Identification and metabolomic analysis of a starch-deficient Crypthecodinium cohnii mutant reveals multiple mechanisms relevant to enhanced growth and lipid accumulation

Abstract

Docosahexaenoic acid (DHA, C22: 6), which can be synthesized by the heterotrophic microalga Crypthecodinium cohnii, has received significant interest recently, due to its important roles in human health. However, the high production cost caused by low productivity and yield is still a major hurdle for industrial production of DHA using C. cohnii. The reduction of starch content has been found to be a promising mean to drive more carbon source to lipid biosynthesis in C. cohnii; however, it usually affects cell growth negatively, which limits its practical application. In this study, atmospheric and room temperature plasma mutagenesis associated with high-throughput screening based on iodine vapour was developed to identify a starch-deficient C. cohnii mutant 16D. The mutant 16D accumulated more biomass, lipid and DHA, with less starch and extracellular polysaccharide. The productivity and the yield of DHA were elevated 1.7 and 1.3 fold, reaching levels of 57.7 mg/L-h and 52.0 mg/g, respectively, in fed-batch fermentation, which are the highest reported so far for DHA production using C. cohnii. LC- and GC–MS based metabolomic analyses revealed multiple possible mechanisms relevant to increased growth and lipid content in mutant 16D, including strengthened Embden-Meyerhof, Krebs cycle and pentose phosphate pathways to provide more CoA, ATP and NADPH for supporting growth and lipid accumulation; and a significant increase in the intracellular abundance of tagatose, which was demonstrated to be involved in lipid content enhancement possibly by activating the triacylglycerol biosynthesis pathway in mutant 16D. The study presents a new strain improvement strategy for DHA-producing C. cohnii and new insights for switching metabolism from starch to growth and lipid in C. cohnii. In addition, the study also demonstrates the inductive effects of tagatose on lipogenesis in heterotrophic microalgae.