Photoproduction of Aviation Fuel β‐Caryophyllene From the Eukaryotic Green Microalga Chlamydomonas reinhardtii

ABSTRACT β‐caryophyllene is a plant‐derived sesquiterpene and is regarded as a promising ingredient for aviation fuels. Microalgae can convert CO 2 into energy‐rich bioproducts through photosynthesis, making them potential platforms for the sustainable production of sesquiterpenes. However, heterologous sesquiterpene engineering in microalgae is still in its infancy, and β‐caryophyllene production in eukaryotic photosynthetic microorganisms has not been reported. In this study, we succeeded in producing β‐caryophyllene in the model eukaryotic microalga Chlamydomonas reinhardtii by heterologously expressing a β‐caryophyllene synthase ( QHS ). Furthermore, overexpressing the key enzyme of the 2‐C‐methyl‐D‐erythritol 4‐phosphate pathway in the QHS ‐expressing strain ( QHS‐DXS‐HDR −18) resulted in a 17‐fold higher β‐caryophyllene production compared to the single expression of QHS ( QHS −28). Additionally, when isopentenyl diphosphate isomerase ( CrIDI ) was overexpressed, the β‐caryophyllene production was up to 480.6 μg/L in QHS‐DXS‐HDR‐CrIDI −16 and increased by 1.8‐fold compared to the parental strain QHS‐DXS‐HDR −18. Under photoautotrophic and photomixotrophic conditions in photobioreactors, the β‐caryophyllene production in QHS‐DXS‐HDR‐CrIDI −16 reached 854.7 and 1016.8 μg/L, respectively. Noticeably, all the β‐caryophyllene‐producing strains generated in this study did not exhibit adverse effects on cell growth and photosynthesis activity compared to the untransformed strain. This study demonstrates the first successful attempt to produce β‐caryophyllene in the eukaryotic microalga C. reinhardtii and develops a novel strategy for increasing sesquiterpene production in eukaryotic photosynthetic microorganisms.