Efficient Production Of 9?-hydroxy Androstenedione By Metabolic Engineering Modification Of Mycobacterium Sp.

The steroid drug is the second-largest drug in the world with important market prospects,the market share of which has increased year by year.9a-Hydroxyandrost-4-ene-3,17-dione(9a-OH-AD)is an important steroid intermediate,which was used in the synthesis of anti-inflammatory and anti-allergic glucocorticoids.At present,the production of 9a-OH-AD is mostly obtained by microbial transformation of phytosterols.Although the concentration of 9a-OH-AD can be increased by strain screening and mutagenesis,there are still some problems such as the lower utilization of the substrate and the bad conversion efficiency.The reasons include that the microbial metabolic pathway of phytosterols and the function of key enzymes in the pathway are not clear.Therefore,a wild strain(Mycobacterium sp.LY-1)screened and preserved in our laboratory was used as the research object in this study,and it could transform phytosterols to 9a-OH-AD.Through the whole genome sequencing,the genes of phytosterol metabolism were analyzed and compared.Combined with the analysis of differential transcriptional level,the related enzymes correlation with the synthesis of 9a-OH-AD were identified.Then,several key enzyme genes involved with the synthesis of 9a-OH-AD in the sterol metabolic pathway were enhanced expression by metabolic engineering.Eventually a high efficient recombinant strain was constructed,and the concentration of substrate and the molar yield of 9a-OH-AD were further increased after optimization of the transformation process.The main research results are listed as follows:(1)Analysis of phytosterol metabolic pathways of Mycobacterium sp.LY-1.Whole genome sequencing of Mycoacterium sp.LY-1 was performed,and 53 contigs were obtained through gene assembly.The results of gene sequence prediction and functional annotation indicated that the functional proteins of Mycobacterium sp.LY-1 are mainly distributed in[Q](synthesis and metabolism of secondary metabolites),[C](energy generation and transportation)and[K](transcription).The gene clusters of sterol metabolism pathways in My cobacterium sp.LY-1 were compared with those in reported sterol-degrading strains Mycobacterium tuberculosis H37Rv and M.neoaurum VKM Ac-1817D.Sterol metabolism related enzymes contained Cho(cholesterol oxidase),17?-Hsd(17?-Hydroxysteroid dehydrogenase/isomerase),HsdA/HsdB(?-hydroxyacyl-CoA dehydrogenase),KshA(3-Ketosteroid-9?-hydroxy oxygenase),KshB(3-Ketosteroid-9a-hydroxy reductase),KstD(3-Ketosteroid-?1-dehydrogenase)and so on.And the sterol metabolism pathway of 9a-OH-AD synthesized from Mycobacterium sp.LY-1 was determined.(2)Determination of key enzymes for phytosterol metabolic pathway.Based on the analysis of phytosterol metabolic pathway,the fermentation process of phytosterols by Mycobacterium sp.LY-1 was compared under two conditions including with or without soybean oil.According to the correlation characteristics between cell growth and product synthesis,the samples were collected for subsequent differential transcription analysis at the three points,namely early stage(60 h),middle stage(84 h)and later stage(168 h)of transformation.The qRT-PCR technique was used to analyze and compare the transcriptional levels of 37 sterol metabolism genes under the two conditions.It is indicated that 9 enzyme genes were significantly different at the transcriptional level and were key enzymes in the sterol metabolism pathway.Two genes were key enzymes for degradation pathway KstD,and the others were key enzymes for the synthesis pathway,including Cho,FadE30,HsdB,KshA2,KshB,Hsdl,and FadA5.(3)Metabolic engineering of Mycobacterium sp.LY-1.In the wild strain Mycoacterium sp.LY-1,single enhanced expression of 7 key enzymes were performed in synthetic pathways,namely ChO,FadE30,HsdB,KshA2,KshB,Hsdl,and FadA5.As a result,the ability of recombinant strain for transforming phytosterols to 9a-OH-AD was increased.Among them,the molar yield of the strain LY-1-kshA2 was the highest.When the substrate concentration was 15 g·L-1,the molar yield of 9a-OH-AD could reach 40.8%.In order to improve the ability of recombinant strain to product 9a-OH-AD further,on the basis of the single gene enhanced expression,the recombinant strain LY-1-kshA2-kshB-hsdB was constructed,which was a combination enhanced expression of three genes,kshA2,kshB and hsdB.The results showed that the molar yield of 9a-OH-AD by the recombinant strain LY-1-kshA2-kshB-hsdB,still reached 43.4%.(4)Optimization of the transformation process for recombinant strains.The recombinant strain LY-1-kshA2-kshB-hsdB was used as the study object.The influence of culture media components such as carbon source,nitrogen source,phosphate and metal ions for transforming phytosterols to 9a-OH-AD were investigated.An orthogonal test was used to optimize the components of the medium.Finally,the optimum medium for the recombinant strains LY-1-kshA2-kshB-hsdB was determined as corn steep liquor 30·g L-1,KNO3 4.0 g·L-1,NaH2PO4 1.2 g·L-1,FeSO4 0.075 g·L-1 and CaCl2 0.10 g·L-1.Based on above work,the culture conditions were optimized,and the optimal conditions for the recombinant strains were determined as pH 8.0,Inoculation amount 2%,temperature 30?,and rotation speed 120 rpm.Under the optimum culture conditions,the concentration of substrate phytosterols increased from 15 g·L-1 to 30 g·L-1,and the yield of 9a-OH-AD was stable at 41%.