Molecular Regulation Mechanism Of Biosynthesis Unsaturated Fatty Acids By Polyketide Synthase Pathway In Marine Microbes

Docosahexaenoic acid?DHA?is an omega-3 unsaturated fatty acid,has been associated with beneficial health effects.It is well known that de novo synthesis of DHA does not occur in the human and it has very important medical applications and great nutritional value.Schizochytrium sp.is a kind of the ideal sources of DHA,which the synthetic pathway is polyketide synthase pathway?PKS?,has important development prospects in industrial production.However,at present stage,there are few studies on its molecular regulation mechanism.Consequently,this paper intends to study molecular regulation mechanism of biosynthesis unsaturated fatty acids by PKS in marine microbes.The results obtained were listed as follows:Sustainable sources have to be developed to meet the increasing consumer demand for DHA.Saccharomyces cerevisiae was the first fungus to be sequenced by the whole genome and has been certified as safe strain by FDA.Due to the maturation of S.cerevisiae genetic manipulation,it has been used as a host for cloning or purifying proteins,and as a tool for determining the function of enzymes and individual modules in PKS clusters.However,there was no report on the biosynthesis of DHA by PKS pathway in Saccharomyces cerevisiae.This study successfully reconstructed the PKS pathway of DHA synthesis of Schizochytrium sp.FJU-512 in Saccharomyces cerevisiae BJ5464-Npg A for the first time,and obtained the target product?DHA?.Then,the protein was purified to obtain a higher purity target protein,and its activity was confirmed in vitro.In addition,the metabolic engineering regulation was used to increase the yield of oil and DHA,and the highest yeild of fatty acids DHA of total fatty acids and DHA yield reached 387.78mg/L,10.21%,and39.57mg/L,respectively,which were at the leading level of heterologous expression.Both Schizochytrium and Saccharomyces were single-celled eukaryotic,and there were numerous metabolic similarities between them.In this study,the results of metabolic regulation of S.cerevisiae engineering strain to enhance DHA biosynthesis were applied to Schizochytrium sp.FJU-512,and it was found that the addition of malate can significantly promote the accumulation of DHA.In order to study its molecular regulation mechanism,the global changes in genes levels of Schizochytrium sp.FJU-512 were systematically analyzed for both lipid accumulation and lipid turnover phase through comparative transcriptome analysis.Transcriptome analysis demonstrated that the genes for oxidative phosphorylation were up-regulated at lipid accumulation phase and were down-regulated at lipid turnover phase when malate was used.Nevertheless,?-oxidation and pentose phosphate pathway were down-regulated at lipid accumulation phase but were up-regulated at lipid turnover phase.Furthermore,it was found that glycolysis and citrate cycle?TCA cycle?were inhibited,and pyruvate catabolism,branched-chain amino acid metabolism,fatty acid and vitamin VB₆ anabolism were heightened in both two stages.Specifically,previous research have misinterpreted the supply of acetyl-Co A and NADPH under some stress treatment,such as Acetyl-Co A carboxylase?ACC?and ATP citrate lyase?ACL?were up-regulated for enhancing the supply of acetyl-Co A,and malic enzyme?ME?was a key enzyme for promoting NADPH.However,this paper provides results showed that pyruvate dehydrogenase E2 component?PDHE2?and acetolactate synthase I/II/III large subunit?ILVB,ILVG and ILVI?were the major provider of acetyl-Co A,while glucose-6-phosphate dehydrogenase?G6PD?was a chief contributor of NADPH,which provides a theoretical basis for the metabolic engineering to enhance biosynthesis of docosahexaenoic acid in Schizochytrium sp..In order to further study the mechanism of fatty acid PKS synthesis,this study used protein crystallography to reveal the catalytic mechanism of key enzyme ER.Through bioinformatics comparative analysis,it was found that in the PKS pathway of Schizochytrium sp.FJU-512,ER is in the Orf B and Orf C subunits respectively,while in Shewanella piezotolerans WP3,ER is Pfa D,which is a single enzyme.Therefore,this study performed a structural analysis of Pfa D in the PKS synthase system derived from Shewanella piezotolerans WP3.Herein,we report the recombinant expression in E.coli BAP1 and purify Pfa A,Pfa B,Pfa C and Pfa D from Shewanella piezotolerans WP3.The crystal structures of Pfa D were solved by molecular replacement.We also presented the 2.0?resolution crystal structure of Pfa D,which was an enoyl reductase.The model of Pfa D was consisted of one homodimer in the asymmetric unit,and each subunit contained three domains:the N-terminal domain,the central domain forming a classic TIM barrel with a single FMN cofactor molecule bound atop the barrel and the C-terminal domain forming a lid above the TIM barrel.We docked NAD and an inhibitor?TUI?molecule into the active site and speculated the inhibition and catalytic mechanism,which provided theoretical basis and technical support for improving LC-PUFA accumulation through protein engineering and metabolic engineering strategies.