Engineering the yeast Yarrowia lipolytica to synthesize very- long-chain fatty acids and the effects of temperature on fatty acids synthesis

Abstract:

[Background] The yeast Yarrowia lipolytica is an oleaginous microorganism and can produce long-chain fatty acids and oils with high cell contents. However, the synthesis of very-long-chain fatty acids by this yeast remains to be investigated. [Objective] To engineer Y. lipolytica to synthesize very-long-chain fatty acids with high values and evaluate the influences of temperature on biomass, oil production, and fatty acids compositions. [Methods] The fatty acids elongation enzyme genes AtFAE1 in Arabidopsis thaliana, BtFAE1 in Brassica tournefortii and CgKCS in Cardamine graeca were codon-optimized according to the frequency of codon usage in Y. lipolytica. The plasmids pYLEX1-AtFAE1, pYLEX1-BtFAE1, pYLEX1-CgKCS and pYLEX1-AtFAE1-BtFAE1-CgKCS were constructed and used to transform the host strain Y. lipolytica Po1g, generating the yeast strains Po1g-AtFAE1, Po1g-BtFAE1, Po1g-CgKCS and Po1g-AtFAE1-BtFAE1-CgKCS. Furthermore, the DGAT1 gene encoding diacylglycerol acyltransferase was overexpressed in the strain Po1g-CgKCS to improve oil production. [Results] The results showed that the overexpression of AtFAE1, BtFAE1, and CgKCS led to the remarkably difference in the composition and content of fatty acid in Y. lipolytica. AtFAE1 mainly catalyzes the synthesis of C20:1, BtFAE1 prefers to synthesize erucic acid (C22:1), and CgKCS can catalyze the synthesis of nervonic acid (C24:1). Nevertheless, co-expression of the three genes in Y. lipolytica did not increase the production of nervonic acid. Overexpression of the gene DGAT1 in Po1g-CgKCS apparently increased the oil content by 50%. The results from the temperature experiment showed that a lower temperature benefited the synthesis of unsaturated fatty acids, while a higher temperature benefited the synthesis of saturated fatty acids in Y. lipolytica in our tests. [Conclusion] The fatty acids elongase CgKCS can directly catalyze the synthesis of the C24:1 fatty acid from the C18:1 fatty acid, and the synthesis of unsaturated fatty acids can be improved by optimizing the culture temperature. This study provides theoretical and technical guidance for the construction of cell factories for very-long-chain fatty acids and the optimization of fermentation.

Key words:Very-long-chain fatty acids, Yarrowia lipolytica, Lipid, Metabolic engineering, Temperature, Fermentation