A genetically engineered strain for producing 9-OHAD, and its preparation method and application

Abstract

The present invention provides a genetically engineered strain for producing 9-OHAD, which is a strain of 3-sterol-1-Δ 1 The coding genes KstD1-5 of ‑dehydrogenase and Opccr of bifunctional reductase are knocked out; 17‑hydroxysteroid / 22‑OH‑BNC‑CoA dehydrogenase Hsd4A and side chain degradation gene FadE28‑29 are overexpressed at the same time; the starting strain of the 9‑OHAD production strain is actinomycetes. The present invention also provides a preparation method and application of the genetically engineered strain. The genetically engineered strain for producing 9‑OHAD of the present invention stably accumulates 9‑OHAD products, reduces the accumulation of by-products, improves the purity and yield of 9‑OHAD in the final product, and has a high conversion rate from raw materials to final products, which can reach more than 83%.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and biochemical engineering, and specifically relates to a high-yield 9α-hydroxyandrostane-4-ene-3,17-dione strain, and a preparation method and application thereof. Background Art

[0002] Steroid drugs are the second largest class of drugs after antibiotics, and play a vital role in the prevention and clinical application of various diseases such as immune system diseases, rheumatism, tumors and inflammation. Actinomycetes show obvious natural advantages in the utilization and metabolism of heterotypic biomass. For example, mycobacteria can use natural sterols or some other steroidal compounds as the only carbon and energy source. The interruption of the mycobacterial phytosterol decomposition pathway will lead to the excessive accumulation of some important intermediates, such as 9-OHAD, AD, BA, etc., which can be used as precursors for the production of steroid hormone drugs.

[0003] 9α-hydroxy androstenedione (9-OHAD) is an important intermediate in the industrial production of glucocorticoid drug synthesis. Due to the presence of the unique 9α hydroxyl group, it is relatively easy to dehydrogenate with the C11 position to generate C9,11-dehydrosteroids, which is more conducive to the introduction of 9α-halogenated-11β-hydroxylated steroids at the C9 position. 9-OHAD is commonly used to synthesize a variety of steroidal APIs such as hydrocortisone, dexamethasone, betamethasone, and triamcinolone. It is often used clinically as an anti-sex hormone, corticosteroid, and contraceptive drug, and has important commercial value. 9-OHAD can be obtained from steroids through two microbial transformation methods. One method is that the hydroxyl group is directly introduced into the C9 position of AD by a microorganism with hydroxylation function. For example, 3-ketosteroid 9α-hydroxylase (KshAB) from Mycobacterium neoaurum JC-12 and Mycobacterium sp. VKMAc-1817D was co-expressed in Bacillus subtilis and Escherichia coli, achieving efficient conversion of AD to 9-OHAD. However, due to the low activity of KSH hydroxylase, the hydrophobicity of AD and its toxicity to cells, the low dose of AD limits its large-scale application. Another method is to directly convert cheap phytosterols into 9-OHAD, which has become a research hotspot due to its simple separation process and economical and efficient production method. Although many microorganisms can convert sterols into 9-OHAD, there are still some defects. For example, the accumulation of by-products, the low utilization rate of phytosterols and the degradation of products have seriously limited the industrial application of these strains.

[0004] Microbial transformation of phytosterols to accumulate 9-OHAD is highly susceptible to 3-sterol-△ 1 -dehydrogenase (3-ketosteroid-Δ 1-dehydrogenase, referred to as KstD). Due to the presence of KstD, the introduction of 9α-hydroxyl into the steroidal polycyclic structure will form an extremely unstable 9-OHADD, which triggers the breakage of the steroidal B ring, so that the C9 hydroxysteroid is completely degraded by the microorganism. On the other hand, due to the complexity of the metabolic pathways, the C22 metabolic pathway and the C19 metabolic pathway often coexist in actinomycetes, so C19 and C22 metabolites accumulate simultaneously during the conversion of phytosterols. For example, Mycobacterium sp. VKMAc-1817D can effectively convert sitosterol into 9-OHAD, while producing some by-products, such as AD, 9-OHHP, 9,24-DHC, etc. These by-products with similar structures not only hinder the purification and refining process of 9-OHAD, but also significantly reduce the yield of 9-OHAD. In the metabolism of phytosterols, 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A and dual-acting reductase mnOpccr are two important gene targets that connect the C22 metabolic pathway and the C19 metabolic pathway. For example, after knocking out Hsd4A and KstD in M.neoaurium ATCC 25795, the C22 intermediate 4-HP was stably accumulated in the conversion of phytosterols. In addition, in the metabolism of sterols in M.neoaurium CCTCC AB2019054, the inactivation or overexpression of mnOpccr changed the metabolic flux between AD and 4-HP. Considering the competitiveness of the C9 and C22 pathways, inhibiting the C22 metabolic flux can reduce C22 byproducts and increase 9-OHAD production. Therefore, it is feasible to functionally modify Hsd4A and mnOpccr to develop a 9-OHAD production strain that can convert phytosterols into 9-OHAD without C22 byproducts.

