Identification And Mechanism Analysis Of A Crypthecodinium Cohnii Mutant With High DHA Productivity

Docosahexaenoic acid(DHA,C22:6),a polyunsaturated fatty acid,can be synthesised by heterotrophic microalgae Crypthecodinium cohnii.Due to its important roles in human health,DHA has received significant attention recently.However,high production cost caused by low productivity and yield is still a major hurdle for industrial production of DHA using C.cohnii.It has been reported that decreasing starch content could be a promising mean to drive more carbon source into lipid biosynthesis in C.cohnii,however,its negative effect on cell growth limits its practical application.In this study,atmospheric room temperature plasma mutagenesis associated with high-through screening based on iodine vapour was developed and applied to identify a starch-deficient C.cohnii mutant 16 D.The mutant 16 D accumulated more biomass,lipid and DHA,while less starch in cells and extracellular polysaccharide.In a fedbatch fermentation process,the productivity and the yield of DHA in C.cohnii mutant16 D were elevated by 1.7-and 1.3-fold,reaching levels of 57.7 mg/L/h and 52.0 mg/g,respectively.The productivity of lipid was elevated by 1.5-fold,reaching a level of185.2 mg/L/h,compared with the wild type strain.The GC-MS and LC-MS based metabolomic analyses were then used to reveal possible mechanisms relevant to the increased growth and lipid accumulation in mutant 16 D.The results showed that multiple mechanisms were involved,including: strengthened Embden-Meyerhof,Krebs cycle and pentose phosphate pathways to provide more Co A,ATP and NADPH for supporting growth and lipid accumulation;up-regulated fatty acids and amino acids to sink excess ATP caused by decreased starch content;at last,the intracellular abundance of tagatose was significantly increased,which was demonstrated to be relevant to lipid content enhancement possibly by activating triacylglycerol biosynthesis pathway in mutant 16 D.The study presents a new strain improvement strategy for DHA-producing C.cohnii and new insights for metabolic switch 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.