beijing Bailingwei Technology Co., Ltd.

[0037] Example 1: Construction of gene knockout plasmid and screening of knockout strains

[0038] (1) Construction of knockout plasmid

[0039] The construction of plasmids includes pNS-ΔKstD1, pNS-ΔKstD2, pNS-ΔKstD3, pNS-ΔOtsA, pNS-ΔTreY, pNS-ΔTreS, pNS-ΔLpqY, pNS-ΔSugC, and pNS-ΔSugA / B / C. Taking the knockout plasmid pNS-ΔOtsA as an example, first locate the region where the target gene OtsA is located, and then select a fragment of about 1000bp in length from the upstream and downstream sequences as the arms of homologous exchange during gene knockout. It is worth noting that a certain length of overlap region must be retained between the homologous exchange arm and the target gene, and the ideal length is about 150bp. A pair of vector primers Pvector f / r and two pairs of knockout primers D OtsA U f / r and D OtsA D f / r were designed. D OtsA U f was a forward primer for the upstream homology arm of the knockout target gene OtsA, D OtsA U r was a reverse primer for the upstream homology exchange arm of the knockout target gene OtsA, D OtsA Df was a forward primer for the downstream homology arm of the knockout target gene OtsA, and D OtsA D r was a reverse primer for the downstream homology exchange arm of the knockout target gene OtsA. There was a homology of about 20 bp between the upstream and downstream homology arm primers, and both had a homology of about 20 bp with the vector connection position, and were convenient for subsequent one-step cloning and connection. The relevant sequence was cloned from the template and matched with the corresponding target fragment after comparison. The sequencing analysis showed that the amplified sequence was the same as the ideal sequence. The nucleotide sequences of the knockout genes OtsA, TreY, TreS, LpqY, SugA, SugB, and SugC are shown in SEQ ID NO. 1 to NO. 6. The primer sequences are shown in Table 1.

[0040] Table 1. Primers for gene knockout plasmid construction

[0041]

[0042] The target fragment and vector fragment after 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-ΔOtsA. See the schematic diagram. Figure 2 . Transform the ligation product into the pre-made DH5α E. coli competent medium. Use Kan resistance screening to select transformants for verification, and then extract the plasmid using a plasmid extraction kit.

[0043] Strain WM00 was derived from the original strain ATCC 25795 by disrupting three endogenous KstD enzymes (3-sterone-Δ1-dehydrogenase), and subsequent mutants WM01-WM07 were all modified on the basis of WM00. The following strains were obtained: WM00: Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3;

[0044] WM01: Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOtsA (abbreviated as ΔOtsA in the figures);

[0045] WM02: Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔTreS (abbreviated as ΔTreS in the figures);

[0046] WM03: Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3TreY (abbreviated as ΔTreY in the figures);

[0047] WM04: Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOtsA / TreS / TreY (abbreviated as ΔOtsATreSY in the figure);

[0048] WM05: Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOtsA / TreS / TreY / LpqY (abbreviated as ΔOtsATreSYY in the figure);

[0049] WM06: Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOtsA / TreS / TreY / LpqY / SugC (abbreviated as ΔOtsATreSYYC in the figure);

[0050] WM07: Mycobacterium neoaurum ATCC 25795 ΔKstD1 / 2 / 3 ΔOtsA / TreS / TreY / LpqY / SugA / B / C (abbreviated as ΔOtsATreSYYABC in the figure).

[0051] (2) Screening of knockout strains

[0052] Mycobacteria were cultured in M1 medium to mid-logarithmic phase, the bacterial solution was collected and the medium was removed by centrifugation, and the new gold mycobacterium competent state was obtained by washing with sterile water and 10% glycerol (three times). After the above knockout plasmid was treated with alkali, it was added to the mycobacterium competent state (dissolved on ice) and allowed to stand at 4°C for 10 minutes; the voltage of the electroporator was set to 2.5kV, the aperture of the electric shock cup was selected to be 2mm, and the electric shock frequency was confirmed to be within the range of 3.5-5ms for two consecutive electric shocks, and then 800μL of pre-cooled LB medium was added. After fully suspending the bottom bacteria, it was transferred to a sterile centrifuge tube. Incubate at 37°C / 180rpm with shaking for 4h, discard the supernatant after centrifugation, resuspend and apply it to Kan solid medium, and invert and culture at 30°C for 3-5 days.

