Study On Metabolic Regulation Of EPA Biosynthesis By Pythium Splendens

Eicosapentaenoic acid(EPA;20:5 ?5,8,11,14,17)is one of many omega-3 long-chain polyunsaturated fatty acids(n-3 LCPUFAs)that can benefit human health which can lower the incidence of cardiovascular disease,atherosclerosis and is of anti-neoplastic and anti-inflammatory functions.EPA is now mainly originated from deep sea fish oil,which can hardly meet the growing demand of the market.Therefore,seeking for alternative sources for EPA production has been a hot spot of current research.EPA production by fungus Pythium splendens is likely to be effective and economical,due to simple cultivate conditions,short fermentation period and potentially high EPA content.However,due to the low carbon utilization rate and the slow microbe growth,EPA yield is not high and can not achieve industrial-scale EPA production.The aim of the present work is to reveal the EPA synthesis pathway and its key enzymes,and increase the growth and EPA yield of P.splendens by metabolic regulation.The findings provides theoretical and experimental basis to the industrialization of EPA production and the strategy employed could be used to increase the production of EPA.The main creative results are as follow:(1)Based on lipid metabonomics analysis,the rate-limiting enzymes of EPA synthesis pathway were revealed.With the method of lipid metabonomics analysis,it is suggested that ?8 desaturase pathway and its intermediate metabolite might exist in EPA synthesis pathway of P.splendens by adding metabolic precursor substances(LA,GLA and ALA).In this pathway,?8 desaturation step appears to operate in the n-3 route,and there is an unusual branch in the n-6 route.More importantly,n-6 route is the main EPA biosynthetic route,and n-3 route and ?8 desaturase pathway are ancillary routes.Based on the analysis of fatty acid metabolism at various cultivation conditions,the results showed that ?6,?12 and ?17 desaturase are the rate-limiting enzymes in EPA biosynthesis.To confirm this result,three transgenic strains were engineered by separately expressing D6,D12 and D17 genes in P.splendens.The results showed that increasing the copy number of the key genes effectively enhanced the n-6 route and improved EPA production while simultaneously decreasing the amount of OA,LA or ARA.EPA yield of D6,D12 and D17 transgenic strain represents 2.2-fold,3.0-fold and 3.6-fold increase over the parent strain,separately.It further indicates that ?6,?12 and ?17 desaturase were the rate-limiting enzymes in this strain.(2)Based on the main EPA synthesis pathway and key enzymes information of P.splendens,the influential factors are investigated and regulated.It is found that under the optimized cultivation conditions,when adding 5 g/L mannitol to the fermentation medium which has 95 g/L glucose,controlling the quantity of inorganic nitrogen source(NaNO3)within 4.5 g/L,and adding 64.45 g/L bean sprout juice,1.50 g/L citric acid,0.19 g/L glutamic acid and 8 g/L linseed oil after 72 h of cultivation,wild-cell got biomass of 39.47 g/L and EPA yield of 1120 mg/L.Compared with before optimization,biomass,EPA yield and the utilization rate of glucose had an increase of 199.6 %,511.4% and 35.6% respectively.These results showed that with the manipulation of adding mannitol adjust osmotic pressure to increase glucose utilization,controlling the quantity of inorganic nitrogen source to promote lipid synthesis,and strengthening CTS and n-6 route,the problem of the slow growth of P.splendens and low utilization of carbon could be effectively solved and hence significantly improved the EPA production.(3)The rheological characteristics of P.splendens suspension culture system is studied.The rheological constitutive equation is established with the analysis of rheological parameters at different fermentation times.The result showed that this suspension culture system is pseudoplastic non-newtonian fluid.Consistency coefficient and rheological index are of exponential and linear relationships with biomass of P.splendens,respectively.(4)Based on the rheological characteristics of P.splendens suspension culture system,the influential factors of batch scale are investigated and regulated.A culturing technology of batch scale which can produce the higher yield EPA is provided.First,according to the analysis of dissolved oxygen demand in different stages,the three-stage dissolved oxygen regulation strategy was established,i.e.,DO0-48 h =15 %,DO48-96 h =20 %,and DO96-168 h =15 %.Second,adding 0.3 g/L NaNO3 when nitrogen source was consumed,and adding 21.5 g/L bean sprout juice after 72 h,120 h and 144 h of cultivation,can not only stimulate ME activity,enhance CTS metabolic flux and NADPH content,but also effectively relieve the shortage of NADPH during the late fermentation.These feed-controlling can remove limiting effect of acetyl-CoA transport and dehydrogenation reaction,and result in a significant increase in lipid and EPA production.Third ME activity caused by the high concentration of NADPH and thus improve the generation of lipid and NADPH and the production of lipid and EPA.This represents a 109.5 % increase in biomass(43.20 g/L)and a 128.6% increase in the EPA yield(1440 mg/L)when grown under above conditions,compared with the fermentation level of shake flask.(5)Based on the gene expression quantity of transgenic strain and its fermentation optimization,D12 gene expression quantity is low,which is the rate-limiting factor of EPA production.Under optimal conditions,D12 transgenic strain eventually made the EPA yield of 1370 mg/L and 1640 mg/L in shake flask and 10 L fermentor,which respectively represent 6.3-fold and 7.5-fold increase over the parent strain before optimization.The above findings can provide theoretical and experimental basis to the industrialization of EPA production.