(Figure 3a). 261 candidate R. opacus PD630 TAGs cycle genes were identified for the TAGs cycle reactions (Figure 3b). The largest gene family in the Rhodococcus TAGs cycle corresponds to the FAD dependant acyl-CoA dehydrogenases that operate in the b- oxidation of fatty acyl-CoAs (EC 1.3.99.3). R. opacus PD630 contains 71 of these acyl-CoA dehydrogenase genes whereas Corynebacterium glutamicum 13032 only contains two genes predicted for this reaction (Figure 3a). Large gene families identified in the Rhodococcus TAGs cycle also include: DGATs (EC 2.3.1.20) resolved in a phylogenetic tree (Figure S6), TAG lipases (EC 3.1.1.3), acyl-CoA synthetases (EC 6.2.1.3), enoyl-CoA hydratases (EC 4.2.1.17), and acetyl-CoA C-acyltransferases (EC 2.3.1.16). Rhodococcus species contained at least 261 genes that could contribute to this metabolic cycle without consideration of membrane transport genes as they have yet to be defined for each catabolic substrate. The TAGs cycle -CoA ligases/synthe- tases that ligate –CoA with carboxylic acids play a role in initiation as well a b-oxidation of lipids have been grouped into 1 category represented as (EC 6.2.1.1/2/3) because we could not resolve the homologous enzymes that vary in their substrate chain length specificity (acetyl- EC 6.2.1.1, propionoyl- EC 6.2.1.2, acyl- EC 6.2.1.3). The largest number of genes in R. opacus PD630 dedicated to the TAGs cycle were mostly attributed to the acetate/acyl-CoA synthetases (EC 6.2.1.1/2/3)(18 genes), acyl-CoA dehydrogenases (EC 1.3.99.3)(71 genes), and enoyl-CoA hydratases (EC 4.2.1.17) (64 genes). These large gene families are central to the b-oxidation pathway suggesting that Rhodococcus can catabolize and extract energy through aerobic respiration from a diverse range of carboxylic acids as well as biosynthesize from these compounds a diverse array of lipid products. The related Actinomycetale C. glutamicum 13032 provided stark contrast to the large number of lipid metabolism genes we observed in Rhodococcus. C. glutamicum 13032 had four pathway holes (Figure 3a) with a total of 19 genes implicated in the TAGs cycle (Figure 3b). A substrate-permissive glycerol-3 phosphate acyl transferase could bypass the EC 2.3.1.15 pathway hole allowing for production of essential phospholipids; however the 3-hydro- xyacyl-CoA dehydrogenase in the b-oxidation of fatty acids (EC 1.1.1.35) is missing in C. glutamicum 13032, a result that is consistent with the observed deficiency in catabolism of fatty acids described below. C. glutamicum 13032 is also missing the DGAT enzyme responsible for the final step in TAGs and WE biosynthesis. A complete table of genes for the twelve species presented in Figure 3 can be found in Table S4. Odd-Carbon Straight-Chain Lipid Accumulation in Rhodococcus During fermentation of glucose, R. opacus PD630 and R. jostii RHA1 produced abundant lipids that were likely odd in carbon number based on their elution profile on gas chromatograph-flame ionizing detector analysis (GC-FID) (Figure 4a and 4b). The odd- carbon lipid species were found in Rhodococcus TAGs that were purified by thin layer chromatography (TLC) prior to conversion into fatty acid methyl esters (FAMEs) for assay by GC-FID (Figure 4c and 4d). During growth on glucose these odd-carbon fatty acids increase in relative abundance accumulating to as much as 30% in R. opacus PD630 and 40% in R. jostii RHA1 of the total lipids detected in the GC-FID assay (Figure 4e). Fermentation analytics of media concentrations of ammonium and glucose as well as cellular total fatty acids, and residual dry weight (Figure S7) indicated glucose depletion at 96 hours further stimulated R. jostii RHA1 to produce higher levels of odd-carbon lipids than R. opacus PD630. Genomics of Rhodococcus for Biofuels Chemical Identity and Structure of Straight-Chain Odd- Carbon