Metabolic Engineering of Yarrowia lipolytica for Enhanced Microbial Production of Medium-Chain α, ω-Diols from Alkanes via CRISPR-Cas9 Mediated Pathway Optimization and P450 Alkane monooxygenase Overexpression

Ye Chan Kim¹Yoon Hwan Choi Won¹Eun-Jung Kim²Byung-Gee Kim³Hyungdon Yun^1*

• ¹Konkuk University, Seoul, Republic of Korea
• ²EGC Therapeutics, Inc., seoul, Republic of Korea
• ³Seoul National University, Gwanak-gu, Republic of Korea

Medium-to long-chain α, ω-diols are essential precursors for polyesters and polyurethanes, but their microbial production from abundant alkane feedstocks remains underdeveloped. To enable de novo production of α, ω-diols directly from alkanes, this study utilized the oleaginous yeast Yarrowia lipolytica Po1g strain, chosen for its natural ability to metabolize hydrophobic substrates and its proven advantages over Escherichia coli for alkane bioconversion.. We employed CRISPR-Cas9 to systematically delete 10 genes responsible for fatty alcohol oxidation (fatty alcohol dehydrogenase, alcohol dehydrogenase 1–8, fatty alcohol oxidase 1) and 4 genes responsible for fatty aldehyde oxidation (fatty aldehyde dehydrogenase 1–4), creating strain YALI17 with reduced over-oxidation activity. This modification increased 1,12-dodecanediol production 14-fold (0.05 mM to 0.72 mM) from 50 mM n-dodecane. Further enhancement was achieved by overexpressing alkane hydroxylase genes, with Alk1 overexpression in YALI17 yielding 1.45 mM 1,12-dodecanediol. Finally, biotransformation using automated pH-controller yielded 3.2 mM 1,12-dodecanediol from 50 mM n-dodecane. These results demonstrate the first successful biotransformation of medium-to long-chain α, ω-diols from alkanes in yeast, establishing Y. lipolytica as a promising platform for alkane-based biomanufacturing through targeted metabolic engineering.