Compartmentalized sesquiterpenoid biosynthesis and functionalization in the Chlamydomonas reinhardtii plastid

Terpenoids play key roles in cellular metabolism, with some organisms
having evolved expanded terpenoid profiles for specialized functions such
as signaling and defense. While heterologous production in microbial hosts
offers an alternative to natural extraction, the development of efficient
biosynthetic platforms remains challenging. Here, we developed a
subcellular engineering approach in the model green alga Chlamydomonas
reinhardtii by targeting both sesquiterpenoid synthases and
cytochrome P450s (CYPs) to the plastid, exploiting its photosynthetic
electron transport chain to drive CYP-mediated oxidation without reductase
partners. Nuclear-encoded sesquiterpenoid synthases were expressed with
farnesyl pyrophosphate synthase fusions and targeted to the plastid, while
CYPs were modified for soluble localization in the plastid stroma by
removing transmembrane domains. The plastid environment supported
hydroxylation, epoxidation, and oxidation reactions, with
functionalization efficiencies reaching 80% of accumulated products.
Carbon source availability influenced product ratios, revealing metabolic
flexibility in the engineered pathways. Overall sesquiterpenoid yields
ranged between 250-2500 µg L–1 under screening
conditions, establishing proof-of-concept for using plastid biochemistry
in complex terpenoid biosynthesis. Living two-phase terpenoid extractions
with different perfluorinated solvents revealed variable performances
based on sesquiterpenoid functionalization and solvent type. This work
demonstrates that photosynthetic electron transport can drive CYP-mediated
functionalization in engineered subcellular compartments. However,
improvements in photobioreactor cultivation concepts will be required to
facilitate the use of algal chassis for scaled production.