Construction Of High-yield Production Mycobacteria Of Dehydroepiandrosterone Through Analysis Of Metabolic Pathways And Gene Knockout

Dehydroepiandrosterone(DHEA)is an important steroidal drug intermediate.It has many medicinal activities and can synthesize a variety of steroidal drugs with high market and economic value.The production of DHEA mainly uses chemical synthesis,while other steroidal intermediates 4-AD and ADD have corresponding microbial transformation strains for biotransformation synthesis,and the yield is much higher than DHEA.There is currently no strain capable of directly transforming DHEA,so the microbial transformation method for DHEA remains to be studied.Our group obtained a strain of industrially produced steroidal intermediate 4-AD.This paper aims to produce DHEA by modifying the metabolic pathway of mycobacteria.The whole genome sequencing of mycobacteria is carried out to analyze and deduct its metabolism.The steroidal pathway,through molecular biology and genetic engineering methods,studied the key enzymes Cho,Hsd,KSI in the pathway,and constructed ChoM1,ChoM2,Hsd multiple gene knockout mycobacteria.The main contents are summarized as follows:1.In this paper,whole-genome sequencing of high-yield 4-AD strains was carried out,and the genome was annotated by bioinformatics and genomics,and the metabolic sterol compound pathway was deduced.The mycobacteria were found to have a sterol nucleus and a branched-chain degrading enzyme system.Sterol alcohol degrading enzymes include cholesterol oxidase(Cho),3?-sterol dehydrogenase(Hsd),isomerase(KSI),1-2 dehydrogenase(KstD),9OH-hydroxylase(Ksh),Branched chain degrading enzymes mainly include monooxygenase and fatty chain ?-oxidoreductase.2.Crystals of KSI291 protein were obtained by protein purification and crystallization screening.The structure data was collected and optimized by X-ray diffraction method.The structure of KSI291 was compared with the structure of homologous protein.It was found that KSI291 and the homologous protein have similar overall structure,and the key amino acids in the active center are more mutated,and the water molecules in the cavity are Residues S16 and Y57 of KSI291 are capable of forming hydrogen bonds with the substrate,so that KSI291 retains the catalytic function.Based on this structural analysis,we propose the catalytic mechanism of KSI291.3.By analyzing the key enzymes in the pathway,we constructed ChoM1,ChoM2 and Hsd knockout vectors,and obtained three single-knockout mycobacteria,?ChoM1,?ChoM2 and ?Hsd by homologous double-crossover recombination.On this basis,three double-knockout type mycobacteria,?ChoM1-ChoM2,?ChoM1-Hsd,?ChoM2-Hsd,and a three-gene knockout type Mycobacterium,?ChoM1-ChoM2-Hsd,were obtained.The fermentation of the three gene knockout mycobacteria showed that other unknown enzymes could participate in the oxidation reaction of the hydroxyl group at the C3 position of the sterol molecule.