Electron transfer engineering of artificially designed cell factory for complete biosynthesis of steroids

• Published in: Nature communications Vol. 16; no. 1; pp. 3740 - 19
• Main Authors: Chen, Qihang, Wei, Wenqian, Chao, Zikai, Qi, Rui, He, Jianhong, Chen, Huating, Wang, Ke, Wang, Xinglong, Rao, Yijian, Zhou, Jingwen
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 21.04.2025; Nature Publishing Group; Nature Portfolio
• Subjects: 119/118; 140/131; 38/70; 631/326/2522; 631/553/552; 631/61/318; 631/92/552; 7-Dehydrocholesterol reductase; 82/80; Adenine; Biosynthesis; Cholesterol; Cholesterol - biosynthesis; Cholesterol Side-Chain Cleavage Enzyme - genetics; Cholesterol Side-Chain Cleavage Enzyme - metabolism; Electron transfer; Electron Transport; Electrons; Engineering; Enzymes; Factories; Humanities and Social Sciences; Industrial engineering; Metabolic Engineering - methods; multidisciplinary; NADP - metabolism; NADPH-diaphorase; Nicotinamide; Nicotinamide adenine dinucleotide; Oxidoreductases Acting on CH-CH Group Donors - genetics; Oxidoreductases Acting on CH-CH Group Donors - metabolism; Pregnenolone; Pregnenolone - biosynthesis; Reductases; Regeneration; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Science; Science (multidisciplinary); Steroid hormones; Steroids; Steroids - biosynthesis; Synthetic Biology - methods
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-025-58926-9

Biosynthesis of steroids by artificially designed cell factories often involves numerous nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymes that mediate electron transfer reactions. However, the unclear mechanisms of electron transfer from regeneration to the final delivery to the NADPH-dependent active centers limit systematically engineering electron transfer to improve steroids production. Here, we elucidate the electron transfer mechanisms of NADPH-dependent enzymes for systematically engineer electron transfer 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). The electron transfer engineering (ETE) process makes the electron transfer chains shorter and more stable which significantly accelerates deprotonation and proton coupled electron transfer process. This study underscores the significance of ETE strategies in steroids biosynthesis and expands synthetic biology approaches. Bioproduction of steroids involves NADPH-dependent enzymes that mediate electron transfer reactions. Here, the author reveal the electron transfer mechanisms of NADPH-dependent enzymes and demonstrate the complete biosynthesis of steroids through electron transfer engineering.