Engineering a malic enzyme to enhance lipid accumulation in Chlorella protothecoides and direct production of biodiesel from the microalgal biomass

Abstract Genetic engineering of the eukaryotic microalga Chlorella protothecoides remains in the early stages of development. There is also an urgent need to develop cost-effective biodiesel production processes from wet and lipid-rich C. protothecoides. Malic enzyme is a potential key enzyme responsible for a rate-limiting step associated with lipid accumulation in some microorganisms. In this study, we genetically engineered C. protothecoides by overexpressing a key malic enzyme to improve lipid accumulation for direct biodiesel production from microalgal biomass. An open reading frame encoding 509 amino acid residues of a putative malic enzyme was obtained and identified. Cloning and overexpression of the malic enzyme gene allowed genetic engineering of C. protothecoides for lipid accumulation and further direct production of biodiesel without the need for lipid extraction steps. The total lipid accumulation in genetically engineered C. protothecoides increased 2.8-fold compared to the wild type. A dehydration step using methanol pretreatment coupled with H 2 SO 4 or lipase catalyzed biodiesel production enabled 93–98% biodiesel yields directly from microalgal biomass without extraction of lipid under optimal conditions. The efficient direct dehydration and chemical and enzymatic transformation processes could significantly reduce the overall cost of biodiesel production from wet and lipid-rich microalgal feedstocks.