Modular Expression of Botryococcus braunii Genes Enhances Isoprenoid Production in the Diatom Phaeodactylum...

Isoprenoids are essential natural compounds with high structural and functional diversity. Among them, carotenoids and triterpenoids such as squalene have high biotechnological value but remain challenging to produce sustainably. The green microalga Botryococcus braunii synthesizes large amounts of triterpenoids through specialized methylerythritol phosphate (MEP) pathway configurations involving distinct 1-deoxy-D-xylulose-5-phosphate synthase (DXS) isoforms, a bifunctional squalene synthase (SQS) and squalene synthase-like (SSL) enzymes working together for the synthesis of the compound. However, its slow growth limits industrial application. In this work, we engineered the isoprenoid metabolism of the fast-growing diatom Phaeodactylum tricornutum with combinations of B. braunii enzymes. Episomal uLoop assembly enabled rapid expression and evaluation of B. braunii DXS and SQS variants, enhancing precursor flux and resulting in the coaccumulation of squalene and carotenoids. This work demonstrates the functional transfer of specialized algal terpenoid enzymes into a tractable diatom host and highlights P. tricornutum's potential as a versatile platform for sustainable, high-value isoprenoid biosynthesis.