| Docosahexaenoic acid(DHA)is a nutritional supplement with important physiological activity for human health,and has important effects on the protection of cardiovascular and cerebrovascular,and the development of human brain and optic nerve.DHA is also used for the prevention and treatment of neurological and cardiovascular diseases such as hypertension,atherosclerosis and Alzheimer’s disease,and is widely used in combination with chemical drugs in the preparation of drug delivery systems for the treatment of cancers such as breast cancer,melanoma and stomach cancer.In the past,DHA was mainly obtained by refining large amounts of deep-sea fish oil,which would seriously pollute the environment and damage the marine ecology.The use of modern fermentation engineering technology to cultivate marine native organisms,Schizochytrium sp.,can realize the large-scale commercial production of DHA.However,the development of this industry is seriously restricted by the problems of easy degradation of strains,low DHA yield and high fermentation cost of Schizochytrium sp.Therefore,research on strain selection,DHA yield enhancement and cost reduction is needed to achieve high yield and low cost fermentation production of DHA,and the main research results were obtained as follows.(1)A high-throughput rapid sorting technique was established to rapidly screen for high-yielding strains of DHA and ensure long-term stable fermentation production.Schizochytrium sp.can differentiate into cell subpopulations with different lipid synthesis abilities during successive passages of culture,leading to a serious decrease in DHA production,which is detrimental to the normal application of this strain in scientific research and industrial production.Strain characterization studies showed that Schizochytrium sp.H016 differentiated into two cell subpopulations with intracellular lipid droplet enrichment and lipid droplet degradation after successive passaged cultures.For this,a rapid screening strategy based on flow cytometric fluorescence sorting technology was established,which can quickly identify and sort out high DHA-producing strains and low DHA-producing strains based on single-cell lipid production capacity,shortening the screening cycle from 15 days to 2 days compared to the previous way of fermentation screening by selecting single colonies,and providing a reliable technical solution to ensure the long-term stability of DHA fermentation production yield.(2)The global regulatory network of DHA synthesis and metabolism in Leptosphaeria fumigatus was elucidated based on the combined transcriptomic and proteomic analyses,revealing a positive correlation between the antioxidant capacity of the microorganisms and DHA production.A subgroup of high and low lipid-producing strains of Schizochytrium sp.provides a research model for studying the metabolic patterns of DHA synthesis.It was shown that enhanced glycolytic pathway and weakened TCA cycle can provide more acetyl coenzyme A precursors for DHA synthesis;pentose phosphate pathway is the main source of NADPH for DHA synthesis;enhanced polyketide synthase pathway facilitates DHA biosynthesis;enhanced glycerolipid synthesis pathway and lipid droplet protein expression facilitates more conversion of fatty acids to triglycerides for storage in intracellular lipid droplets The enhancement of peroxisome is a key factor influencing DHA synthesis and accumulation.This provides important theoretical and practical guidance for the subsequent promotion of DHA synthesis and reduction of lipid peroxidation.(3)The synthesis and accumulation of DHA were significantly promoted by enhancing the antioxidant capacity of Schizochytrium sp.The effects of different antioxidants on the DHA production of Schizochytrium sp.were screened and evaluated.The addition of 1 m M sesamol to the fermentation medium significantly increased the lipid production and DHA production of Schizochytrium sp.by 53.52% and 78.30%,respectively.Metabolic mechanism studies showed that sesamol could induce the gene expression of polyketide synthase and antioxidant enzyme system,increase the total antioxidant capacity of Schizochytrium sp.,reduce the peroxidation of intracellular polyunsaturated fatty acids during the fermentation process,and facilitate the synthesis and accumulation of DHA.In addition,sesamol could inhibit malic enzyme activity and promote glucose-6-phosphate dehydrogenase activity enhancing the supply of NADPH of the pentose phosphate pathway,thus substantially promoting DHA synthesis.Further,the results of batch supplementation fermentation showed that the addition of sesamol significantly increased DHA production by 90.76% to 17.55 g/L,indicating that improving the antioxidant capacity of the organism can significantly promote DHA synthesis and can be an important strategy to improve DHA production in industrial fermentation of Schizochytrium sp.(4)The use of the starch saccharification liquid of Dioscorea zingiberensis tubers(SSLD)instead of glucose as a carbon source significantly improved DHA production and reduced fermentation costs in Schizochytrium sp.The carbon source is the largest and most costly part of the fermentation medium of Schizochytrium sp.,and screening for efficient and cheap carbon sources can significantly enhance the competitive advantage of the industry.SSLD is a hydrolysate produced by enzymatic starch digestion during the industrial extraction of Dioscorea zingiberensis,which is rich in glucose and nutrients such as polyphenols and flavonoids,with the characteristics of high sugar and rich in antioxidant substances.Here,SSLD was used as a novel potential carbon source to promote DHA biosynthesis in Schizochytrium sp.for high value utilization.When the total sugar concentration in SSLD was optimized to 90 g/L,the biomass and DHA production of Schizochytrium sp.increased by 32.1% and 36.92%,respectively,compared with glucose as the carbon source culture condition.Gene expression analysis showed that SSLD significantly increased the expression of antioxidant enzyme genes and polyketide synthase genes in Schizochytrium sp.,which effectively improved the cellular antioxidant capacity and DHA production.Therefore,SSLD is an efficient and economical carbon source for DHA production in Schizochytrium sp.In summary,this study achieved the rapid sorting of high yielding strains of DHA,and constructed a regulatory network for DHA synthesis and metabolism by comparing the physiological metabolic characteristics of high and low lipid yielding strains,and determined the mechanism of increasing antioxidant properties to promote the accumulation of DHA synthesis,and finally achieved a significant increase of DHA yield and reduction of fermentation cost by screening high sugar and antioxidant carbon sources. |
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