Genome-scale overexpression screen reveals membrane homeostasis as a key determinant for free fatty acids overproduction in Escherichia coli

Genome-scale target identification is essential for optimizing microbial biosynthesis due to the highly complex and interconnected nature of cellular metabolism. Free fatty acids (FFAs), valuable precursors for biofuels and industrial chemicals, have been extensively studied in Escherichia coli. However, genome-wide exploration of beneficial targets that promote FFAs production remains limited, hindering efforts to fully unlock the potential of microbial biosynthetic capabilities. In this study, we performed genome-scale screening of upregulation targets for FFAs overproduction in E. coli by leveraging the ASKA (A Complete Set of E. coli K-12 ORF Archive) library in combination with fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). We found that overexpression of rfaY, encoding a lipopolysaccharide (LPS) core heptose II phosphokinase, led to a 207.8% increase in FFAs production by enhancing membrane stability, as evidenced by reduced permeability and improved integrity. Further investigation revealed that overexpressing additional LPS biosynthesis-related genes similarly improved membrane robustness and FFAs production. Through iterative screening, yafL and rimM were identified as synergistic partners with rfaY, and subsequent integration of fadR overexpression ultimately yielded the optimal strain rfaY⁺-yafL⁺-fadR⁺, which achieved a FFAs titer of 39.6 g/L under fed-batch fermentation—the highest reported to date in E. coli. This study highlights the significance of genome-scale mining potential genetic determinants for enhancing the biosynthesis of desired products.