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Overexpression of ‘tesA Gene and Effect on Conversion of Fatty acyl-CoA to Fatty acid

Overview :

We aimed to overexpress the ‘tesA gene (which codes for thioesterase I) in E. coli to (1) enhance the conversion of fatty acyl-CoA to free fatty acid, and (2) bypass the β-oxidation pathway for fatty acid metabolism. To facilitate this strategy, we have subcloned the ‘tesA coding sequence downstream of the PBAD promoter in the pSB1C3 vector. E. coli TOP10 cells (Invitrogen) was used as the host to overexpress ‘tesA. Expression of ‘tesA gene was measured by quantitative RT-PCR and the effect of ‘tesA overexpression (leading to a possible increase in thioesterase I activity) was indirectly tested by determining the oleic acid concentrations in control and ‘tesA recombinant E. coli cells by GC-MS.

Results :

Quantitative real-time PCR analyses showed that ‘tesA mRNA overexpression was observed only in recombinant E. coli Top10 cells and not in the DH5α host. Expression of ‘tesA was 2.5-fold higher in the recombinant ‘tesA E. coli TOP10 cells than the control cells (Figure 1).

For the fatty acid analysis in E. coli TOP10 cells, the levels of both oleic acid and palminitic acid were elevated in recombinant ‘tesA TOP10 cells as compared to the control host cells (Figures 2A and 2B). The results suggested that the increase in oleic acid and palmitic acid levels in the recombinant TOP10 cells may be due to increased expression of the ‘tesA gene (and hence thioesterase I activity) in these E. coli cells. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future in order to determine the statistical significance of the data.