Organelle-Targeted Multi-Enzyme Engineering Enables Medium-Chain Fatty Acid Production in Chlamydomonas reinhardtii

Medium-chain fatty acids (MCFAs, C8-C12) are valuable bio-based feedstocks, but their production in photosynthetic microorganisms remains limited by low yields, cytotoxicity, and poor scalability. Here, we engineered Chlamydomonas reinhardtii (Chlamydomonas) for MCFA production by introducing heterologous lipid biosynthetic enzymes targeted to distinct subcellular compartments. A plastid-targeted medium-chain-specific acyl-ACP thioesterase from Umbellularia californica and endoplasmic reticulum-targeted acyltransferases from Cocos nucifera and Elaeis guineensis were co-expressed from the nuclear genome, reflecting the compartmentalized organization of fatty acid (FA) synthesis and triacylglycerol (TAG) assembly in microalgae. Stepwise GC-based screening identified multiple MCFA-producing transformants, among which MCFA #17 exhibited the highest MCFA accumulation under nitrogen-depleted conditions. Medium-scale cultivation (3 L) revealed sustained MCFA production over time, with MCFAs detected in both total FA and TAG fractions. Elevated temperature (30°C) further enhanced MCFA accumulation, accompanied by increased transgene expression driven by a heat shock protein Hsp70A promoter. Importantly, large-scale cultivation in a 50 L photobioreactor demonstrated stable growth and maintenance of MCFA production under scale-up conditions. Together, these results establish an organelle-targeted multi-enzyme strategy that enables robust and scalable MCFA biosynthesis in Chlamydomonas.