Identification of crucial roles of transcription factor IhfA on high production of free fatty acids in Escherichia coli

Transcription factor engineering has unique advantages in improving the performance of microbial cell factories due to the global regulation of gene transcription.Omics analyses and reverse engineering enable learning and subsequent incorporation of novel design strategies for further engineering.Here,we identify the role of the global regulator IhfA for overproduction of free fatty acids(FFAs)using CRISPRi-facilitated reverse engineering and cellular physiological characterization.From the differentially expressed genes in the ihfALstrain,a total of 14 beneficial targets that enhance FFAs production by above 20% are identified,which involve membrane function,oxidative stress,and others.For membrane-related genes,the engineered strains obtain lower cell surface hydrophobicity and increased average length of membrane lipid tails.For oxidative stress-related genes,the engineered strains present decreased reactive oxygen species(ROS)levels.These gene modulations enhance cellular robustness and save cellular resources,contributing to FFAs production.This study provides novel targets and strategies for engineering microbial cell factories with improved FFAs bioproduction.