[0005] At present, the problems of microbial transformation of plant sterols to produce 9-OHAD include low transformation ability of strains, a large variety of by-products and degradation, which seriously affect the production efficiency of 9-OHAD. Therefore, it is urgent to develop efficient and specific production strains to meet production needs. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a genetically engineered strain for producing 9-OHAD, which is used to solve the problems of low conversion ability and many types of by-products in the existing strain technology. Compared with the starting strain of the 9-OHAD production strain, the 3-sterol-△ 1-dehydrogenase encoding genes KstD1, KstD2, KstD3, KstD4 and KstD5 and bifunctional reductase Opccr encoding genes are knocked out; 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A and side chain degradation genes FadE28-29 are overexpressed; wherein the starting strain of the 9-OHAD production strain is actinomycetes. The genetically engineered strain of the present invention can stably accumulate 9-OHAD.

[0007] Preferably, the encoding genes of the 3-sterol-△1-dehydrogenase (KstD1, KstD2, KstD3, KstD4 and KstD5) have the nucleic acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, wherein the inactivation or knockout of these five genes can be achieved by common technical means such as CRISPR or homologous recombination.

[0008] Preferably, the coding sequence of the bifunctional reductase Opccr has the nucleotide sequence shown in SEQ ID NO:8 or a sequence with more than 80% homology to the nucleotide sequence shown in SEQ ID NO:8, wherein the inactivation or knockout of the Opccr gene can be achieved by common technical means such as CRISPR or homologous recombination.

[0009] As certain embodiments of the present invention, in the genetically engineered bacteria of the present invention, the actinomycetes are mycobacteria, nocardia, rhodococcus or arthrobacter. As certain embodiments of the present invention, in the genetically engineered bacteria of the present invention, the actinomycetes are Mycobacterium fortuitum ATCC 35855 (ATCC 35855) or Mycobacteriumfortuitum ATCC 6842 (ATCC 6842).

[0010] As certain embodiments of the present invention, in the genetically engineered bacteria of the present invention, the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase gene Hsd4A and the side chain degradation gene FadE28-29 overexpressed in the genetically engineered bacteria are recombined with another copy of the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase gene Hsd4A and the side chain degradation gene FadE28-29. As certain embodiments of the present invention, the gene sequence of the recombined 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase gene Hsd4A and the side chain degradation gene FadE28-29 is from M.neoaurum DSM 44074. As certain embodiments of the present invention, the overexpressed 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene sequence is shown in SEQ ID NO:7 or its degenerate sequence, and the overexpressed side chain degradation gene FadE28-29 gene sequence is shown in SEQ ID NO:10 or its degenerate sequence. Preferably, the coding sequence of the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A has a nucleotide sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology with the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:7. The Hsd4A may be derived from Mycobacterium, Rhodococcus, Arthrobacter or Nocardia. More preferably, the Hsd4A gene is derived from Mycobacterium neoaurum DSM 44074.

[0011] Preferably, the coding sequence of the side chain degradation related gene FadE28-29 has the nucleotide sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology with the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 10. The FadE28-29 can be derived from Mycobacterium, Rhodococcus, Arthrobacter or Nocardia. More preferably, the FadE28-29 gene is derived from Mycobacterium neoaurum DSM 44074.

[0012] As certain embodiments of the present invention, the gene sequences of the genes KstD1, KstD2, KstD3, KstD4 and KstD5 encoding 3-sterol-△1-dehydrogenase in the starting strain of the 9-OHAD production strain are respectively as shown in SEQ ID NO:2-SEQIDNO:6 or their degenerate sequences, and the gene sequence of the gene encoding the bifunctional reductase Opccr in the starting strain of the 9-OHAD production strain is as shown in SEQ ID NO:8 or its degenerate sequence.

