Fang Lixia, Hao Xueyan, Fan Jie, Liu Xiaolei, Chen Yaru, Wang Lian, Huang Xiaoying, Song Hao, Cao Yingxiu
State Key Laboratory of Synthetic Biology, Tianjin University, Tianjin, China.
Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
Nat Commun. 2025 Mar 29;16(1):3060. doi: 10.1038/s41467-025-58368-3.
Microbial physiology plays a pivotal role in construction of superior microbial cell factories for efficient biosynthesis of desired products. Here we identify that pcnB repression confers improved physiology for overproduction of free fatty acids (FFAs) in Escherichia coli through genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). The repression of pcnB can enhance the stability and abundance of the transcripts of genes involved in the proton-consuming system, thereby supporting global improvements in membrane properties, redox state, and energy level. Based on pcnB repression, further repression of acrD increases FFAs biosynthesis by enhancing FFAs efflux. The engineered strain pcnB-acrD-fadR achieves 35.1 g L FFAs production in fed-batch fermentation, which is the maximum titer reported to date in E. coli. This study highlights the significance of uncovering hidden genetic determinants that confer improved microbial physiology for enhancing the biosynthesis of desired products.
微生物生理学在构建用于高效生物合成所需产品的优质微生物细胞工厂中起着关键作用。在此，我们通过结合荧光激活细胞分选（FACS）和下一代测序（NGS）的基因组规模CRISPRi调控，确定了pcnB抑制赋予大肠杆菌中游离脂肪酸（FFA）过量生产更好的生理学特性。pcnB的抑制可增强质子消耗系统相关基因转录本的稳定性和丰度，从而支持膜特性、氧化还原状态和能量水平的整体改善。基于pcnB抑制，进一步抑制acrD可通过增强FFA外排来增加FFA生物合成。工程菌株pcnB - acrD - fadR在分批补料发酵中实现了35.1 g/L的FFA产量，这是迄今为止大肠杆菌中报道的最高滴度。本研究突出了揭示隐藏的遗传决定因素对于改善微生物生理学以增强所需产品生物合成的重要性。
