Metabolic engineering for improved heterologous pinene production in the chloroplast of Phaeodactylum tricornutum

BACKGROUND: The marine diatom Phaeodactylum tricornutum is a promising platform for the sustainable production of terpenoids. This is due to its robust photosynthetic growth and natural accumulation of terpenoids, mainly including photosynthetic pigments. P. tricornutum harbors the methyl-erythritol-phosphate (MEP) pathway in the chloroplast, a dedicated route used for the production of terpenoid-like photosynthetic pigments. Despite its natural predisposition for terpenoid production in the chloroplast, previously reported titers of heterologously produced terpenoids in P. tricornutum are relatively low. RESULTS: In this study, we used a metabolic engineering strategy to enhance the production of the terpenoid pinene by increasing the production of their precursors. We episomically co-expressed either an isopentenyl diphosphate isomerase (IDI), a geranyl diphosphate synthase (GPPS), or both, along with a pinene synthase (PinS) in the chloroplast of P. tricornutum. We found that the combination of both IDI and GPPS leads to the hyperaccumulation of the monoterpenoid pinene, compared to the strain with only pinene synthase or IDI and GPPS expressed individually. Furthermore, the integration of all three genes in the genome resulted in a strain with a 92-fold higher pinene production, compared to the strain expressing only the pinene synthase. Lastly, we cultivated one of the high-performing transgenic strains at different light intensity regimes and found that the production of pinene increased at elevated light intensities. CONCLUSION: In this study, we performed metabolic engineering in the chloroplast of P. tricornutum by expressing heterologous IDI and GPPS together with a pinene synthase. We showed that this approach considerably boosted pinene production, especially in strains where the genes were randomly integrated in the genome. Moreover, we further increased pinene titers by modulating the light intensity during cultivation. Overall, we demonstrated the potential of combining metabolic engineering with optimized cultivation parameters, specifically light intensity, to enhance the production of monoterpenoids in the chloroplast of P. tricornutum.