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

Area of Science:

• Biotechnology
• Synthetic Biology
• Microalgal Engineering

Background:

• Medium-chain fatty acids (MCFAs) are valuable bio-based feedstocks.
• Production in photosynthetic microorganisms is limited by low yields, cytotoxicity, and poor scalability.

Purpose of the Study:

• To engineer *Chlamydomonas reinhardtii* for enhanced medium-chain fatty acid (MCFA) production.
• To develop a scalable and robust biosynthesis strategy for MCFAs in microalgae.

Main Methods:

• Co-expression of heterologous lipid biosynthetic enzymes (plastid-targeted thioesterase, ER-targeted acyltransferases) in *Chlamydomonas reinhardtii*.
• Utilized organelle-specific targeting to mimic natural fatty acid synthesis and triacylglycerol assembly pathways.
• Optimized production through nitrogen depletion, elevated temperature (30°C), and scale-up cultivation in a 50 L photobioreactor.

Main Results:

• Identified MCFA-producing transformants, with MCFA #17 showing highest accumulation.
• Demonstrated sustained MCFA production in total fatty acid and triacylglycerol fractions during medium-scale cultivation.
• Observed enhanced MCFA accumulation and transgene expression at elevated temperatures.
• Confirmed stable growth and MCFA production during large-scale cultivation in a 50 L photobioreactor.

Conclusions:

• An organelle-targeted multi-enzyme strategy enables robust and scalable medium-chain fatty acid (MCFA) biosynthesis in *Chlamydomonas reinhardtii*.
• This approach overcomes key limitations in microalgal-based MCFA production, paving the way for industrial applications.