[0013] To achieve the above-mentioned purpose and other related purposes, the present invention provides a method for preparing the genetically engineered bacteria, wherein the method comprises inactivating the genes encoding 3-sterol-△1-dehydrogenase KstD1-5 and the gene encoding bifunctional reductase Opccr in the genome of Mycobacterium fortuitum ATCC 35855 or Mycobacteriumfortuitum ATCC 6842, and simultaneously overexpressing the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and the side chain degradation gene FadE28-29.

[0014] The present invention achieves high-yield 9-OHAD by solving the degradation of 9-OHAD and reducing the content of by-products. All five KstDs were knocked out in ATCC 35855 to achieve stable accumulation of 9-OHAD. By knocking out Opccr and overexpressing Hsd4A, the metabolic flow of the C22 metabolic pathway was reduced, the accumulation of the by-product 9-OHHP was reduced, and the content of the by-product 9,24-DHC was reduced by overexpressing the side chain metabolic gene FadE28-29. Finally, the yield of 9-OHAD in the modified Mycobacterium fortuitum reached 12.21 g / L, and the molar yield was 84.41%.

[0015] As certain embodiments of the present invention, in the method of the present invention, by knocking out the 3-sterone-△ 1 -dehydrogenase encoding genes KstD1-5 and bifunctional reductase Opccr encoding genes to inactivate them. As certain embodiments of the present invention, the 3-sterone-△ 1 -dehydrogenase encoding genes KstD1~5 and the bifunctional reductase Opccr encoding gene.

[0016] As certain embodiments of the present invention, in the method described in the present invention, the overexpression of 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and side chain degradation gene FadE28-29 is to recombinant another copy of 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and side chain degradation gene FadE28-29 in the genome of Mycobacterium fortuitum ATCC 35855 or Mycobacterium fortuitum ATCC 6842.

[0017] As certain embodiments of the present invention, in the method described in the present invention, the recombinant another copy of the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and the side chain degradation gene FadE28-29 are derived from M. neoaurum DSM44074.

[0018] As certain embodiments of the present invention, the nucleotide sequence of the recombinant 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene is as shown in SEQ ID NO:7 or its degenerate sequence, and the nucleotide sequence of the recombinant side chain degradation gene FadE28-29 is as shown in SEQ ID NO:10 or its degenerate sequence.

[0019] To achieve the above-mentioned purpose and other related purposes, the present invention provides the use of the genetically engineered bacteria in the fermentation production of 9-OHAD.

[0020] As certain embodiments of the present invention, in the application of the present invention, the fermentation medium produced by the fermentation contains 10-20g / L corn steep liquor powder, 8-15g / L glucose, 0.5-1g / L diammonium hydrogen phosphate, 5-8g / L sodium nitrate, 1-3g / L Tween 80, and 10-30g / L of a mixture of phytosterols and hydroxypropyl-β-cyclodextrin. As certain embodiments of the present invention, the fermentation medium produced by the fermentation contains 15g / L corn steep liquor powder, 10g / L glucose, 0.7g / L diammonium hydrogen phosphate, 6g / L sodium nitrate, 2.0g / L Tween 80, phytosterols and hydroxypropyl-β-cyclodextrin are mixed in a mass ratio of 1:3, and the pH is 7.5. As certain embodiments of the present invention, the fermentation medium contains 10g / L of phytosterols and hydroxypropyl-β-cyclodextrin mixed in a mass ratio of 1:3.

[0021] Preferably, the phytosterols in the fermentation medium are pre-mixed with hydroxypropyl-β-cyclodextrin in a certain ratio, stirred and ultrasonically treated to a uniform state, and then added to the medium. More preferably, the mass ratio of phytosterols to hydroxypropyl-β-cyclodextrin is 1:3.

[0022] As certain embodiments of the present invention, in the application of the present invention, the fermentation production conditions are fermentation temperature of 30°C-37°C, pH of 7-8, and fermentation time of 4-6 days. As certain embodiments of the present invention, the fermentation temperature is 30°C. As certain embodiments of the present invention, the pH of the fermentation is 7.5. As certain embodiments of the present invention, the fermentation time is 5 days.

