Characterization of key triacylglycerol biosynthesis processes in rhodococci

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production

Genomics of Rhodococcus

Members of the genus Rhodococcus have metabolic versatility and unique adaptation capacities to fluctuating environmental conditions, enabling the colonization of a wide variety of environments; they also play an important role in nutrient cycling and have potential applications in bioremediation, biotransformations and biocatalysis. Rhodococcus spp. are mainly distributed in soil, water and marine sediments, although some of them are also pathogens for humans, animals and plants. Consistent with the wide catabolic diversity, Rhodococcus spp. possess large and complex genomes (up to 10.1 Mbp), which contain a multiplicity of catabolic genes, high genetic redundancy of biosynthetic pathways and large catabolic plasmids, the latter encoding peculiar metabolic and physiological traits. Recently, the progress in sequencing technology led to a dramatic increase in the number of sequenced Rhodococcus genomes, which have been investigated through diverse bioinformatic approaches. In particular, whole-genome comparative and genome-based functional studies were associated to omic technologies for the study of the global Rhodococcus cell response with the aim of providing insight into the genetic basis of specific catabolic capacities and phenotypic traits. Lastly, genome-based advances in Rhodococcus engineering led to the first design of molecular toolkits for tunable and targeted genome editing. Besides this, genome-based metabolic models were developed to make metabolic predictions of the Rhodococcus cell response to specific growth conditions. Both the synthetic and system approaches offered new opportunities for genome-scale rational design of Rhodococcus cell for environmental and industrial applications