| Currently, new energy source are being investigated because of the exhausted stocks of fossil fuels and atmospheric pollution. As one of renewable, biodegradable, nontoxic bio-energy, biodiesel is becoming the focus in energy field. Heterotrophic alge cells can reach higher biomass concentration and accumulate moderate lipid content, but the low lipid productivity can not meet the feedstock demands of large-scale biodiesel production due to the accumulation of biomass and lipid is in different growth stage. It has been demonstrated that ion stress strategy could effectively enhance the lipid productivity to realize the industrial production of biodiesel. The paper investigated the lipid accumulation and metabolic pathway of Chlorella protothecoides under Cu2+/Fe2+ induction. The main contents and results were as follows:(1)The optimized biomass and lipid yield were achieved by 6.47 g/L and 5.78 g/L with Cu(Ⅱ) stressed strategy. The fatty acids compositions(almost 100% of them are C15 to C20) are ideal for preparing high quality biodiesel. Further, 30 differentially expressed proteins response to Cu(Ⅱ) stressed strategy fell in 12 major biological process groups. Among them, seven proteins participated in carbohydrate metabolic process, comprising triosephosphate isomerase(spot A75), transaldolase(spot A107), enolase(spot A133), pfkB family kinase(spot A144), glucose-1-phosphate adenylyltransferase(spot B9), glycoside hydrolase(spot B30), and glucose-6-phosphate isomerase(spot B92). One up-regulated protein, ribulose bisphosphate carboxylase large chain(spot A89) was involved in Calvin cycle. 3-hydroxyacyl-[acyl-carrier-protein] dehydratase(spot A12), glycerol-3-phosphate acyltransferase(spot A25), and acyl-CoA-binding protein(spot A134) were up-regulated proteins which participated in lipid metabolism process. Three down-regulated proteins were assigned with TCA cycle, i.e. malate dehydrogenase(spot B26 and B105) and succinyl-Co A synthetase subunit alpha(spot B108). The up-regulated protein spot A58(ATP-dependent Clp protease proteolytic subunit) and both of down-regulated protein spot B46(Peptidyl-prolyl cis-trans isomerase) and spot B59(60S ribosomal protein L5) participated in protein metabolic activities, including protein biosynthesis, degradation, and folding. Five up-regulated proteins were designated as regulators and transporters of biological processes, including kinase-like protein(A01), kinase-like protein(A34), casein kinase I(A138), ATP synthase subunit alpha(A126), and nucleoside transporter(A37). Two down-regulated proteins, DNA-directed RNA polymerase subunit beta(spot B83) and RNA-dependent RNA-polymerase(spot B42) were involved in RNA biosynthesis. The up-regulated 5’-nucleotidase domain protein(spot A42) and the two down-regulated inosine-5’-monophosphate dehydrogenase(spot B122) and adenylate kinase(spot B91) were associated with nucleotide metabolism. Additionally, nucleoside diphosphate kinase II(spot B61) and superoxide dismutase(spot B62) participated in ATP biosynthesis and ROS scavenging and defense activities, respectively.(2)The lipid accumulation up to 62 % and a maximum lipid productivity of 820.17 mg/L/d was achieved with iron(II) induction strategy. The induced cells produced significantly elevated levels of 16:0, 18:1Δ9, and 18:2Δ9, 12 fatty acids(over 90 %). Furthermore, 31 altered proteins in iron(II) induced algal cells were successfully identified. These differentially expressed proteins were assigned into 9 molecular function categories, including carbohydrate metabolism, lipid biosynthesis, calvin cycle, cellular respiration, photosynthesis, energy and transport, protein biosynthesis, regulate and defense, and unclassified. The down-regulated 3-phosphoshikimate 1-carboxyvinyltransferase(L19) participated in carbohydrate metabolism. Three up-regulated 3-oxoacyl-[acyl-carrier-protein] synthase(H44), S-acyltransferase(H16) and malic enzyme(H09) were responsible for lipid biosynthesis. One up-regulated ribulose bisphosphate carboxylase large chain(H22) was involved in Calvin cycle. Two up-regulated cytochrome c biogenesis protein CcsA(H33) and cytochrome b6-f complex iron-sulfur subunit(H37) participated in respiration and photosynthesis. Three up-regulated ATP synthase(H12, H14, and H15) and one down-regulated ATP-dependent(S)-NADH-hydrate dehydratase(L36) were assigned as energy and transport. Five up-regulated proteins, elongation factor(H08), 60 S ribosomal protein L37a(H17), adenosylhomocysteinase(H23), mitochondrial GTPase 1(H47), and 40 S ribosomal protein S27(H88) were involved in protein biosynthesis. Six up-regulated proteins, proteasome subunit beta type(H10), serine/threonine-protein phosphatase(H28), V-type H+ ATPase subunit A(H48), tubulin beta chain(H06), heat shock protein 70B(H01), and MATE efflux family protein(H19) participated in regulate and defense, respectively.The expression abundance of malate dehydrogenase, ATP synthase, ribulose bisphosphate carboxylase and ribosomal protein in Cu2+/Fe2+ induced C. protothecoides cells were all changed. It is expected to lay the foundation for breeding and genetic engineering strategies targeting enhanced lipid accumulation for industrial applications of based-microalgae biodiesel. |
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