[0023] The specific application and conditions can be adjusted according to actual requirements. The fermentation inoculation amount and seed solution concentration of the BA production bacteria. As certain embodiments of the present invention, the OD of the seed solution of the 9-OHAD production bacteria is600 The OD of the seed solution of the 9-OHAD producing bacteria is 8-12, and the volume of the seed solution is 10%-20% of the fermentation culture solution. 600 is 10.

[0024] As described above, the genetically engineered strain for producing 9-OHAD, and the preparation method and application thereof of the present invention have the following beneficial effects:

[0025] 1. The genetically engineered strain provided by the present invention can stably accumulate 9-OHAD products, solving the problem of 9-OHAD degradation during fermentation;

[0026] 2. The genetically engineered strain provided by the present invention simultaneously reduces the accumulation of byproducts 9-OHHP and 9,24-DHC, and improves the product purity and yield of 9-OHAD;

[0027] 3. It has realized the direct production of 9-OHAD from phytosterols through biotransformation. Compared with ATCC 35855, the 9-OHAD yield of MF-FA5020 has increased by 53.85%. Moreover, 20 g / L of phytosterols accumulated 12.21 g / L of 9-OHAD in 144 hours, and the molar yield of 9-OHAD was 83.68%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Shown is a schematic diagram of the suicide delivery plasmid pKADel of the present invention;

[0029] Figure 2 Schematic diagram showing the fermentation process and liquid chromatography analysis results of the KstD-deficient mutant strain MFΔkstD of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0031] The following describes the embodiments of the present invention through 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 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.

[0032] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0033] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0034] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0035] Example 1: Transformation of knockout plasmid

[0036] The present invention uses gene homologous recombination to edit the gene of ATCC 35855. The specific principle is described in the literature "Parish T, Stoker NG. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology. 2000, 146: 1969-75". In order to simplify the knockout plasmid construction steps, the inventors first combined the plasmids p2NIL and pGoal19 into a knockout plasmid pKADel. The pKADel plasmid is shown in Figure 1 , the specific steps are as follows:

[0037] 1. Circular PCR was performed on plasmid p2nIL using primers p2nIL-F / R to eliminate the redundant fragment between the PstI and SalI restriction sites and introduce a new AlfII restriction site to form plasmid p2nIL-1.

[0038] 2. Use primers p2n3-F / R to amplify the screening marker gene fragments ScaB and LacZ of plasmid pGoal19, and then insert the amplified marker gene fragments into the PacI and EcoRI restriction sites of plasmid p2nIL-1 to form plasmid pDCO-1.

[0039] 3. In order to avoid environmental microbial contamination and improve the efficiency of recombination screening, the sequence of the apramycin gene fragment (Pamic-apramycin) is shown in SEQ ID NO: 1, synthesized by GENEWIZ, and the fragment is amplified with primers pDCO-F / R and inserted into the SphI digestion site of plasmid pDCO-1 to generate a knockout plasmid pKADel, which is used for gene editing of ATCC35855. All PCR primer sequences involved in the present invention are shown in Table 1.

[0040] Table 1. Primer sequences involved in the present invention

[0041]

[0042]

[0043] Example 2 Construction of KstD gene knockout plasmid

[0044] 3-sterone-△ 1-The coding gene sequences of dehydrogenases (KstD1, KstD2, KstD3, KstD4 and KstD5) are shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the knockout plasmid of KstD was constructed according to the following method.

[0045] The nucleotide sequences of KstD1-6 are shown in SEQ ID NOs: 2-6, respectively. PCR amplification was performed using the genome of ATCC 35855 as a template. The upstream and downstream homology arm fragments of the gene to be knocked out were amplified using up-F / R and down-F / R, respectively. The amplified upstream and downstream homology arm fragments were then connected by Overlap PCR, and finally the connected homology arm large fragments were inserted into the AlfII restriction site of the knockout plasmid pKADel and the connection products were transformed into DH5α competent cells, coated with Kan and Apr double resistance LB plates (tryptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L, agar: 1.5%, Kan: 50 μg / ml, Apr: 50 μg / ml), and cultured overnight at 37°C. Single clone colonies were picked for colony PCR to verify the correct bands, and then cultured and extracted plasmids for sequencing to ensure that there were no mutations in the plasmid construction. Finally, five KstD gene knockout plasmids pKAdelΔk1, pKAdelΔk2, pKAdelΔk3, pKAdelΔk4 and pKAdelΔk5 were obtained.

