High-throughput screening with insertional mutagenesis site sequencing identifies targets participating in enhanced sustainable aviation fuel (SAF) production

Abstract

Rhizosphere saprophytic bacteria are emerging microbial chassis for the sustainable synthesis of valuable chemicals, including free fatty acids (FFAs) and biofuel precursors. However, current titers remain insufficient for industrial deployment. Therefore, identifying genes associated with elevated free fatty acid production remains a critical objective. We have constructed a comprehensive, genome-wide insertional mutagenesis library. Selection of high FFAs mutants through sequential rounds of cell sorting, followed by rapid amplification and identification of the disrupted loci, will be performed next. In parallel, four enzymes—both heterologous and homologous—are being screened to further channel carbon flux into the FFA branch. Planned transcriptomic and computational analyses are expected to elucidate the regulatory and metabolic shifts accompanying enhanced fatty acid biosynthesis and central carbon metabolism. The genetic determinants and enzymatic interventions uncovered in this study will furnish a rational framework for engineering robust, high-yield bacterial platforms for renewable oleochemicals production.