Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Heterogeneous Expression of △~6-Fatty Acid Desaturase Gene in Yeast and Construction of Novel Expression Vectors for Oleaginous Yeast

Author WangPing
Tutor JiangMuLan
School Chinese Academy of Agricultural Sciences
Course Microorganism
Keywords Polyunsaturated Fatty Acid △~6-Fatty Acid Desaturase Gamma-Linolenic Acid (GLA) novel expression vector Oleaginous Yeast
Type Master's thesis
Year 2009
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Gamma-Linolenic Acid (GLA) is one of the important polyunsaturated fatty acids and an essential fatty acid for human. It has a series of biological functions, such as regulation of lipid metabolism, strengthening of nutrition, inhibiton of ulcers, enhancing insulin, anti-tumor, helping weight loss and so on. Therefore, development of genetically engineered strains which can produce high levels of GLA is of great significance. Oleaginous Yeast is an excellent strain for genetic improvement of lipdic characters because of not only its low cost, short fermentation cycle, small negative impact to environment, but also high content of oils and linoleic acid. Heterogenous expression of genes in Oleaginous Yeast has not been reported untill now.In the previous work, we have already cloned two△6-fatty acid desaturase genes from Cunninghamella echinulata MAIN6 and Rhizopus stolonifer YF6. In this study, on the one hand, the expression vectors with these two genes were constructed respectively, which can be used in Pichia pastoris GS115 and Saccharomyces cerevisiae INVscI to verify the functions of these two△6-fatty acid desaturase genes. On the other hand, two novel transformation systems for Oleaginous Yeast Lipomyces kononenkoae and Trichosporon fermentans were constructed. All these work are helpful for the further experimental and theoretical studies of Oleaginous Yeast.Results indicated that recombinant P. pastoris GS115 with△6-fatty acid desaturase gene from C. echinulata MAIN6 could accumulate GLA to 3.0% of total fatty acids content, while recombinant P. pastoris GS115 with△6-fatty acid desaturase gene from R. stolonifer YF6 could not accumulate GLA. Both of S. cerevisiae INVscI recombinants with△6-fatty acid desaturase gene from C. echinulata MAIN6 and R. stolonifer YF6 could not accumulate GLA, which could be as a result of S. cerevisiae INVscI was not suitable for the expression of more than 30 kDa protein. Alternatively, these genes could not be expressed on the minimum growth medium.With hybrid promoter hp4d from pINA1296, the hygromycin B resistance gene(HYG)and green fluorescent protein gene(GFP), we have successfully constructed a novel expression vector for genes expressed in L. kononenkoae and T. fermentans. Results showed that HYG and GFP have both been expressed in L. kononenkoae due to the novel expression vector. However, only HYG has been expressed in T. fermentans. We noticed that T. fermentans itself has obvious fluorescence.

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