BioProject

Microbial physiology plays a pivotal role in construction of a superior microbial cell factory for efficient production of desired products. More...

Microbial physiology plays a pivotal role in construction of a superior microbial cell factory for efficient production of desired products. Here we identified pcnB repression through genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS), which confers improved physiology for free fatty acids (FFAs) overproduction in Escherichia coli. The repression of pcnB could improve the stability and abundance of the transcripts involved in proton-consuming system, conferring a global improvement on cell membrane, redox state, and energy level. These physiological advantages facilitated further identification of acrD repression enhancing FFAs efflux. The engineered strain pcnBi-acrDi-fadR+ achieved 35.1 g l−1 FFAs production in fed-batch fermentation, which is the maximum titer in E. coli reported to date. This study underscores the significance of hidden genetic determinants in microbial biosynthesis and sheds light on the role of microbial physiologies in boosting microbial biosynthesis. Overall design: In this study, we carried out genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS) to identify genetic determinants for FFAs overproduction in Escherichia coli. Two rounds of screening were carried out. In the first round, the transformed cell library (named Tra1) carrying the pCF plasmid and the genome-scale sgRNA expression plasmid library was induced by IPTG and then cultivated for 40 h to sample the cell library before sorting (BS) (named Cul2). Then, BS was dyed with Nile red and sorted by FACS, obtaining the cell library after sorting (AS) (named Sor3). In the second round, the cultured cell library before sorting (BS) (named B-cul2) carrying the pCF-pcnBi plasmid and the genome-scale sgRNA expression plasmid library was sorted by FACS, obtaining the cell library after sorting (AS) (named B-sor3-P5). The Tra1, Cul2, Sor3, B-cul2, and B-sor3-P5 were applied for NGS to get the reads of sgRNA sequence. Less...