Heterologous Reconstitution Of Polyunsaturated Fatty Acid DHA Biosynthetic Pathway In Saccharomyces Cerevisiae

Polyunsaturated fatty acids (PUFAs) are unbranched fatty acids containing more than one double bond, including DHA (C22:6n-3). They have a number of important nutritional and pharmaceutical values. They are involved in the human inflammatory response, cardiovascular events, adipogenesis, immune response, blood pressure regulation, cholesterol metabolism, development of allergies and infant retinal structures. Recently, their potential functions of reducing heart disease, improving vision sensitivity and reading ability have been verified. Currently, the principal source of PUFAs is deep-sea fish. However, it is considered insufficient and endangered due to an expanding market and environmental pollution. Marine microalgae, the primary producers of PUFAs, are being explored as an alternative source for yielding these essential elements of health.Desaturases and elongase system are critical enzymes involved in biosynthesis of PUFAs and occur in most living cells. Rencently, more and more genes encoding enzymes involved in PUFAs biosynthesis of microalgae, including desaturases and elongases, have been identified. And the genes of desaterase and elongase systems have important biotechnological appeal from genetic engineering point of view. The identification and prospecting of these genes becomes a novel method for enhanced PUFAs production. As we all know, there are two pathways of synthesizing PUFAs, that is the common A6desaturation pathway and the alternative△8desaturation pathway. We focus on reconstitution of DHA biosynthesis pathway in Saccharomyces cerevisiae, therefore it can put some experimental evidence for transgenic crops producing PUFAs.Isochrysis galbana and Pavlova viridis, marine microalgae, are rich in PUFAs, which accumulates up to30%of the total fatty acids. This marine microalgae were used for representing an attractive production system for PUFAs such as DHA. In this dissertation, Isochrysis galbana and Pavlova viridis have-been as useful sources for cloning fatty acid desaturase and elongase genes. The results and significance of this study are summarized as follows: (1) A novel A4-fatty acid desaturase gene which we named IgFAD4-2was isolated from Isochrysis galbana via LA-PCR. IgFAD4-2was1302bp in length, encoding a protein of433amino acids with an estimated molecular weight of48.1kDa. Sequence analysis indicated that the predicted amino acid of IgFAD4-2was similarity to other A4-fatty acid desaturase from other microalgae and fungi, including three conserved histidine boxes and a cytochrome b5domain at its N-terminus. By adding heterologous substrate Docosapentenoic acid (DPA) in the culture medium, we confirmed the predicted enzyme function that catalyzed the conversion of DPA into DHA in Saccharomyces cerevisiae. The conversion rate was34.2%.(2) LA-PCR was used for cloning the gene of A5-fatty acid elongase from Pavlova viridis. A novel A5-fatty acid elongase gene was isolated and we named it as PvElo5, which was840bp in length, encoding a protein of279amino acids. The results of the deduced amino sequence indicated that it contained a typical conserved histidine motif and a double lysine domain located in the endoplasmic reticulum in its C-terminals. The heterologous expression in S. cerevisiae demonstrated that PvElo5encoded a△5-fatty acid elongase that mediated the elongation of Eicosapentaenoic acid (EPA) to DPA and Arachidonic acid (AA) to Adrenic acid (ADA), the conversion rate for EPA to DPA and ARA to ADA are26.9%and26.3%, respectively.(3) The A4-fatty acid desaturase gene IgFAD4-2and the A5-fatty acid elongase gene PvElo5were used for constructing the S. cerevisiae co-expressing A8pathway vector pYAE5-E5-D4and A6pathway vector pYMM5-E5-D4. The transformants containing pYAE5-E5-D4and pYMM5-E5-D4were cultivated in induction medium with exogenous substrate ALA and inducer galactose under the optimum culture conditions. GC analysis of fatty acids extracted from the transformants confirmed that all the genes were expressed and the substrates were converted into DHA. Two engineered strains YAE5-E5-D4and YMM5-E5-D4could produce3.1%and1.6%DHA.(4) Real-time PCR results and fatty acid analysis demonstrated that PUFAs production by the△8pathway might be more effective than that by the A6pathway. Otherwise, Response surface methodology for YAE5-E5-D4showed that the maximum DHA yield of20.74mg/L/d was obtained when the substrate concentration of104.2μmol/L, induction temperature of23.4℃, induction time of56.7h, respectively, the maximum DHA yield of17.74mg/L/d was obtained for YMM5-E5-D4when the substrate concentration of167.7μmol/L, induction temperature of24.2℃, induction time of49.6h, respectively.