Author Summary Biofuels research is focused on understanding the energy- related metabolic capabilities of a broad range of biological species. To this end we sequenced the genome of Rhodococcus opacus PD630, a bacterium that accumu- lates close to 80% of its cellular dry weight in oil, a rare trait in the prokaryotic and eukaryotic kingdoms. R. opacus PD630 has a large 9.27 Mb genome that contains many homologous genes dedicated to lipid metabolism. The number and novelty of these predicted genes presents a challenge to the complete and accurate metabolic reconstruction of this species’ metabolism based only on genome sequence. To refine our sequence-based meta- bolic reconstruction, we developed a multidisciplinary approach that included integrating the identification of abundant yet uncommon straight-chain odd-carbon lipid biosynthesis and the results of a catabolic screen for growth substrates. Comparative analysis of the R. opacus PD630 genome sequence with those of a group of related species provided a view into how this bacterium became such a remarkable TAGs producer and led to the identification of a set of biofuels target genes for this group of bacteria. Our synthesis of genome sequence and phenotypic information supports a model for the genetic basis for prokaryotic oleaginy and provides key insights for the engineering of next-generation biofuels with genes that are conserved in both prokaryotic and eukaryotic kingdoms. enzymatic production of the very-long and highly-modified extracellular lipids characteristic of Actinomycetales. Lipid metabolism in the genus Mycobacterium has been a major focus of scientific research due to the effect pharmacological inhibitors of lipid biosynthesis such as isoniazid [17,18,19], thiolactamycin, and pyrazinamide [20] have on killing pathogenic mycobacteria. Whole-genome views of lipid metabolism in mycobacteria reveal these bacteria have developed several lipid biosynthesis systems and a large number of genes to support diverse and abundant lipid biosynthesis. In Mycobacterium an order to the enzyme activity of the lipid synthases has been established through genetic, biochemical, and pharmacological evidence; wherein lipids are biosynthesized de novo by the multifunctional FAS type 1a enzyme followed by further elongation via the FAS II system and the multifunctional MAS-family type 1b synthases. Collectively these 3 fatty acid synthase systems produce 2 classes of fatty acyl- CoAs that differ in chain length. One class contains lipids ,20 carbons (C20) that are components of the plasma membrane and the storage lipids TAGs and WEs. Another class contains lipids .C26 that are built by multiple synthases and can reach lengths as long as C90 in some Mycobacterium species [19,21] but only C60 in Rhodococcus opacus [22] and C54 molecules have been observed in R. equi [23]. The longer chain length lipids are used in Actinomycetales to build a protective extracellular coat that helps these bacteria survive in harsh environments whether it be the phagosome of a macrophage or contaminated soil enriched in toxic organic compounds. The interplay between multiple lipid biosynthesis systems in Actinomy- cetales [19] requires genetic understanding for engineering the flow of carbon to desired lipid types. Results Metabolic Reconstruction of Rhodococcus opacus PD630 To establish a genetic model of Rhodococcus metabolism, we generated a high quality draft sequence of the Rhodococcus opacus Genomics of Rhodococcus for Biofuels PD630 genome. DNA sequencing with 454 shotgun and 3 kb paired-end reads resulted in 16 large scaffolds containing 9.27 Mb of assembled DNA sequence. We stitched the gapped genome scaffolds based on extensive chromosomal-synteny with the complete genome sequences of related Rhodococcus species R. jostii RHA1 and R. opacus B4 (Figure S1). The R. opacus PD630 genome contained 8632 genes that underwent metabolic reconstruction using pathway tools software [24] resulting in a model containing 1735 metabolic reactions. Enzymes were connected to metabolic reactions based on enzyme commission numbers (EC#) that were assigned by the EFICAz2 algorithm [25] and by gene-name recognition within pathway tools software. This automated EC# assignment allowed for multiple genomes to undergo metabolic reconstruction in parallel. Comparisons between metabolic reconstructions for a set of 8 phylogenetically related and one outlier species Ralstonia eutropha H16 that were assembled in this way can be browsed at (http:// tinyurl.com/opacuscyc14-5-comparative). The resulting initial metabolic reconstruction of R. opacus PD630 Opacuscyc14.5_ comparative contains 400 metabolic pathways and 135 transport reactions. A more complete metabolic reconstruction of R. opacus PD630 by pathway hole filling using the pathway tools 14.5 software, additional EC # assignments made with the database at Kyoto Encyclopedia of Gene and Genomes KEGG (http://www. genome.jp/kegg/), and limited manual curation (outlined in Figure S2) resulted in a metabolic reconstruction containing an additional 282 metabolic reactions, 44 metabolic pathways, and eight transport reactions. Opacuscyc_14.5 was improved by refining the metabolic model of TAGs biosynthesis and degrada- tion by metabolic product characterization of uncommon fatty acids that accumulate to high levels in Rhodococcus. The results of a screen for bacterial growth on 190 metabolic compounds were used as a multi-genic test of the reconstruction, described in more detail below. Comparison of the results of the catabolic screen with the metabolic pathway predictions revealed that precision was 65% before refinement and 71% after refinement (Table S1). The current working model of Opacuscyc14.5 contains 2017 metabolic reactions, 444 metabolic pathways, and 143 transport reactions that can be browsed at (http://tinyurl.com/4dv5m32). Rhodococcus Oleaginy Resulted from Key Genes That Emerged in Actinomycetales Several eubacteria of the order of Actinobacteria including Rhodococcus, Corynebacterium, Mycobacterium, and Bifidobacterium are distinguished for having both the type 1a fatty acid synthase (FAS) (Figure 1a) and a FAS II lipid biosynthesis system. FAS is a polyketide synthase related protein containing all of the necessary enzymatic activities for de novo lipid biosynthesis (Figure 1b). In mycobacteria FAS has been shown to elongate C2 and C3 carbon acyl-CoAs into C16, C18, C24, and C26 fatty acyl-CoAs [26,27]. In the suborder of bacteria Corynebacterineae, the FAS II system of lipid biosynthesis elongates FAS product fatty acyl-CoAs through the enzymatic activity of 6 families of enzymes (Figure 1c) for biosynthesis of mycolic acids and related cell wall associated lipids. The FAS II system in these Actinomycetales operates on C16–C26 length fatty acyl-CoAs not the usual short chain lipid biosynthesis substrates characteristic of the related enzymes in bacteria, chloroplasts, and mitochondria. The number of unique FAS genes is shown in Figure 1a in the context of a phylogenetic tree of genera built by AMPHORA [28]. Unlike the widespread taxonomic representation of the FAS II genes, the FAS type 1a gene in Rhodococcus has genus representation in prokaryotes is limited to only within Actinomycetales. The FAS gene is likely to PLoS Genetics | www.plosgenetics.org 2 September 2011 | Volume 7 | Issue 9 | e1002219