Liu Ying, Yue Shengjie, Bilal Muhammad, Jan Malik, Wang Wei, Hu Hongbo, Zhang Xuehong
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian 223003, China.
Biology (Basel). 2022 Feb 24;11(3):363. doi: 10.3390/biology11030363.
Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate (MEP) pathway as the precursor, which shows good antimicrobial activity against several Gram-positive bacteria and fungi. However, the highest yield of endophenazine A was about 20 mg/L in , limiting its large-scale industrial development. P3, possessing an efficient PCA synthesis and MEP pathways, is a suitable chassis to synthesize endophenazine A. Herein, we designed an artificial biosynthetic pathway for the synthesis of endophenazine A in P3. Primarily, the prenyltransferase PpzP from 9663 was introduced into P3 and successfully synthesized endophenazine A. Another phenazine compound, endophenazine A1, was discovered and identified as a leakage of the intermediate 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP). Finally, the yield of endophenazine A reached 279.43 mg/L, and the yield of endophenazine A1 reached 189.2 mg/L by metabolic engineering and medium optimization. In conclusion, we successfully synthesized endophenazine A and endophenazine A1 in P3 for the first time and achieved the highest titer, which provides a reference for the heterologous synthesis of terpenoid phenazines.
内吩嗪A是一种萜类吩嗪，以前体吩嗪-1-羧酸（PCA）和源自2-甲基-D-赤藓糖醇-4-磷酸（MEP）途径的二甲基烯丙基二磷酸（DMAPP）为原料，对多种革兰氏阳性细菌和真菌具有良好的抗菌活性。然而，内吩嗪A在[具体环境]中的最高产量约为20 mg/L，这限制了其大规模工业化发展。具有高效PCA合成和MEP途径的P3是合成内吩嗪A的合适底盘。在此，我们设计了一条在P3中合成内吩嗪A的人工生物合成途径。首先，将来自[具体菌株]9663的异戊烯基转移酶PpzP引入P3，并成功合成了内吩嗪A。另一种吩嗪化合物内吩嗪A1被发现并鉴定为中间体4-羟基-3-甲基-2-丁烯焦磷酸（HMBPP）的泄漏产物。最后，通过代谢工程和培养基优化，内吩嗪A的产量达到279.43 mg/L，内吩嗪A1的产量达到189.2 mg/L。总之，我们首次在P3中成功合成了内吩嗪A和内吩嗪A1，并实现了最高滴度，为萜类吩嗪的异源合成提供了参考。
