Improving Lipid Content in the Diatom by the Knockdown of the Enoyl-CoA Hydratase Using CRISPR Interference.

The diatom shows potential as a source for biofuel production because of its considerable lipid content. Fatty acid β-oxidation plays a critical role in lipid breakdown. However, we still have a limited understanding of the role of fatty acid β-oxidation in lipid content in this microalga. In our study, we utilized a CRISPR interference method to reduce the expression of enoyl-CoA hydratase (PtECH), which is involved in the hydration of trans-2-enoyl-CoA to produce 3-hydroxyacyl-CoA during the β-oxidation pathway. Using this method, we developed two transgenic lines, PtECH21 and PtECH1487, which resulted from interference at two different sites of the gene, respectively. RT-qPCR analysis confirmed that the mRNA levels of PtECH in both mutants were significantly lower compared to the wild type. Surprisingly, the lipid content of both mutants increased notably. Additionally, both knockdown mutants exhibited higher chlorophyll content and improved photosynthetic efficiency of the photosystem II compared to the wild type. This study introduces a new approach for enhancing lipid content in and expands our knowledge of the functions of enoyl-CoA hydratase in microalgae.