[0053] The transformants obtained on the plate were placed in a 37°C / 180rpm shaker for 12 hours, and 100 μL was transferred to a sucrose plate (LB without NaCl containing 8% sucrose) to screen the double exchange transformants, and incubated at 30°C for 3-5 days. The transformants with good colony status were selected and copied on Kan resistance plates and non-resistance plates at the same time. The transformants that did not grow on Kan plates and grew on non-resistance plates were screened by PCR, and the PCR products were verified by electrophoresis and sequencing (such as Figure 3 ). Finally, a knockout strain was obtained.

[0054]

Performance test

[0055] Mycobacterium neoformans cell permeability assay

[0056] (1) Fluorescence labeling method to test the total permeability of Mycobacterium neoformans cells

[0057] The total cell permeability of Mycobacterium neogoldensis can be tested by the fluorescein diacetate (FDA) labeling method. Mycobacterium neogoldensis was inoculated in MM medium and cultured until the cell OD600 reached 0.4-0.5, then the cells were collected and washed with phosphate. The obtained cells were resuspended in 4.5 mL of phosphate buffer, mixed with 0.5 mL of FDA acetone solution (2 mg / mL), and incubated at 32°C for 30 minutes. The detection was performed using an ELISA reader, with the maximum excitation wavelength of 485 nm and the emission wavelength of 538 nm. The results of cell permeability detection of different mutant strains are shown in Figure 2. Figure 4 and Figure 6 shown.

[0058] (2) 4-Cholesten-3-one intracellular content method to test the cell-specific permeability of Mycobacterium neoformans

[0059] The cell permeability of Mycobacterium neogoldensis to steroidal compounds can be characterized by testing the cell permeability of 4-cholestene-3-one. The stored Mycobacterium neogoldensis was inoculated into 30 mL of M2 medium containing 2.0 g / L 4-cholestene-3-one. Then, after 12 hours of culture, 5 mL of the culture was centrifuged at 12,000 rpm for 10 minutes and then washed twice with 1.0 mL of double distilled water (ddH2O). The residual 4-cholestene-3-one in the culture medium was removed by washing with 1.0 mL of a mixture of petroleum ether and ethyl acetate (6:4, v / v). The cells (50 mg, wet weight) were then resuspended in 1.0 mL of a mixture of acetonitrile and ddH2O (7:3, v / v), and the cell structure was disrupted using a FastPrep-24 instrument. The supernatant was collected by centrifugation and the intracellular content of 4-cholestene-3-one was determined by testing the supernatant. A high performance liquid chromatography (HPLC) system equipped with a reverse phase C18 column (250 mm × 4.6 mm) was used for the detection. The detection wavelength was 210 nm, and the mobile phase was a mixture of methanol and water (7:3, v / v). Figure 4 and Figure 6 shown.

[0060] Analysis on the changes of acid layer components of Mycobacterium neogoldensis

[0061] (1) Extraction of bacterial acid components

[0062] Taking any of the mutant strains WM00-WM07 as an example, 50 mg of new gold mycobacterium cells were centrifuged and collected, and a mixture of 0.5 mL of methanol and chloroform (2:1, v / v) was added to resuspend the collected cells. The uniform mixture was then incubated at 60 ° C for 2 hours and then centrifuged at 12000 rpm for 10 minutes. Polar lipids including TMM and TDM were dissolved in the supernatant by the above measures. Next, 500 μL of 10% tetrabutylammonium hydroxide was added to the above-mentioned defatted cells and heated at 100 ° C overnight. After cooling, 500 μL of dd H2O, 250 μL of dichloromethane, and 62.5 μL of iodomethane were added to the mixture. Then, the mixture was stirred for 30 minutes and then centrifuged to remove the upper solution. The lower solution was washed with hydrochloric acid and dd H2O. After dehydration, the mixture was dried, and the residue was redissolved in a mixture of toluene (0.2 mL) and acetonitrile (0.3 mL), and then incubated for 1 hour at 4° C. MAME was obtained by centrifugation at 12,000 rpm for 10 minutes.

