Genetic and metabolic engineering of medium chain fatty acids synthesis in yeast : role of thioesterases.

• Main Author: Lee, Benedict Jia Hong.
• Other Authors: Chen Wei Ning, William
• Format: Theses and Dissertations
• Language: English
• Published: 2013
• Subjects: DRNTU::Engineering
• Online Access: http://hdl.handle.net/10356/54064
• Institution: Nanyang Technological University

In this project, we demonstrated the genetic engineering of POX deletion strains of S. cerevisiae (baker’s yeast) through the heterogeneous expression of thioesterases to produce medium chain fatty acids in S. cerevisiae cells. The thioesterases expressed in S. cerevisiae were chosen for its specificity for medium chain fatty acids phenotype in order to terminate fatty acid synthesis at the desired chain length of C8-C12. The cDNA sequences coding for these enzymes were first chemically synthesized and cloned into expression vectors for transformation into the POX deletion strains of S. cerevisiae. Yeast colony PCR was performed to establish that the gene constructs were present in the transformed yeast cells. Yeast colony PCR showed that the vectors containing the gene constructs were successfully cloned into the POX deletion strains of S. cerevisiae. Galactose induction were then performed to express the enzymes coded by the genes in these deletion strains and it was noted that POX1 deletion strain with the rabbit thioesterase II did not grow upon galactose induction. This could be protein toxicity coupled with the effect of POX1 gene deletion. Western blot analysis using His-tag antibody were performed on the other POX deletion strains of S. cerevisiae to detect the presence of thioesterase II proteins. The fatty acid profile of the cloned yeast cells was then analysed by GCMS. The GCMS analysis showed a significant increase of 1.1 ~1.7 fold in production of lauric acid (C12 fatty acid) and myristic acid (C14 fatty acid) within the cells when compared to negative controls containing empty vectors without thioesterase II genes. The proportion of these medium chain fatty acids in the total fatty acid profile of S. cerevisiae had also increased by 6.7% to 18.94%.