Characterizing and Engineering Triacylglycerol Biosynthesis in Rhodococcus

Bacteria-derived triacylglycerols (TAGs) have been proposed as a potential alternative for the production of biodiesel. TAGs from Actinobacteria, such as rhodococci, are of particular interest since these bacteria can grow on lignocellulosic biomass and do not compete with the human food supply, in contrast to TAGs derived from crops. Moreover, strains such as Rhodococcus jostii RHA1 accumulate over 60% of their dry weight in TAGs under conditions of nutrient stress, such as nitrogen limitation (N-). Studies of TAG biosynthesis in RHA1 have been confounded by the occurrence of multiple homologs of Kennedy pathway enzymes. We are elucidating the physiological relevance of these homologs under various conditions. A quantitative analysis of the transcripts of these genes revealed that two diglyceride acyltransferases, atf8 and atf10, were more abundant under N- conditions, while atf6 and atf9 were the most abundant transcripts under nitrogen saturating conditions (N+). Time-point analysis of the expression of these genes revealed that the former were transcribed upon ammonia exhaustion, while the latter were transcribed once the carbon source was consumed. Accordingly, under N- conditions, TAGs began to accumulate after ammonia was depleted. Additionally, our bioinformatic analyses predict three members from the haloacid dehalogenase superfamily (HADs) to be involved in the Kennedy pathway. Targeted deletion of these genes, along with their overexpression in wild-type RHA1 greatly perturbed TAG accumulation, highlighting the role of these homologs in catalyzing the last steps of the Kennedy pathway. Overall, our studies facilitate the engineering of RHA1 as a platform to produce a biodiesel precursor.