Lipids Stored in TAGs The FAS type 1a enzyme purified from Mycobacterium phlei was shown to convert malonyl-CoA and the C3 substrate propionoyl- CoA into undefined fatty acids without propionoyl-CoA carbox- ylase in vitro [27], suggesting that straight-chain odd-carbon lipids could be made by the type 1a FAS. We analyzed the chemical identity and structure of the Rhodococcus stored odd-carbon fatty acids to evaluate whether these lipids are methyl-branched or straight-alkyl chains in order to provide insight into the enzyme(s) responsible for this uncommon lipid biosynthesis. The known substrate preferences of the FAS type 1a enzymes are straight- chain substrates (acetyl-CoA, propionyl-CoA, and malonyl-CoA [26,27]) while the MAS-family type 1b synthases incorporate the C3 methyl-branched lipid substrate methylmalonyl-CoA [35,37,39,43,44]. To determine the identity and chemical structure of the putative Rhodococcus odd-carbon lipids we purified them as FAMEs by taking advantage of their enrichment when R. opacus PD630 and R. jostii RHA1 were grown on propionate as the sole carbon source (Figure 4f). Propionate can be converted intracellularly to propionoyl-CoA through the activity of propionoyl-CoA ligase (EC 6.2.1.2)(Figure 1b) allowing for degradation via the methylci- trate cycle [45] or incorporation into lipids via two routes. In the straight-chain lipid biosynthesis pathway diagramed in Figure 1b, propionoyl-CoA is a substrate in the initial condensation reaction with malonyl-ACP. By contrast, in the branched-lipid biosynthesis pathway diagrammed in Figure 1d, propionoyl-CoA is first converted into methylmalonyl-CoA prior to incorporation into lipids by the MAS-family type 1b synthases. FAMEs from propionate grown cells were purified via reverse phase HPLC then analyzed on a coupled gas chromatograph/ electron ionization-mass spectrophotometer GC-EI-MS. We identifed ions with masses that corresponded to the methyl esters of pentadecanoic acid m/z 256 Da C15:0 (Figure S8), heptadeca- noic acid m/z 284 Da C17:0 (Figure S9), and heptadecenoic acid m/z 282 Da C17:1 (Figure S10). Fragmentation of these full length FAMEs resulted in ions that also matched previously reported spectra for electron ionized ions with these odd-carbon chain length FAMEs [46]. To discriminate between structural isomers with the same molecular weight (straight-chain and methyl-branched lipids), we performed 1H-NMR on the HPLC-purified FAMEs from Rhodococcus (Figure S11). We saw evidence of the methyl esters as expected from the transesterification of cellular lipids with methanol at 3.68 ppm; however, the FAMEs purified from Rhodococcus showed no evidence of methyl-branching at 0.86 ppm along the aliphatic chain of the C15:0, C17:0, and C17:1 fatty acids as compared to the branched control 16-methylhepta- decanoate. The NMR spectra in addition to the mass spectra demonstrate that these odd-carbon lipids contained straight-chain alkanes and a cis-alkene, not the methyl-branched forms expected from a methylmalonyl-CoA intermediate generated by propionoyl carboxylase in reaction EC 6.4.1.3. The absence of methyl- branched lipids in the shorter odd-carbon storage lipids indicated that de novo biosynthesis of methyl-branched lipids in Rhodococcus if present is restricted to the longer chain length lipids as previously described for the cell wall associated lipids in Mycobacterium. The identification of the stored pentadecanoic acid, heptadecanoic acid, and heptadecenoic acid odd-carbon straight-chain fatty acids could result from the FAS type 1a enzyme that is known to incorporate the three carbon molecule propionoyl-CoA and malonyl-CoA [27]. The odd-carbon fatty acids isolated from Rhodococcus grown on glucose contained predominantly seventeen PLoS Genetics | www.plosgenetics.org 7 September 2011 | Volume 7 | Issue 9 | e1002219