Cloning and Characterization of Diacylglycerol Acyltransferase in Escherichia coli and Rhodococcus opacus PD630

Abstract

Biodiesels are increasing in popularity as prices of petroleum have increased over the past two decades and triacylglycerols (TAG) are an attractive alternative as a feedstock to produce biodiesel. Rhodococcus opacus PD630 is an environmental organism well characterized for its ability to accumulate TAG to a high percentage of its cell dry weight. It is known that the accumulation of TAG by R. opacus PD630 is dependent on a high carbon to nitrogen ratio. To improve the attractiveness of R. opacus PD630 as a source of TAG, cheaper waste substrates such as food wastes must be utilized, however, these substrates are likely to be inhibitory to TAG accumulation. Glycerol is a cheap low nitrogen feedstock derived from the production of biodiesel from TAG. Growth of R. opacus PD630 on glycerol as a sole carbon source was examined and the bacterium was unable to grow in the broth culture. Diacylglcyerol acyltransferase (DGAT) is the rate-limiting enzyme in the accumulation of Triacylglcyerols. If TAG accumulation could be controlled independently of host regulation, a wide range of waste substrates could be utilized. To address this, a recombinant E. coli, containing vector pDGAT harboring an IPTG inducible His-tagged DGAT gene, was characterized for its protein expression and lipid accumulation. An optimized electroporation protocol was developed to clone pDGAT into R. opacus PD630 and the expression of DGAT examined. E. coli DGAT was able to produce DGAT protein when induced with IPTG while R. opacus DGAT A, an erythromycin resistant transformant produced DGAT constitutively. A second erythromycin resistant transformant was unable to produce DGAT and was shown to not harbor pDGAT. Sequencing of pDGAT revealed it was never successfully constructed and that this plasmid was in fact pQE-80L-DGAT.