Efficient photoproduction of a high‐value sesquiterpene pentalenene from the green microalga Chlamydomonas reinhardtii

SUMMARY Microalgae can convert CO 2 into energy‐rich bioproducts through photosynthesis, emerging as promising platforms for sustainable and light‐driven terpenoid production. Pentalenene, a tricyclic sesquiterpene originally identified as an antibiotic metabolite in Streptomyces species, has recently emerged as a promising candidate for advanced aviation fuels. However, pentalenene biosynthesis does not naturally occur in photosynthetic microorganisms, limiting its cost‐effective production. In this study, we established a photosynthetic platform for pentalenene production by expressing a heterologous pentalenene synthase ( penA ) in the microalga Chlamydomonas reinhardtii . To enhance pentalenene production, we engineered the methylerythritol phosphate (MEP) pathway by overexpressing rate‐limiting enzymes deoxyxylulose 5‐phosphate synthase (DXS) and 4‐hydroxy‐3‐methylbut‐2‐enyl‐diphosphate reductase (HDR). We also introduced isopentenyl diphosphate isomerase (IDI) to improve the equilibrium between the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The engineered penA ‐ DXS‐HDR‐IDI strain achieved a titer of 2.86 mg/L, which is a 10.2‐fold increase compared with the parental strain expressing penA alone. The penA‐DXS‐HDR‐IDI strain achieved a pentalenene titer of 13.65 mg/L under photomixotrophic conditions in photobioreactors. In addition, metabolite profiling revealed elevated levels of MEP pathway intermediates (DXP, MEP, ME‐cPP) and precursors glyceraldehyde 3‐phosphate (G3P) and farnesyl diphosphate (FPP) as critical drivers of high pentalenene yields. Notably, the engineered pentalenene‐producing strains exhibited cell growth and photosynthetic activity comparable to the untransformed strain. This study represents the first successful attempt to produce pentalenene in a photosynthetic host and provides a rational engineering strategy for the production of other sesquiterpenes in microalgae and other industrial microorganisms.