Compartmentalized sesquiterpenoid biosynthesis and functionalization in theChlamydomonas reinhardtiiplastid

Abstract Terpenoids play key roles in cellular metabolism, with some organisms having evolved expanded terpenoid profiles for specialized functions such as signaling and defense. Many terpenoids have applications in pharmaceuticals, fragrances, and agriculture, but their harvest from natural sources can be challenging. Heterologous production of specialty terpenoids in microbial hosts offers an alternative, using terpene synthases and further enzymatic decoration to expand chemical complexity and functionality. Here, we explored the heterologous production of 10 different sesquiterpenoids (STPs, C15) and their further biofunctionalization mediated by cytochrome P450s (CYPs) in the green alga Chlamydomonas reinhardtii. STP synthases were expressed from the nuclear genome and localized to the algal plastid, coupled with co-expression of selected CYPs. STP production in the plastid was supported by farnesyl pyrophosphate synthase fusions to STP synthases, and CYPs were modified for soluble localization in the plastid stroma by removing transmembrane domains. Various target CYPs were screened for STP functionalization in the alga, and different product ratios were generated based on trophic modes. Overall STP yields ranged between 250-2500 µg L−1 under screening conditions, with CYP-mediated functionalization reaching up to 80% of accumulated heterologous STP products. Living two-phase terpenoid extractions with different perfluorinated solvents revealed variable performances based on sesquiterpenoid functionalization and solvent type. This work demonstrates the feasibility of generating heterologous functionalized terpenoid products in alga using soluble, plastid-localized CYPs without reductase partners. However, overall improvements in photobioreactor cultivation concepts will be required to facilitate the use of algal chassis for scaled production. Significance Statement This study demonstrates the feasibility of producing and modifying heterologous terpenoid products in the algal plastid using sesquiterpene synthases (STPS) and soluble cytochrome P450s (CYPs). We show that algal cultivation conditions influence the composition and ratios of functionalized terpenoid products, which can be extracted through solvent-based ‘milking’ during growth. The reducing equivalents that enable CYP activity in the plastid appear derived from photosynthetic electrons without requiring the co-expression of a cytochrome P450 reductase (CPR) partner, simplifying engineering strategies. As algae can be cultivated with minimal inputs (trace elements, light, and CO2) from sources like wastewater, this approach offers the potential for sustainable production of complex specialty terpenoid chemicals. Competing Interest Statement The authors have declared no competing interest.