Mutation Breeding And Transcriptome Analysis Of Schizochytrium With High-yield DHA Production

Docosahexaenoic acid(DHA)is an important omega-3 series of polyunsaturated fatty acids.Much attention has been paid to its benefits to the human body.It is widely used in infant milk powder,DHA supplements for pregnant women and health products.There are many limitations that extracted DHA by traditional method,at the same time,the yield is difficult to meet the needs of the market.It has many advantages to produce DHA by microbial fermentation,However,the yield is low,and the mechanism of DHA synthesis is not clear.The aim of this research is to improve the yield of DHA in Schizochytriu,m by ARTP mutagenesis,and to reveal the mechanism of high-yield DHA through transcriptome analyse.The main contents of this research is as follows:(1)The original strain SR21 was mutated with ARTP for 15 s.Screened the high-yield strain by plates with different concentrations of malonic acid,iodoacetic acid,and malonic acid-iodoacetic acid mix.A high-yield DHA strain named NA2 with stable characters was obtained by the screening plate with 1.2 g·L-1 malonic acid.Fermented 120 h in shake flask,its biomass,total fatty acid,and DHA yield was 36.00 g·L-1,22.24 g·L-1,6.59 g·L-1,respectively,which increased by 18.11%,14.87%,and 46.11%,respectively,compared with the original strain.(2)Compared the fermentation characteristics of the original strain SR21 and the mutagenic strain NA2 on the 5 L bioreactor,the final DHA production of NA2 was 27.5%higher than that of SR21.Furthermore,optimized the substrate feeding strategy of NA2 in 5 L fermentor,and compared the fermentation performance of four substrate feeding strategies:1)the carbon source(glucose)was intermittently fed,nitrogen source wasn't added;2)the carbon source was intermittently fed,at the same time,nitrogen source was intermittently fed;3)maintain glucose concentration,nitrogen source was intermittently fed;4)DO feed-back feeding strategy.The results showed that under the third strategy NA2 obtained the best fermentation performance.The final biomass,total fatty acid,and DHA yield was 81.83 g·L-1,53.23 g·L-1 and 13.66 g·L-1,respectively.(3)Sequenced the original strain SR21 and mutant strain NA2.Annotated the function of genes,and classificated them to different metabolic pathways.Looked for different expression genes and classified these genes into related metabolic pathways to discusse the possible mechanism of the increase of DHA yield in NA2.The results showed that:Compared with the original strain,the expression of the ECHS1 gene involved in amino acid degradation and the tpiA gene involved in energy metabolism increased,providing a large amount of precursor substance,reducing power,and energy for the synthesis of DHA.Quantitative Real-time PCR technique was applied to virefy the differentially expressed genes,and the results were consistent with the transcriptome analyse.