| Isochrysis sphaerica and Phaeodactylum tricornutum, rich in DHA (Docosahexaenoic acid, C22:6 n-3) and EPA (Eicosapentaenoic acid, C20:5 n-3) respectively, are two kinds of the most important marine microalgae. They are primary producers of the very long chain polyunsaturated fatty acids (PUFAs), thus can be used as the alternative source for commercial production of PUFAs. As their positive effects on people's health are becoming more and more evident, these two kinds of marine microalga attract more attention nowadays. In this dissertation, by using I.sphaerica and P.tricornutum as research materials, we investigated the effect of environmental factors on growth and fatty acid composition and content of I.sphaerica, cloning and functional characterization ofΔ4-fatty acid desaturase andΔ5-fatty acid elongase from I.sphaerica and P.tricornutum, co-expression of isfad4 and ptfae5 and so on. The results and significance of this study are summarized as follows:(1) Growth curve and fatty acids composition of I. sphaerica under different light intensity and temperatures were examined in this study. Results showed that light intensity significantly affected the growth rates. The higher the light intensity, the faster the growth of I. sphaerica. Contents of PUFAs (Polyunsaturated fatty acids) were affected if the linght intensity were higher than 58μmol/(m~2·s) and lower than 16μmol/(m~2·s). Proportion of DHA reached to 11.77% of total fatty acids when light intensity was 24μmol/(m~2·s). The optimal growth temperature for I. sphaerica was between 20℃to 25℃. Low temperature was beneficial to TUFAs (Total unsaturated fatty acids) accumulation. Under 20μmol/(m~2·s) of linght intensity, the TUFAs proportions reached to its highest of 64.99% at 20℃, and the DHA proportions reached its highesof 12.19% at 15℃.(2) A novelΔ4-fatty acid desaturase gene which we named isfad4 was isolated for the first time from I.sphaerica via degenerate PCR, Genome walking and RT-PCR. isfad4 was 1284bp in length, encoding a protein of 427 amino acids with an estimated molecular weight of 48 kDa. Sequence analysis indicated that the predicted amino acid of isfad4 was similarity to otherΔ4-fatty acid desaturase from other microalgae and fungi, including three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. By adding heterologous substrate DPA(n-3) in the culture medium, we confirmed the predicted enzyme function that catalyzed the conversion of DPA(n-3) into DHA in Pichia pastoris. The conversion rate was 79.8%.(3) Based on a function unknown gene fragment, extension primer PCR was used for cloning the gene ofΔ5-fatty acid elongase from P.tricornutum. A novelΔ5-fatty acid elongase gene was isolated and we named it as ptfae5, which was 1110bp in length, encoding a protein of 369 amino 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 P.pastoris demonstrated that ptfae5 encoded aΔ5-fatty acid elongase that mediated the elongation of EPA to DPA (Docosapentaenoic acid, C22:5 n-3) and ARA (Arachidonic acid, C20:4 n-6) to DTA (Adrenoic acid, C22:4 n-6), the conversion rate for EPA to DPA(n-3) and ARA to DTA is 91.8% and 81.1%, respectively. In the condition of keeping the core regions of ptfae5, the shorten gene was transformed into the P.pastoris. The heterologous expression in P.pastoris indicated that the N-terminals lacking ofΔ5-fatty acid elongase from P.tricornutum did not changed the substrate specificity but reduced the activity of the enzyme.(4) TheΔ4-fatty acid desaturase gene isfad4 and theΔ5-fatty acid elongase gene ptfae5 was used for constructing the co-expressing vector pAOd4e5, which was then integrated into P.pastoris. Functional analysis through Gas Chromatograph (GC) indicated that the recombinant strain had the ability of transforming EPA into DHA, the results showed that both the isfad4 and ptfae5 were efficiently expressed. The conversion rates for EPA and DPA(n-3) were both 90.8%, the conversion rates for ARA and DTA were 83.2% and 90.9%, respectively.
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