Organelle–Targeted Multi–Enzyme Engineering Enables Medium–Chain Fatty Acid Production in <i>Chlamydomonas reinhardtii</i>

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 <i>Chlamydomonas reinhardtii</i> (<i>Chlamydomonas</i>) for MCFA production by introducing heterologous lipid biosynthetic enzymes targeted to distinct subcellular compartments. A plastid-targeted medium-chain-specific acyl-ACP thioesterase from <i>Umbellularia californica</i> and endoplasmic reticulum-targeted acyltransferases from <i>Cocos nucifera</i> and <i>Elaeis guineensis</i> 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 <i>Hsp70A</i> 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 <i>Chlamydomonas</i>.