Study On Plant Sterol Transfer And Side Chain Degradation In The Synthesis Of 9-OHAD In Mycobacterium

With the emergence of more and more diseases,steroid hormones have gradually developed into an important category of Chinese medicine.As we all know,steroid hormone is an important signal molecule in human body.It can regulate the normal physiological function of cells through binding with specific receptor proteins.Once missing,it will lead to dysfunction of organism.9-OHAD is an important class of steroidal precursors,which is an important prerequisite for the availability of various steroid drugs and can be obtained by microbial degradation of phytosterols.However,the hydrophobicity of phytosterols and the complexity of the side chain degrading enzyme catalysis limit the yield of 9-OHAD.In this paper,mycobacterium MS136 was used as the starting strain,and the phytosterol transport system and the side chain degradation process were studied by genetic engineering.At the same time,the effects of the passage and fermentation conditions on the transformation of phytosterols were studied and the efficient transformation of phytosterols was realized.Phytosterols transport processes require energy and transmembrane proteins to assist.The energy and transmembrane protein related genes,linked to the vector pmv261,were constructed to construct phytosterol transport systems.The 9-OHAD yield of mceG-yrbE4A-yrbE4 B,RmceG-sup4 A was 6.02 g /L,which was 19% higher than the original strain and the yield of the mixture was 13.2 g / L.The results showed that the transport system can improve the mass transfer process of phytosterols.Sterol side chain degradation is a multi-enzyme catalysis process involving more than 20 enzymes involved.Eleven different genetic engineering strains were constructed by searching for the key genes involved in the degradation of the side chains in the steroid metabolic gene cluster.Fermentation analysis showed that the yield of 9-OHAD was increased by 16%-18% compared with the original strain by fermentation of Rj125-fad19 and fad19.The results showed that the key genes of overexpression of lateral chain degradation could increase the ability of cell to transform phytosterols.The mycobacteria need to be passaged before they are activated by fermentation.The fermentation of the original strain proves that the yield of 9-OHAD reaches the maximum when it reaches the four generation,but there are no definite terms after the modification of the bacteria belongs to the first few generations.In the experiment,the transformation of the bacteria 3 times,recorded as two,three,four generations,and its were carried out fermentation transformation,the results found that the transformation of bacteria in the third generation,already have the strongest conversion capacity,and The fourth generation has no significant change relative to the third generation.