[0046] Example 3: Construction and screening of KstD knockout strains

[0047] The constructed KstD gene knockout plasmid was electroporated into ATCC 35855 strain competent cells, incubated with shaking at 37°C for 2 h, and then spread on Kan and Apr double resistance LB plates (tryptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L, agar: 1.5%, Kan: 50 μg / ml, Apr: 50 μg / ml, X-Gal 400 μg / ml), and cultured overnight at 37°C for 72 h. Pick a blue single colony from the solid culture dish, use the primers up-F / down-R of the corresponding knockout gene for colony PCR verification, transfer the single clone that shows a long and a short double PCR band to 2ml LB liquid culture medium (tryptone: 10g / L, yeast extract: 5g / L, sodium chloride: 10g / L, Tween 80: 2g / L) and shake culture at 37℃ for 24h, then take part of the bacterial solution and streak culture on LB solid culture medium containing 20% ​​sucrose and 400μg / ml X-gal for the second screening. Pick a white colony on the sucrose plate and use the primers up-F / down-R for colony PCR verification, transfer the single clone that shows a single short PCR band to liquid LB culture medium, and take part of the bacterial solution for storage after culturing at 37℃ for about 36 hours. If the gene is successfully knocked out, the PCR product should be a single fragment of about 2.5kbp. The single gene knockout strains were electro-transfected and then screened for the next gene knockout strain, and finally five KstD1-5 all-knockout KstD-deficient strains MFΔkstD were obtained.

[0048] Example 4: Shake flask fermentation verification of KstD-deficient strain MFΔkstD

[0049] Seed culture medium: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L, Tween 80 2.0g / L, pH value adjusted to about 7.0, high pressure sterilization at 115℃ for 15min.

[0050] Fermentation medium: corn syrup powder 15g / L, glucose 10g / L, diammonium phosphate 0.7g / L, sodium nitrate 6g / L, Tween 80 2.0g / L, add phytosterol 10g / L, adjust pH to about 7.5, autoclave at 115℃ for 15min. During the preparation process, phytosterol and hydroxypropyl β-cyclodextrin should be mixed evenly with a small amount of culture medium in a mass ratio of 1:3, stirred for 20min, ultrasonically dissolved for 30min, and then packaged and sterilized.

[0051] Fermentation method:

[0052] The LB solid medium was cultured at 37° C. for 72 hours to activate the strains, and the MFΔkstD strain obtained in Example 3 and the wild-type Mycobacterium strain (ATCC 35855) were activated respectively; colonies with better growth status were selected from the activation plate and inoculated into the seed medium, and cultured at 180 rpm and 37° C. for 2 days to obtain a seed solution; the seed solution was transferred to a 250 mL baffled bottle containing 30 ml of fermentation medium at an inoculum amount of 10% (v / v), and fermented at 200 rpm and 30° C., and samples were taken every 24 hours during the entire fermentation process, and the fermentation culture was carried out for 120 hours.

[0053] like Figure 2 As shown in a, with a feed amount of 10 g / L phytosterols, ATCC 35855 can accumulate 3.87 g / L 9-OHAD in 48 h, and then 9-OHAD is rapidly degraded. When all five KstDs were inactivated, MFΔkstD could accumulate 5.28 g / L 9-OHAD in 48 h, and the yield of 9-OHAD increased by 36.43% compared with the starting strain ATCC 35855. More importantly, the product 9-OHAD did not decrease with the extension of fermentation time.

[0054] Figure 2 b is the fermentation liquid phase chromatogram of the MFΔkstD strain. It can be seen that in addition to the main product 9-OHAD, there are many by-products (9-OHHP, 9,24-DHC, etc.), which seriously affects the purity and yield of the product and is not conducive to subsequent separation and purification.

[0055] Example 5: Elimination of C22 byproduct 9-OHHP

[0056] The accumulation of C22 byproduct 9-OHHP indicates that some phytosterols flow toward the C22 metabolic pathway. From the following phytosterol metabolic pathway, it can be seen that Hsd4A and Opccr are two known key enzymes that determine the direction of phytosterol metabolic flow.

[0057]

[0058] In the above pathways: Phytosterol refers to plant sterols; Side chain degradation refers to the side chain degradation process; C19steroids refers to 19-carbon steroid compounds; C22steroids refers to 22-carbon steroid compounds.

