Abstract
Squalene, a lipophilic triterpene with multifaceted bioactivities, faces bioproduction bottlenecks in microbial hosts due to inefficient biosynthetic pathways and limited storage capacity. Here, we address these challenges through systems metabolic engineering integrating redox-balanced 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) variants and membrane lipid remodeling. By developing a hybrid HMGRs system combining NADPH-dependent and NADH-preferred enzymes, squalene production reached 852.06 ± 28.95 mg/L with balanced cofactor utilization. Subsequent engineering of membrane morphology and lipid metabolism generated lipid-enriched elongated cells, through the overexpression of dgs, murG and plsC, boosting squalene production to 970.86 ± 55.67 mg/L. Implementation of delayed induction strategies coupled with 10 % dodecane overlay as an in situ recovery system achieved a final squalene titer of 1267.01 mg/L in a 3 L bioreactor. Mechanistic studies revealed fatty acid (FA) and phosphatidylethanolamine (PE) as key reservoirs for squalene in E. coli, with dgs overexpression specifically promoting cellular elongation. This article provides comprehensive insights into engineering strategies and mechanistic perspectives, establishing a universal framework for hydrophobic metabolite biomanufacturing in prokaryotic hosts.