文献类型：期刊文献

英文题名：Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes

作者：Xiong, Liang-Bin[1];Liu, Hao-Hao[1];Xu, Li-Qin[1];Sun, Wan-Ju[1];Wang, Feng-Qing[1];Wei, Dong-Zhi[1]

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2017

卷号：16

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000401777500002)】；

基金：This work was supported by the National Natural Science Foundation of China (Grant No. 31370080) and the National Special Fund for State Key Laboratory of Bioreactor Engineering.

语种：英文

外文关键词：Mycobacterium; 22-Hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC); mmpL3; choM; cyp125; fadA5; Sterol catabolism

摘要：Background: The strategy of modifying the sterol catabolism pathway in mycobacteria has been adopted to produce steroidal pharmaceutical intermediates, such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), which is used to synthesize various steroids in the industry. However, the productivity is not desirable due to some inherent problems, including the unsatisfactory uptake rate and the low metabolic efficiency of sterols. The compact cell envelope of mycobacteria is a main barrier for the uptake of sterols. In this study, a combined strategy of improving the cell envelope permeability as well as the intracellular sterol metabolism efficiency was investigated to increase the productivity of 4-HBC. Results: MmpL3, encoding a transmembrane transporter of trehalose monomycolate, is an important gene influencing the assembly of mycobacterial cell envelope. The disruption of mmpL3 in Mycobacterium neoaurum ATCC 25795 significantly enhanced the cell permeability by 23.4% and the consumption capacity of sterols by 15.6%. Therefore, the inactivation of mmpL3 was performed in a 4-HBC-producing strain derived from the wild type M. neoaurum and the 4-HBC production in the engineered strain was increased by 24.7%. Subsequently, to enhance the metabolic efficiency of sterols, four key genes, choM1, choM2, cyp125, and fadA5, involved in the sterol conversion pathway were individually overexpressed in the engineered mmpL3-deficient strain. The production of 4-HBC displayed the increases of 18.5, 8.9, 14.5, and 12.1%, respectively. Then, the more efficient genes (choM1, cyp125, and fadA5) were co-overexpressed in the engineered mmpL3-deficient strain, and the productivity of 4-HBC was ultimately increased by 20.3% (0.0633 g/L/h, 7.59 g/L 4-HBC from 20 g/L phytosterol) compared with its original productivity (0.0526 g/L/h, 6.31 g/L 4-HBC from 20 g/L phytosterol) in an industrial resting cell bio-transformation system. Conclusions: Increasing cell permeability combined with the co-overexpression of the key genes (cyp125, choM1, and fadA5) involved in the conversion pathway of sterol to 4-HBC was effective to enhance the productivity of 4-HBC. The strategy might also be useful for the conversion of sterol to other steroidal intermediates by mycobacteria.