Effects Of Oxidative Respiratory Chain Pathway And Pyruvate Metabolism Enhancement On Sterol Transformation In Mycobacterium Neoaurum

Androstenedione（AD）is the core intermediate of steroid drug synthesis.In the process of degradation of phytosterol side chain by microbial fermentation to produce AD,there are problems such as imbalance of cofactor metabolism,low electron transfer efficiency and toxic effects of pyruvate accumulation,which lead to long transformation cycle of bacteria and decreased production intensity.Because of the complexity of the sterol metabolic pathways and the lack of awareness of sterol metabolic pathway,the enhanced productivity phytosterol production strain method is aimed at the key enzyme gene modification of metabolic pathways,concentrated in a single enzyme and a few on the increase or decrease of enzyme function,ignoring the overall regulation of metabolic pathways are often the cause of the decrease in the strength of production,or by-products.In this paper,Mycolicibacterium Neoaurum TCCC 11978（MNR M3）,which is an excellent AD producing strain,is taken as the research object,and the practical problems of too long transformation cycle and low production intensity are focused on the regulation of the electron transport chain of the strain and the metabolism of pyruvate,the by-product.A coordinated regulation strategy based on electron transport and pyruvate metabolism was established.On this basis,metabolic engineering,fermentation engineering and other technologies were integrated to enhance strain production intensity and product generation rate,providing new ideas for the construction of AD production strains.The research results of this paper are as follows.Through bioinformatics analysis,key enzymes of NADH respiratory chain and FADH₂respiratory chain in two electron transport pathways of sterol metabolism pathway were found.The effects of enhancing NADH oxidase and SDHX succinate dehydrogenase on bacterial growth and phytosterol biotransformation were compared and analyzed.Overexpression of NADH oxidase and SDHX（succinate dehydrogenase）increased the expression levels of complexⅠand complexⅡ.The overexpression of complexⅠrelated（poxA）NADH oxidase increased the activity of complexⅠby 1.53 times,the yield of AD increased by 27.39%,and the conversion of phytosterol was 91.06%.In SDH dehydrogenase related to complexⅡ,overexpression of sdh B subunit increased the enzyme activity of final complexⅡby 1.13 times,AD production increased by 24.42%,and the final conversion rate of phytosterol was 88.09%.It was found that sterol metabolism was correlated with electron transport chain metabolic pathway,and strengthening key enzymes in electron transport chain pathway was beneficial to plant sterol biotransformation of Mycolicibacterium neoaurum MNR M3,in which the respiratory chain of complexⅠwas more significantly affected.