Metabolomics Analyses Of Glucose Tolerance And DHA Accumulationin In Crypthecodinium Cohnii

Crypthecodinium cohnii has attracted great attention in DHA production due to its ability to accumulate lipids with high fraction and high quality of DHA.It has been reported that other types of polyunsaturated fatty acids account for only less than 1% of the total lipids in C.cohnii.However,pevious studies showed that high concentration of glucose could inhibit the cell growth of DHA-producing C.cohnii.So a glucose-tolerant strain of C.cohnii would be important for large-scale application,which increases the economic viability of this system.In this study,a glucose-tolerant C.cohnii strain was firstly obtained by Adaptive laboratory evolution(ALE)after 650 days with gradually increased glucose concentration.The results showed that glucose tolerance and DHA-rich lipids accumulation in the evolved strain were significant improved.The average doubling times of strain ALE3 grown on 45 g/L glucose were 31.9% shorter than that of the original strain S0.The specific growth rate and the glucose depletion rate of ALE3 strain were 2.8 and 2.6-fold higher than that of S0 strain under an initial glucose concentration of 45 g/L respectively.Lipid content could increase by 15.49% at 45 g/L glucose concentrations.Metabolic profiles were compared to reveal mechanisms related to glucose tolerance of C.cohnii through ALE.The results showed that hub metabolites including glycerol,glutamic acid,malonic acid and succinic acid were positively regulated during ALE.Metabolomics mechanisms related to glucose tolerance in C.cohnii provided usefull information for further strain improvement.To characterize the growth and DHA production in C.cohnii,fed-batch cultivation was established in a 7.5 L fermenter.The results showed that the highest biomass of 42.9 g/L(DHA yield 5.7 g/L)and DHA productivity of 47.5 mg/L/h were obtained during the fed-bacth fermentation.There was significant increase in lipids accumulation,about 11.4% improvement from 48 h to 72 h,while DHA accumulation improved by 17.8% from 72 h to 96 h.LC-MS metabolomic approach was then employed to decipher possible mechanisms responsible for the growth and DHA production in C.cohnii.Heat map analysis identified metabolites potentially related to growth and DHA accumulation,such asG6 P,GAP,AKG,OXA and DHAP.Oxygen supply is an important factor in aerobic fermentation that could affect not only biomass and product yield but also various cellular functions including carbon metabolism,antibiotic production and stress response.To date,there is few studies about the intrinsic correlation between dissolved oxygen(DO)concentration and C.cohnii metabolism during DHA fermentation process.In this study,the responses of DHA production to diffrenet DO levels in C.cohnii were first evaluated,including specific growth rate,specific glucose consumption rate,fatty acid compositions and DHA productivity.And volcano plot analyses were also used to discriminate and assess the responsive metabolites associated with different DO levels.The results showed that the growth rate and glucose consumption rate under 30% oxygen supply condition were much higher than that under the other condtions tested.However,the highest biomass 49.5 g/L and lipid yield 24.6 g/L were observed under 20% oxygen supply condition.Significant differences under different oxygen supply conditions were observed at the metabolite level.Volcano plot analyses revealed that carbon skeleton for the growth and synthesis of lipid in C.cohnii were mainly from glycolysis and TCA cycle.Glutamic acid and inositol could be related to low dissolved oxygen stress.The results also showed that short-chain fatty acids were used as precursor for longer chains.Ribose-5-phosphate was shown to act as a key factor in DHA accumulation.The results provided useful insights into the functional metabolic relationship between DO levels and DHA production,and valuable information for further improving DHA production in C.cohnii.