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Enhancement of Fatty acid Uptake by Overexpression of FadL and FadD in Escherichia coli.

Overview :

In an attempt to increase fatty acid uptake in E. coli, we constructed a tetracycline-repressible fadL-fadD expression system (BBa_K142606). As we were aiming to ensure significant co-expression of the fadL-fadD genes in E.coli, we subcloned the fadL-fadD gene cassette to the IPTG-inducible pSNAP tag (T7)-2 expression vector to test the effect of FadL-FadD overexpression on oleic acid uptake. Expression of the fadL and fadD genes in recombinant and control E. coli host cells was measured by qRT-PCR assays, and fatty acid uptake measured by GC-MS analysis.

Results :

Quantitative real-time PCR analyses showed that fadL and fadD mRNAs were increased in recombinant fadL-fadD E. coli as compared to the control cells in the absence and presence of IPTG induction (Figures 1A and 1B). For the fatty acid uptake analysis, oleic acid concentration in recombinant and controls cells was measured by GC-MS. Although no difference in oleic acid uptake was observed in the fadL-fadD recombinant and control cells at 0 µM and 40 µM of IPTG, the oleic acid concentration in the fadL-fadD transformant was 2-fold higher at 400 µM IPTG than the control cells (Figure 2). Our overall results, show no apparent correlation in fadL and fadD mRNA expression levels and oleic acid uptake in E. coli. However, due to time constraint, these experiments were performed only once, and further replicates will need to be carried out in future studies in order to determine the statistical significance of the data.