Heterologous production of β-pinene in the chloroplast of the marine diatom Phaeodactylum tricornutum

Microalgae are considered promising hosts for the sustainable production of plant-derived secondary metabolites, such as terpenoids. In particular, the model marine diatom Phaeodactylum tricornutum is a promising candidate for the heterologous production of such chemicals due to its robustness, relatively fast growth, and high natural accumulation of terpenoids. In addition, P. tricornutum possesses two separate pathways for the synthesis of terpenoid precursors, the MEP pathway in the chloroplast and the MVA pathway in the cytosol. In this study, we explored the two pathways for the heterologous β-pinene production in P. tricornutum by episomally expressing pinene synthase in either the chloroplast or cytosol for the first time. Our results show that the chloroplast expression from episomes led to β-pinene titers of up to 10.27 ± 1.45 µg · L -1 . No β-pinene synthesized from the cytosolic MVA pathway precursors was detected, however the expression and functionality of the pinene synthase was confirmed both in the cytosol and in the chloroplast. Furthermore, to enhance production in the chloroplast, we developed more stable transgenic lines with random chromosomal integration of two different pinene synthase genes. We observed higher titers compared to the episomal mutants, up to 19.35 ± 1.42 µg · L -1 with the pinene synthase from Abies grandis and 20.07 ± 0.51 µg · L -1 with the pinene synthase from Citrus limon . All the β-pinene producing strains used in this study also produced α-pinene as a side product, which accounted for 20–25% of total monoterpenoid production. Overall, this study represents a fundamental step in microalgal engineering towards the synthesis of monoterpenoids. • The monoterpenoid β-Pinene was produced heterologously in Phaeodactylum tricornutum for the first time, with titers up to 10.27 ± 1.45 µg·L -1 . • Production of heterologous terpenoids was demonstrated in the chloroplast of P. tricornutum for the first time. • The chloroplast is more suitable than the cytosol for the heterologous synthesis of β-Pinene in the tested laboratory conditions. • Transgenic lines with randomly integrated chromosomal expression (RICE) produced up to 3-fold higher β-Pinene titers compared to transgenic lines with Episomal Expression (EE).