5-12: Biodiesel synthesis through in vivo transesterification by Gordonia KTR9

Members of the Actinomycete bacterial group are a potential platform for biodiesel production. Several strains within this group accumulate high levels of triacylglycerol (TAG), a precursor to biodiesel. Gordonia KTR9, an oleaginous actinomycete, was grown in media supplemented with methanol, ethanol, or propanol. Cultures were able to grow in the presence of up to 4% methanol and ethanol and 2% propanol. Alcohol concentrations between 0.5-1% did not effect growth but at 4% growth rates were reduced by 50% and at 8% no growth occurred. KTR9 grew on ethanol as the sole carbon source but was unable to grow on methanol and propanol as the sole carbon source. Production of methyl, ethyl, and propyl esters was observed. C16:0 was the predominate hydrocarbon, accounting for 80-90% of the ester content in cultures grown with methanol and ethanol while C18:1 was the primary propyl ester hydrocarbon. TAG synthesis is mediated by a wax esterase/diacylglycerol acyltransferase, which is also capable of catalyzing transesterification. This enzyme is encoded by homologs of the atf gene. Analysis of the KTR9 genome identified seven putative atf homologs with significant homology to the atf genes of Rhodococcus jostii RHA1, Three of these homolog’s contain the HHxxDG active site motif and are potentially responsible for TAG accumulation in KTR9. Expression of these genes was confirmed by microarray analysis. Currently we are exploring the role of these atf homologs in TAG synthesis and ester formation by overexpression and gene knockout analysis.