Improving Phytosterol Biotransformation By Co-expression Of MceG And VHb Of Mycobacteria

Steroid drugs have significant anti-allergy,anti-infection,anti-shock,and other physiological and pharmacological functions,which are just needed in anti-inflammatory and endocrine regulation,with a vast market prospect.China is the world’s largest producer of steroid hormone drugs and the world’s largest producer of steroid raw materials,of which 70% is used for export,with an annual output value of more than 10 billion yuan.Androstenedione(AD)as the core raw material medicine of steroid drugs,can synthesize most of the steroid hormone drugs.The establishment of a microbial transformation technology system with phytosterols as the substrate has laid a solid foundation for the industrial production of AD.Mycobacterium neoaurum has a strong metabolism ability of phytosterols,which has been transformed into an important strain for the industrial production of AD.Transforming phytosterols by M.neoaurum to produce AD is a complex metabolic process involving various cofactors,and its main metabolic step is theβ-like oxidative degradation process of the phytosterol side-chain.The transport efficiency of phytosterol production strains and dissolved oxygen supply are the main limiting factors for the efficient production of AD.In this thesis,the AD production strain Mycobacterium sp.LZ2(Msp)was used as a research object to improve the AD production efficiency by improving the phytosterol transport capacity and intracellular dissolved oxygen supply of the strain using genetic engineering.Mycobacteria relies on the Mce4 transporter for phytosterol uptake,and Mce G provides energy to Mce4 to play an important role in the active transport of phytosterols.The codon-optimized Vitreoscilla hemoglobin(VHb)and sterol-transporting ATPase(Mce G)genes are co-expressed in Msp.Studies on the productive performance of the recombinant tandem strain Msp-vgb/mce G showed that the tandem expression of Mce G and VHb genes promoted the growth,biotransformation capacity,and adaptation to dissolved oxygen limitation of the strain;it was able to reduce intracellular ROS levels by increasing intracellular catalase activity to ensure that the recombinant strain could achieve high cell viability.The study results on the transcript levels of various key enzyme genes of sterol metabolism showed that the expression levels of genes related to sterol transport,side-chain degradation,and propionyl-Co A metabolism in Msp/vgb-mce G were up-regulated at 48 h and 72 h compared to Msp.Among them,the expression of mce G was most significantly up-regulated at 48 h,which was 6.76-fold higher than that of Msp,indicating that mce G was effectively expressed.The immobilized repetitive fractionation fermentation method was used,and sugarcane bagasse was used as the immobilization vehicle.The conversion of Msp vgb-mce G reached 93.45% when the amount of bagasse was 5 g/L,which was the optimal amount to use.The immobilized replicate batch fermentation was carried out with 5 g/L bagasse as the immobilization carrier.The experimental results showed that the immobilized repetitive batch fermentation could advance the cycle of product generation,thus improving the production efficiency.Compared with single-batch immobilized fermentation,immobilized repetitive batch fermentation could shorten the total fermentation time from 60 days to 37 days.The average conversion efficiency was 0.069g/L/h,which was 1.77 times higher than free cells(0.039 g/L/h).