Screening And Fermentation Optimization Of High DHA Accumulation Mutants In Schizochytrium Sp. S31

Docosahexaenoic acid(DHA)is an omega-3 polyunsaturated fatty acid that is important to the human body.It has attracted wide attention due to its various effects such as preventing cardiovascular diseases and anti-inflammatory.The Schizochytrium sp.,a marine eukaryotic microalga,has been used widely for industrial production of DHA.Compared with traditional source,such as fish oil,the production of DHA by Schizochytrium sp.is unaffected by seasons and regions.In addition,it has no uncomfortable smell.However,high cost resulted from low DHA concentration and productivity limites its industrial application.Therefore,it is important to increase DHA productivity in Schizochytrium.In this study,a combination of atmospheric and room temperature plasma mutagenesis and screening methods based on inhibitor iodoacetic acid and dehydroepiandrosterone resistance were used to obtain high-producing DHA Schizochytrium.After two rounds of screening,three mutants named M-6-8,M-6-16 and M-6-23 all with increased growth and DHA accumulations were sucessfully achieved.The DHA yield of mutant M-6-23 was increased by 58.48% in shake flask cultivation.In addition,the inoculated amount and carbon nitrogen ratio was optimized in a 5-L fermentor.It was found that the optimized condiction included inoculation amount of 10%(v/v),a carbon nitrogen ratio of 4:1 from 0 to 40 hour and then adjusted to 7:1 from 40 to 80 hour in order to achieve high DHA production.This strategy was applied to M-6-23.The DHA titer and productivity of the mutant M-6-23 reached41.35g/L and 430.73mg/L/h,respectively,which is 71.64% and 35.37% higher than the starting strain.LC-MS metabolomic analysis revealed the increased metabolism in PPP and EMP as well as the decreased TCA cycle,which increased the mutant strain’s ATP,Ac-Co A and NADPH content,which might be relevant to the increased growth and DHA biosynthesis in the mutants.Consistently,the enzyme activity analysis showed that the activities of glyceraldehyde 3-phosphate dehydrogenase and glucose 6-phosphate dehydrogenase increased by 29.18% and 45.94%,respectively.This study presents a new strategy for achieving high DHA-producing Schizochytrium,and establishes a two-stage carbon-nitrogen source ratio feeding strategy in a 5 L fermentor,which significantly increases the DHA production.In addition,the differential regulation at the metabolite level between the starting and mutant strains was investigated,the results provide valuable information for engineering Schizochytrium with improved DHA production.