Xiong Liang-Bin, Liu Hao-Hao, Xu Li-Qin, Sun Wan-Ju, Wang Feng-Qing, Wei Dong-Zhi
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.
Microb Cell Fact. 2017 May 22;16(1):89. doi: 10.1186/s12934-017-0705-x.
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.
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.
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.
已采用改造分枝杆菌中甾醇分解代谢途径的策略来生产甾体药物中间体，如22-羟基-23,24-双降胆甾-4-烯-3-酮（4-HBC），该中间体在工业上用于合成各种甾体。然而，由于一些固有问题，包括甾醇摄取率不令人满意和代谢效率低，生产率并不理想。分枝杆菌紧密的细胞包膜是甾醇摄取的主要障碍。在本研究中，研究了一种提高细胞包膜通透性以及细胞内甾醇代谢效率的联合策略，以提高4-HBC的生产率。
编码海藻糖单分枝菌酸跨膜转运蛋白的MmpL3是影响分枝杆菌细胞包膜组装的重要基因。在新金色分枝杆菌ATCC 25795中破坏mmpL3可使细胞通透性显著提高23.4%，甾醇消耗能力提高15.6%。因此，在源自野生型新金色分枝杆菌的4-HBC生产菌株中进行了mmpL3的失活，工程菌株中4-HBC的产量提高了24.7%。随后，为了提高甾醇的代谢效率，在工程化的mmpL3缺陷菌株中分别过表达了参与甾醇转化途径的四个关键基因choM1、choM2、cyp125和fadA5。4-HBC的产量分别提高了18.5%、8.9%、14.5%和12.1%。然后，在工程化的mmpL3缺陷菌株中共同过表达更有效的基因（choM1、cyp125和fadA5），与工业静止细胞生物转化系统中的原始生产率（0.0526 g/L/h，20 g/L植物甾醇产生6.31 g/L 4-HBC）相比，4-HBC的生产率最终提高了20.3%（0.0633 g/L/h，20 g/L植物甾醇产生7.59 g/L 4-HBC）。
提高细胞通透性并联合过表达参与甾醇向4-HBC转化途径的关键基因（cyp125、choM1和fadA5）可有效提高4-HBC的生产率。该策略可能也有助于分枝杆菌将甾醇转化为其他甾体中间体。