[0059] Overexpression of Hsd4A will increase the metabolic flow of the C19 pathway, while knocking out Opccr will block the flow of some phytosterols to the C22 metabolic pathway. Therefore, the inventors overexpressed and / or knocked out these two key genes in an attempt to eliminate the accumulation of 9-OHHP.

[0060] 1. Overexpression of Hsd4A in the MFΔkstD mutant strain

[0061] In order to stably express the exogenous gene in the host bacteria, the integration plasmid pMV40 (see patent: 2022107293262) was selected to construct the exogenous gene expression system. The M.neoaurium ATCC 44074 genome was used as a template, and the primers hsd4a-F / R were used to amplify the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase (Hsd4A, the gene nucleic acid sequence is shown in SEQ ID NO: 7) from the M.neoaurium ATCC 44074 genome; after obtaining the gene fragment with 15bp homology arms on both sides of the AlfII and HindIII sites of the plasmid pMV40, it was infused and recombined with the linear plasmid pMV40 after digestion with AlfII and HindIII to obtain the recombinant expression integration plasmid p40-hsd4A. After sequencing verification, the plasmid p40-hsd4A was introduced into the competent strain MFΔkstD to obtain the Hsd4A overexpression strain MFΔkstD_Hsd4A.

[0062] The nucleotide sequence of Hsd4A is shown in SEQ ID NO:7.

[0063] 2. Knockout of Opccr in the MFΔkstD mutant strain

[0064] Through homology comparison, the inventors found an isoenzyme of mnOpccr (called Opccr) in the ATCC 35855 genome, and the coding gene sequence is shown in SEQ ID NO: 8. The inventors obtained the knockout plasmid pKAdelΔOpccr of Opccr using the gene knockout plasmid construction method described in Example 2. Similarly, the mutant strain MFΔkstDΔOpccr was obtained according to the gene knockout strain construction and screening method described in Example 3.

[0065] The nucleotide sequence of Opccr is shown in SEQ ID NO:8.

[0066] 3. In order to control the flow of phytosterols to the C19 metabolic pathway to the greatest extent, reduce the sterol metabolic flow of the C22 pathway, and remove the by-product 9-OHHP, the inventors overexpressed Hsd4A in the Opccr-deficient strain and obtained the strain MFΔkstDΔOpccr_Hsd4A.

[0067] According to the fermentation method of Example 4, the mutant strains MFΔkstD_Hsd4A, MFΔkstDΔOpccr and MFΔkstDΔOpccr_Hsd4A were fermented, and the liquid chromatography analysis results of the three mutant strains after fermentation for 120 hours are shown in Table 2. The inventors can see that 9-OHHP accounts for 5.46% of the total product in the fermentation product of the strain MFΔkstD, while the proportion of the byproduct 9-OHHP in the fermentation broth of the mutant strains MFΔkstD_Hsd4A and MFΔkstDΔOpccr did not decrease significantly, which shows that overexpressing Hsd4A alone or knocking out the Opccr gene does not reduce the metabolic flow of the C22 metabolic pathway and reduce the content of the byproduct 9-OHHP. However, in the product analysis of the mutant bacteria MFΔkstDΔOpccr_Hsd4A, it was found that the content of 9-OHHP was significantly reduced to only 1.02%, which shows that knocking out Opccr to block the C22 pathway and overexpressing Hsd4A to increase the metabolic flow of the C19 metabolic pathway can effectively remove the accumulation of C22 byproducts.

[0068] Table 2 Liquid chromatography analysis results of three mutant strains after 120 h of fermentation

[0069] strain 9-OHAD 9-OHHP 9,24-DHC other MFΔkstD 78.29 5.46 5.24 11.01 MFΔkstD_Hsd4A 79.46 4.87 4.77 10.09 MFΔkstDΔOpccr 77.13 5.04 4.98 12.85 MFΔkstDΔOpccr_Hsd4A 79.68 1.02 4.31 14.99

