Specific Mutagenesis And Screening Methods For Breeding Higher DHA Producer Of Schizochytrium Sp. And The Related Mechanism Analysis

Docosahexaenoic Acid?DHA?is an?-3 polyunsaturated fatty acid?PUFA?,which has functions of promoting and maintaining the nervous system,preventing cardiovascular diseases,anti-inflammatory,anti-cancer,etc.Schizochytrium is a unicellular eukaryote with high DHA content.Compared with the traditional DHA source of fish oil,DHA production by fermentation of Schizochytrium has advantages of low cost,low pollution,no seasonal variation,simple fatty acids composition,and no fishy smell.It has been widely used in the industrial production of DHA,and is becoming the main source of DHA oil.Considering of the large genome of Schizochytrium and the unclear mechanism of DHA biosynthesis pathway,the correlations between cell morphological changes and DHA biosynthesis during fermentation process of Schizochytrium sp.31 were investigated,a dual mutagenesis and screening method for breeding of high DHA producer was designed,and the mechanism of higher DHA yield was explained by transcriptomics study.Based on the above research,the DHA fermentation of Schizochytrium was optimized.The main results are as follows:1.Based on the cell morphological changes observation by scanning electron microscope?SEM?and the intracellular lipid body accumulation analysis by fluorescent staining and laser scanning confocal microscope?LSCM?,four fermentation stages were clarified as lag stage,balanced growth stage,lipid accumulation stage,and lipid turnover stage.The differences of cell proliferation manners,intracellular lipid body distributions and fatty acid biosynthesis pathways predominance were discovered.During balanced growth stage,cells mainly proliferated as successive binary cell divisions,lipid bodies spread evenly in the cell,fatty acid synthase?FAS?products kept increasing and polyketide synthase?PKS?products kept decreasing.When fermentation process came into lipid accumulation stage,cells mainly appeared as zoosporangia,the lipid bodies started to merge with each other and form larger ones;during this stage,the PKS products started to increase and the FAS products started to decrease.Through microscopic tracking of cell structure changes,the cell morphology was associated with the main fermentation parameters,and the fermentation stages can be quickly and promptly determined.Then an easy method for determination of the key points of DHA synthesis during fermentation period was established.2.Atmospheric&Room Temperature Plasma?ARTP?mutagenesis was applied for Schizochytrium mutation,and a dual screening method was designed that screening of high lipid content cells and high DHA percentage cells in two steps.By evaluation of 3 mutation methods:ultraviolet?UV?,ethylmethylsulfone?EMS?and ARTP,ARTP mutagenesis,which had the highest positive mutation rate and best genetic stability of mutants,was selected as the optimal mutagenesis method for Schizochytrium sp.S31 mutation.Based on the strategy that gradually increasing the cell lipid content and DHA percentage in total fatty acids to improve the final DHA yield,a breeding method was designed that after the first round of ARTP mutagenesis,a succinic dehydrogenase competitive inhibitor malonic acid?MA?was used to screen high lipid content mutants,which were further used for the second round of ARTP mutagenesis and screened by glycopeptide antibiotics zeocin for high DHA content mutants.A high DHA yield mutant mz-17 was finally obtained,with lipid content,DHA content and DHA yield of 59.5%,44.2%and 27.2%,respectively in 250-mL flask scale fermentation,which were improved by 30.8%,22.1%and 87.6%compared with that of the wild-type strain S31.3.The comparative transcriptional analysis was carried out on the high DHA yield mutagenesis strain mz-17 and the wide-type strain S31.The differential expressed genes?DEG?of two strains during the balanced growth stage and lipid accumulation stage were analyzed from four levels,including carbohydrate metabolism,amino acid metabolism,fatty acid biosynthesis pathways and phosphorylation levels,and the metabolic mechanism of high DHA yield in mz-17 was revealed.During fermentation,the glycolysis pathway?EMP?,leucine?Leu?,and isoleucine?Ile?degradation pathways of mz-17 were enhanced compared with S31,especially in lipid accumulation period,with log2 ratio between 1.08 and 1.58,which increased the acetyl CoA accumulation.Besides,the hexose monophosphate pathway?HMP?,the tricarboxylic acid cycle?TCA?,Leu and Ile synthesis pathways,and valine?Val?degradation pathway were all significantly inhibited in mz-17,the log2 ratio of which were all less than-1 at 24 h,reducing the non-fatty acid synthesis consumption of acetyl CoA.The down-regulation of FAS genes?log2 ratio<-1?and up-regulation of PKS genes?log2 ratio>1?led the fatty acid biosythesis to the direction of PUFAs and accumulation of DHA was enhanced.The oxidative phosphorylation level was inhibited in mz-17,which related gene's expression decreased to log2 ratio of-4.03,as a result the low dissolved oxygen?DO?tolerance was improved,providing an advantage for the fatty acid PKS synthesis pathway.4.Based on the fermentation characteristics of mz-17 and the DEG analysis of metabolic pathways,the fermentation optimization was carried out by supplementation of promotion factor for DHA synthesis,adjustment of pH,and control of DO.A Fe²⁺supplementation strategy was designed,in which Fe²⁺concentration was controlled at 1.0 mmol·L^-11 for the first 48 h of fermentation,than supplemented to 1.5 mmol·L^-1,and the DHA production was enhanced by 18.9%;The effect of different pH values to the cell growth and lipid accumulation was investigated,and a two-stage pH control strategy was proposed,in which pH was controlled at 7.0 for the first 60 h of fermentation,and then shifted to 5.0 for the rest of the fermentation.Under this pH control strategy,the DHA production reached 19.7 g·L^-1,which improved by 22.2%.Finally,based on the RNA-seq analysis result that the oxidative phosphorylation level decreased and the characteristic that PKS pathway,which biosynthesis of DHA in Schizochytrium,need no consumption of oxygen,a DO gradually reduction strategy that the DO was decreased from 40%to 20%and 5%at 48 h and 60 h,respectively was designed.The biomass,lipid content and DHA percentage were all increased,and the final DHA production was increased by 15.6%.After the fermentation optimization,the DHA production of mz-17 reached 22.8 g·L^-1,which improved by 68.0%.