(191ay) Engineering a Rubisco-Deletion Crypthecodinium Cohnii for Increased Growth and Lipid Accumulation 

The heterotrophic dinofagellate microalga Crypthecodinium cohnii is well known for its ability to accumulate lipids with a high fraction of polyunsaturated fatty acid, such as docosahexaenoic acid (DHA). However, it has been challenging for genetic modification in this valuable microorganism. In this study, we developed a new genetic transformation methodology for DHA-producing C. cohnii by evaluating suitable selected marker and optimizing transformation protocol. In addition, a gene potentially involved in CO₂ fixation under autotrophic condition, ribulose 1,5-bisphosphate carboxylase (rubisco), was selected as the target for construction of gene knock-down mutant, the results demonstrated that the constructs can be successfully inserted into the C. cohnii chromosome by homologous recombination, which was further confirmed by sequencing, decreased gene expression enzymatic activity, and Western blotting analysis. Interestingly, comparative analysis showed that the deletion of the rubisco gene promoted cell growth and increased lipid content of C. cohnii under heterotrophic condition when compared with the wild type. To explore the possible mechanism related to the enhanced growth and lipid accumulation in the rubisco-deletion mutants, a LC-MS based metabolomic analysis was conducted, and the results showed that the metabolites involved in energy metabolism were up-regulated, suggesting the deletion of the rubisco gene may contribute to the re-direction of more carbon or energy towards growth and lipid accumulation under heterotrophic condition.