[0070] Example 6: Elimination of byproduct 9,24-DHC

[0071] FadE28-29 is a key enzyme gene in the degradation process of steroid side chains. Its lack of activity will lead to the accumulation of incomplete degradation byproducts of steroid side chains. The coding gene sequence of FadE28-29 in the ATCC35855 genome is shown in SEQ ID NO: 9. According to the experimental methods described in Examples 2 and 3, a knockout plasmid pKAdelΔFadE28-29 gene was constructed, and a FadE28-29-deficient strain MFΔkstDΔOpccrΔFadE28-29 was successfully constructed based on the MFΔkstDΔOpccr strain. In addition, the overexpression plasmid p40-fadE28-29 of FadE28-29 (from M.neoaurium ATCC 44074, the gene nucleic acid sequence is shown in SEQ ID NO: 10) was constructed according to the gene overexpression method described in Example 5, and the FadE28-29 overexpression strain MFΔkstDΔOpccr_FadE28-29 was obtained by electroporation into the MFΔkstDΔOpccr strain. Finally, the two strains were fermented and verified. Table 3 shows the results of fermentation liquid chromatography analysis of FadE28-29 gene knockout and overexpression strains. It can be seen that, relative to the strain MFΔkstDΔOpccr, when the FadE28-29 gene was knocked out, the accumulation of the main product 9-OHAD was reduced, and the content of the byproduct 9,24-DHC increased from 5.24% to 16.79%. In the transformation product of MFΔkstDΔOpcc_FadE28-29 strain, the content of 9,24-DHC was only 2.04%, and the purity of 9-OHAD increased from 78.6% to 81.46%.

[0072] Table 3 Fermentation liquid chromatography analysis results of FadE28-29 gene knockout and overexpression strains

[0073] strain 9-OHAD 9-OHHP 9,24-DHC other MFΔkstDΔOpccr 77.13 5.04 4.98 12.85 MFΔkstDΔOpccrΔFadE28-29 44.25 11.95 16.79 27.01 MFΔkstDΔOpccr_FadE28-29 81.46 4.89 2.04 11.61 MF-FA5020 85.12 0.89 2.13 11.86

[0074] The nucleotide sequence of FadE28-29 (from the ATCC35855 genome) is shown in SEQ ID NO: 9. The nucleotide sequence of FadE28-29 (from the DSM 44074 genome) is shown in SEQ ID NO: 10.

[0075] Example 7: Simultaneous reduction of byproducts 9-OHHP and 9,24-DHC and high-concentration fermentation

[0076] In Example 5 and Example 6, the contents of byproducts 9-OHHP and 9,24-DHC were reduced respectively. In order to improve the purity of the 9-OHAD product and reduce the influence of these two byproducts, the FadE28-29 gene fragment with SD sequence was amplified by primer 4aFad-F / R using plasmid p40-fadE28-29 as a template and inserted into the AflII / SalI restriction site of plasmid p40-Hsd4A to construct the co-expression plasmid p40-hsd4A&fadE28-29 of Hsd4A and FadE28-29. This co-expression plasmid was electroporated into MFΔkstDΔOpccr to obtain the final 9-OHAD production strain MF-FA5020. Table 3 shows the product content analysis results of strain MF-FA5020. It can be seen that in the fermentation broth, the accumulation of byproduct 9-OHHP was significantly inhibited, and the content of 9,24-DHC was also significantly reduced.

[0077] Example 8: Evaluation of the production capacity of mutant strain MF-FA5020

[0078] In order to evaluate the ability of MF-FA5020 to convert phytosterols into 9-OHAD, the inventors fermented with 10, 20 and 30 g / L of phytosterol feed respectively. Table 4 shows the conversion effect at different phytosterol concentrations. Specifically, MF-FA5020 can completely convert 10 g / L phytosterols into 6.12 g / L 9-OHAD within 96 hours, and the molar yield of 9-OHAD is 84.18%. Compared with ATCC 35855, the 9-OHAD yield of MF-FA5020 increased by 53.85%. 20 g / L phytosterols accumulated 12.21 g / L of 9-OHAD within 144 hours, and the molar yield of 9-OHAD was 83.68%; importantly, phytosterols were also completely consumed. However, when phytosterols increased to 30 g / L, even if the fermentation process was prolonged, phytosterols were not completely converted, and only 17.62 g / L of 9-OHAD was accumulated, the molar yield of 9-OHAD was 80.53%, and the molar conversion rate of phytosterols was 83.36%.

