Electron transfer engineering of artificially designed cell factory for complete biosynthesis of natural products

Biosynthesis of natural products (NPs) by artificially designed cell factories often involves numerous nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymes that mediate electron transfer reactions. However, the mechanisms of electron transfer from regeneration to the final delivery to the active centers of various types of NADPH-dependent enzymes remain elusive, limiting our ability to systematically engineer electron transfer (ETE) to improve NPs production. Here, we elucidated the electron transfer mechanisms of NADPH-dependent enzymes, which were further contributed to systematically ETE of Saccharomyces cerevisiae , including step-by-step engineering the electron transfer residues of 7-Dehydrocholesterol reductase (DHCR7) and P450 sterol side chain cleaving enzyme (P450 _scc ), electron transfer components for directing carbon flux, and NADPH regeneration pathways, for high-level production of the cholesterol (1.78 g/L) and pregnenolone (0.83 g/L). Additionally, computational chemical analysis indicated that the ETE process make the electron transfer chains shorter and more stable which significantly accelerated proton coupled electron transfer process. This study underscores the significance of ETE strategies in NPs biosynthesis and expands synthetic biology approaches.