[0063] (2) Preliminary detection of components by TLC

[0064] The obtained bacterial acid components were analyzed by TLC experiment. The treated samples were sucked with a capillary pipette (10 cm long) with an inner diameter of 0.3 mm and spotted on a silica gel chromatography plate. The samples were spotted 1 cm away from the edge of the chromatography plate, with a spacing of 0.7 cm between the spots. Before development, the chromatography cylinder needs to be saturated with a developing agent for 30 minutes. The developing agent used was ethyl acetate: methanol = 8:2. The results of the abundance of each sample point detected by TLC were analyzed, and the changes in the TMM:TDM components in the obtained mutant strains were more obvious. The results are as follows Figure 8 The results verified that the increase in TMM content in the cell wall will directly affect the distribution of 9-OHAD inside and outside the cell, ultimately leading to an increase in the extracellular production of 9-OHAD from 12% (WM00) to 32.1% (WM06), which is convenient for subsequent separation and purification.

[0065] Example 2: Application of different mutant strains in the preparation of 9α-hydroxyandrost-4-ene-3,17-dione by microbial fermentation

[0066] Take a loop of bacterial liquid from the glycerol tubes containing strains WM00-WM07, streak on LB solid medium, and culture at 30oC for 48h; pick a single clone in 5mL LB liquid, shake culture at 30℃ for 36h; transfer to 90mL M2 medium at a ratio of 2%, shake culture at 30℃ for 12h, add 10mL of 200g / L sterol solution emulsified with hydroxypropyl cyclodextrin, shake culture at 32℃-180rpm; sample after 96h and 120h of reaction, and the reaction lasts for 168h. When discussing the overall production efficiency of 9-OHAD, the cell transformation suspension is extracted with an equal amount of ethyl acetate. When discussing the production of intracellular 9-OHAD, the cell centrifugation resuspension product is extracted with an equal amount of ethyl acetate after the cells are centrifuged and resuspended. When discussing the production of extracellular 9-OHAD, the supernatant is obtained by centrifugation and then extracted with an equal amount of ethyl acetate. The above three samples are dried to obtain crude products. Add 5 mL of ethyl acetate to the obtained 1 mL sample for extraction, shake and mix for 30 minutes, take 200 μL of the upper organic phase in an EP tube, heat and evaporate, and add 0.8 mL of methanol to dissolve. 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 (7:3, v / v). The conversion results are as follows: Figure 4 , Figure 6 and Fig. 9 The cell permeability of strain WM06 was further improved compared with that of WM04, and its 9-OHAD production increased from 44.7% (WM00) to 65.7%, reaching 9.6 g / L when the phytosterol concentration in the shake flask was 20 g / L.

[0067] The embodiments described above are merely descriptions of preferred implementation modes 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 improving the permeability of Mycobacterium neogoldensis cells, characterized in that: include: Using new Mycobacterium aureum as the chassis bacterium, the KstD1 / 2 / 3 genes in the chassis bacterium genome were knocked out, the endogenous synthesis pathway of trehalose in the chassis bacterium was destroyed, the exogenous circulation ability of trehalose in the chassis bacterium was destroyed, and the new Mycobacterium aureus engineered bacteria with high cell permeability were constructed.

2. The method according to claim 1, characterized in that The method for destroying the endogenous synthesis pathway of trehalose in the chassis bacteria comprises: knocking out one or more of the OtsA gene, the TreS gene, and the TrsY gene in the chassis bacteria genome.

3. The method according to claim 1, characterized in that The method for destroying the exogenous trehalose circulation ability in the chassis bacteria comprises: knocking out one or more of the LpqY gene, the SugA gene, the SugB gene, and the SugC gene in the chassis bacteria genome.

4. The method according to claim 1, characterized in that include: Using Mycobacterium aureum as the base bacterium, the KstD1 / 2 / 3 genes, OtsA gene, TreS gene, TrsY gene, and LpqY gene in the base bacterium genome were knocked out to construct a new Mycobacterium aureum engineered bacterium with high cell permeability.

5. The method according to claim 4, characterized in that Also includes: Knock out one or more of the SugA gene, SugB gene, and SugC gene in the genome of the chassis bacteria.

6. The method according to claim 1, characterized in that The new gold mycobacterium is Mycobacteriumneoaurum ATCC 25795.

7. The new engineered Mycobacterium aureus strain constructed by the method according to any one of claims 1 to 6.

8. The new Mycobacterium aureus engineered bacterium according to claim 7, characterized in that: The engineered Mycobacterium neoaurum is Mycobacterium neoaurum ATCC 25795ΔKstD1 / 2 / 3ΔOtsA / TreS / TreY / LpqY / SugC.

9. Use of the new engineered Mycobacterium aureus strain as claimed in claim 7 in the preparation of 9α-hydroxyandrost-4-ene-3,17-dione by microbial fermentation.

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

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