[0079] Table 4 Conversion effects of different substrate concentrations

[0080] Sterol concentration / (g / L) Sterol conversion rate / % 9-OHAD yield / (g / L) 9-OHAD molar yield / % 10 100％ 6.12 84.18％ 20 99.62％ 12.21 83.68％ 30 83.36％ 17.62 79.14％

[0081] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A genetically engineered strain for the production of 9-OHAD, characterized in that, Compared with the starting strain of the 9-OHAD production strain, the 3-sterone-△ 1 -dehydrogenase encoding genes KstD1, KstD2, KstD3, KstD4 and KstD5 and the encoding gene of the bifunctional reductase Opccr are knocked out; at the same time, 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A and side chain degradation gene FadE28-29 are overexpressed; wherein the starting strain of the 9-OHAD production strain is an actinomycete, and the actinomycete is Mycobacterium fortuitum ATCC 35855, the overexpressed 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene sequence is shown in SEQ ID NO:7 or its degenerate sequence, and the overexpressed side chain degradation gene FadE28-29 gene sequence is shown in SEQ ID NO:10 or its degenerate sequence.

2. The genetically engineered bacterium according to claim 1, characterized in that, The 3-sterol-△ 1 -dehydrogenase encoding genes KstD1, KstD2, KstD3, KstD4 and KstD5 have gene sequences as shown in SEQ ID NO:2-SEQ ID NO:6 or their degenerate sequences, respectively, and the gene encoding gene of the bifunctional reductase Opccr in the starting strain of the 9-OHAD production strain has a gene sequence as shown in SEQ ID NO:8 or its degenerate sequence.

3. A method for preparing the genetically engineered bacterium according to claim 1 or 2, characterized in that, The method is to convert 3-sterone-△ 1 The genes encoding KstD1-5 of the -dehydrogenase and Opccr of the bifunctional reductase were inactivated, while the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and the side chain degradation gene FadE28-29 were overexpressed.

4. The method according to claim 3, characterized in that, By knocking out the 3-sterone-Δ 1 -dehydrogenase encoding genes KstD1~5 and the bifunctional reductase Opccr encoding genes are inactivated.

5. The method according to claim 3, characterized in that, Knockout of the 3-sterone-Δ by CRISPR or homologous recombination 1 -dehydrogenase encoding genes KstD1~5 and the bifunctional reductase Opccr encoding gene.

6. The method according to claim 3, characterized in that, The overexpression of the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and the side-chain degradation gene FadE28-29 is the recombination of another copy of the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and the side-chain degradation gene FadE28-29 in the genome of Mycobacterium fortuitum ATCC 35855.

7. The method according to claim 6, characterized in that, The recombination of another copy of the 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene and the side-chain degradation gene FadE28-29 is derived from Mycobacterium neoaurum DSM44074.

8. The method according to claim 6, characterized in that, The nucleotide sequence of the recombinant 17-hydroxysteroid / 22-OH-BNC-CoA dehydrogenase Hsd4A gene is as shown in SEQ ID NO:7 or its degenerate sequence, and the nucleotide sequence of the recombinant side-chain degradation gene FadE28-29 is as shown in SEQ ID NO:10 or its degenerate sequence.

9. The application of the genetically engineered strain according to claim 1 or 2 in the fermentation production of 9-OHAD.

10. The application according to claim 9, characterized in that, The fermentation medium for the fermentation production contains 10-20 g / L of corn steep liquor powder, 8-15 g / L of glucose, 0.5-1 g / L of diammonium hydrogen phosphate, 5-8 g / L of sodium nitrate, 1-3 g / L of Tween 80, and 10-30 g / L of a mixture of phytosterol and hydroxypropyl-β-cyclodextrin.

11. The application according to claim 10, characterized in that, The fermentation medium for the fermentation production contains 15 g / L of corn steep liquor powder, 10 g / L of glucose, 0.7 g / L of diammonium hydrogen phosphate, 6 g / L of sodium nitrate, 2.0 g / L of Tween 80, and a mixture of phytosterol and hydroxypropyl-β-cyclodextrin in a mass ratio of 1:3, with a pH of 7.

5.

12. The application according to claim 10, characterized in that, The fermentation medium contains 10 g / L of a mixture of phytosterol and hydroxypropyl-β-cyclodextrin in a mass ratio of 1:

3.

13. The application according to any one of claims 9 to 12, characterized in that, The conditions for the fermentation production are a fermentation temperature of 30°C - 37°C, a pH of 7 - 8, and a fermentation time of 4 - 6 days.

14. The application according to claim 13, characterized in that, The fermentation temperature is 30°C.

15. The application according to claim 13, characterized in that, The pH was 7.

5.

16. The use according to claim 13, It is characterized in that The fermentation time is 5 days.

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