Metabolic Engineering Modification Of Mycobacteria For Androstadienedione Efficient Production From Phytosterols

Steroid drugs are the second largest class of drugs after antibiotics.They play an irreplaceable role in the treatment of allergic diseases,rheumatoid arthritis,contraception and other diseases,and have broad application market.Androstadienedione(ADD)is an important precursor for the synthesis of steroid hormone drugs,which can be obtained from the transformation of phytosterols by the microorganism Mycobacterium.At present,although researchers have used metabolic engineering modification to improve the level of ADD produced by Mycobacteria,there are still problems such as low substrate conversion rate and low product yield.The reasons might be the low solubility of the substrate,the weak uptake of sterols by cells,and the low efficiency of strain metabolic engineering and regulation.Therefore,we screened the endogenous expression elements of Mycobacteria,knocked out the β-ketoacyl acyl carrier protein synthase gene related to cell wall synthesis,and used high-strength expression elements to express sterol metabolism-related enzyme genes.At the same time,the heterologous expression of the hemoglobin gene of Vitreoscilla was performed to explore the limiting factors affecting the ability of strain to synthesize ADD.On this basis,the factors that significantly increase the concentration of ADD were combined and expressed to construct high-yielding recombinant strains.And the ability of the recombinant strains to produce ADD was further improved through the optimization of the transformation process.The main research results of this work are as follows:(1)Screening of endogenous expression elements of Mycobacteria.According to the genome transcriptome level data of Mycobacteria,combined with the promoter online prediction website BDGP,the endogenous expression elements were screened.The selected endogenous expression elements were characterized by sequence analysis and strength characterization.The results showed that all of the 23 endogenous expression elements had bacterial conserved-10 and-35 regions,and the intensity of two expression elements were stronger than the reported hsp60 expression elements.This work could provide a molecular element library for the expression and regulation of key enzyme genes in Mycobacteria.(2)Knockout of a key enzyme β-ketoacyl acyl carrier protein synthase gene(KasB)for cell wall synthesis of Mycobacteria.The enzyme gene KasB is related to the cell wall synthesis of Mycobacteria,and knocking out the KasB gene can increase the uptake rate of the substrate phytosterol by cells.In this study,the KasB gene of Mycobacterium neoauricum LY-2 was knocked out by traditional knockout method,and the growth and ADD production characteristics of the mutant strain were studied.The results showed that the growth of mutant bacteria was not affected,and the concentration of ADD also increased from 3.51 g/L to 4.25g/L.(3)Determination of the key enzymes that limit the rate of ADD synthesis in Mycobacterium and heterologous expression of hemoglobin gene(vgb)from Vitreoscilla.In Mycobacterium neoauricum LY-2,the key enzymes and regulators of sterol metabolism including cholesterol oxidase(chom),C27 monooxygenase(smo),hydroxy-Co A dehydrogenase(hsd4A),3-sterol-delta 1-dehydrogenase(kstd),propyl-Co A carboxylase(pcc),transcriptional activator of PRP operon(prp R),cofactor of NADH dehydrogenase(ndh)were enhanced using high-strength expression elements to explore the rate-limiting steps of ADD synthesis by Mycobacterium neoauricum.The results showed that in addition to chom and smo,the enhanced expression of other key enzymes and regulatory factors had certain promoting effects on the increase of ADD concentration.Among them,the enhanced expression of hsd4 A and pcc significantly improved the concentration of ADD to 4.43 g/L and 4.30 g/L,which were26.21% and 22.5% higher than those of the original strain,respectively.Heterologous expression of hemoglobin gene from Vitreoscilla in Mycobacterium neoauricum LY-2 can effectively alleviate the dissolved oxygen limitation.When 10 g/L of phytosterol was used as the substrate,the ADD concentration of vgb heterologous expression strain reached 4.42 g/L.(4)Construction and process optimization of recombinant strains with efficient production of ADD.In order to further increase the concentration of ADD,based on the enhanced expression of a single gene in Mycobacterium neoaureus LY-2,a multi-gene expression plasmid of hsd4 A,pcc and vgb was constructed and electroporated into a mutant strain to construct a high-yielding recombinant strain LY-2-ΔKasB-hsd4A-pcc-vgb.The transformation results from phytosterols showed that the ADD concentration of the high-yielding recombinant strain increased to 4.97 g/L.Taking the high-yielding recombinant strain LY-2-ΔKasB-hsd4A-pcc-vgb as the research object,medium,types of co-solvents,and the ratio of sterols and co-solvents were optimized.The results showed that the concentration of ADD reached 5.46 g/L in the fermentation medium of Mycobacterium neoaureus B.When hydroxypropyl-β-cyclodextrin with a molar ratio of 2 to sterol was added into the fermentation medium of Mycobacterium neoaureus B,the ADD concentration reached 6.4 g/L,and when the substrate dosage was 15g/L,the ADD concentration of the strain reached 9.7 g/L,which is the highest level of ADD production in the current shaking flask.