likely increase intracellularly during nitrogen and glucose starvation resulting from elevated protein and amino acid degradation as the physiology of these cells adapt to these nutrient limitations. Elevated propionoyl-CoA has been proposed to explain increased production of the branched-lipid PDIM when mycobacteria is grown on cholesterol as a sole source of carbon [59]. The FAS type 1a protein in the genus Rhodococcus is highly related to the mycobacterial protein that has been demonstrated to incorporate propionoyl-CoA during de novo lipid biosynthesis and produce lipids with the same lengths observed in the stored lipids TAGs and WEs. Our genomic analysis indicates conservation of all three interconnected lipid biosynthesis systems in Nocardiacae and Mycobacterium namely FAS type 1a, MAS-family type 1b, and FAS II. This group of bacteria also shares the ability to store lipids in high abundance. This conserved lipid biosynthetic network provides means for extrapolating functional information derived from decades of lipid metabolism research in Mycobacterium to the elaborate lipid metabolism in Rhodococcus. Pharmacological studies in combination with enzyme activities performed with purified systems demonstrated that Mycobacterium uses FAS type 1a to convert two and three carbon organic acids into straight-chain fatty acids containing 16, 18, 24, and 26 carbons [20,26,27]. FAS type 1a products are released as the – CoA derivative of a fatty acid due to the dual activity of the FAS type 1a malonyl palmitoyl transferase (MPT) domain [21]. The FAS acyl-CoA products can then proceed through multiple lipid biosynthetic pathways. Studies of mycobacterial lipid metabolism have established a genetic model for this related oleaginous bacterium that connects multiple biosynthetic systems for the building of storage, plasma membrane, as well as very long chain cell wall-associated lipids. The close phylogenetic relationship between the genus Mycobacterium and Rhodococcus resulted from common ancestry wherein these genera share many features of their elaborate lipid metabolisms. Much of what is known about Actinomycetales odd-carbon lipid metabolism comes from work that described longer chain extracellular lipids that are methyl-branched. Prior to this study, it was unclear whether the ,C20 odd-carbon lipids in Rhodococcus are methyl-branched as a result of incorporating methylmalonyl- CoA [16]. Using 1H-NMR to analyze purified lipids we could distinguish between the structural isomers of branched and straight-chain fatty acids demonstrating that the stored odd- carbon lipids in Rhodococcus are straight-chain. The odd-carbon storage lipids in Rhodococcus could result from propionoyl-CoA and malonyl-ACP condensing in the initiation phase of FAS type 1a biosynthesis (Figure 1b). The substrate requirements for produc- tion of straight-chain odd-carbon lipids matches the reported substrate specificity of the multifunctional FAS type 1a enzyme and its product chain lengths. The combination of metabolic product identification and metabolic pathway mapping through genomic sequence supports a serial order to the elaborate network of lipid biosynthesis in Rhodococcus, similar to Mycobacterium, wherein FAS type 1a initiates and elongates fatty acids releasing –CoA products that are in the range of C16–C18 and C24–C26. FAS produced fatty acyl-CoAs can be further elongated in the type II system or by the MAS-family type 1b synthase. The Rhodococcus type 1b synthase OPAG_06239, like the homologous PKS12 gene from Mycobacterium, contains two modules consisting of 6 protein domains in a direct repeat (Figure 1d). The PKS12 enzyme displays dual specificity for malonyl-CoA and methylmalonyl-CoA within the same polypep- tide and oligomerizes in a tail-to-head fashion to perform multiple elongation cycles on C16- and C18-CoA molecules resulting in C30– 34 multiply methyl-branched fatty acids. Enzyme assays with single Genomics of Rhodococcus for Biofuels modules (6 domains) of PKS12 and site-specific mutations within the PKS12 acetyl transferase (AT) domains demonstrated that the N-terminal module incorporates methylmalonyl-CoA whereas the C-terminal module incorporates malonyl-CoA [43]. This mode of biosynthesis results in branched fatty acids that contain a methyl- branch at every fourth carbon from the point of initial condensation by the type 1b enzyme. This alternating of elgonation substrates mechanism explains the curious methylation at every fourth carbon in the mannosyl-b-1-phosphomycoketide (MPM) molecules isolated from Mycobacterium [36]. In Mycobacte- rium, the enzymes of the type 1b MAS-family and FAS II system share a common preference for longer chain length fatty acyl- CoAs resulting in the observed order in biosynthesis enzyme activity. The order of conserved domains and sequence homology of the Rhodococcus type 1b synthase OPAG_06239 suggests that this enzyme will function similarly in some aspects of branched lipid biosynthesis that is characteristic of the MAS-family enzymes in the related Mycobacterium species; however no methyl-branched lipids have been identified in Rhodococcus to date. In species such as Mycobacterium that have both FAS type 1a and 1b synthases, there is a distribution in labor amongst the synthases that is dictated by chain length; wherein FAS type 1a functions as the initiating synthase and the type 1b synthase incorporates methyl branch substrate methylmalonyl-CoA during lipid biosyn- thesis of longer chain length lipids. In more distantly related Actinomycetales, the genera Saccharopolyspora, Salinspora, Frankia, and Streptomyces we observed a type 1b but no type 1a synthase (Figure 1a); thus preventing the distribution of labor observed in Nocardiacae and Mycobacterium. Streptomyces have been reported to store abundant methyl-branched lipids [41] that are shorter in chain length than those isolated from Mycobacterium. The observation of odd-carbon lipids in the TAGs from Rhodococcus intrigued us as a possibility that type 1b synthase activity was contributing to the accumulated TAGs. The chemical analysis we performed indicated the stored TAGs were straight-chain lipids of the appropriate length to have been built by type 1a FAS through iterative biosynthesis with two of this enzymes known substrates (propionyl-CoA and malonyl-CoA). How is the strict distribution of labor observed between type 1a and the other type II and 1b synthases maintained in Nocadiacae and Mycobacterium? The crystal structure of the related FAS from S. cerevisae [62] revealed this synthase is a hexamer complex of apoenzymes. A hexamer complex also explains the behavior in ultracentrifugation studies of the FAS type 1a complex from mycobacteria [26]. The structural studies of fungal FAS provided a structure-based model wherein fatty acids are biosynthesized within the cavity of a 2.6 MDa b barrel structure. The enzyme FAS-ACP domain with growing acyl chain accesses individual catalytic domains shuttling biochemical intermediates from one active site to the next within the FAS type 1a hexamer complex [62]. Fatty acid products of the appropriate length are released as acyl-CoAs by the MPT domain of FAS type 1a. The FAS hexamer in Mycobacterium display a bimodal distribution of product chain lengths that are (C16–18 and C24–26) [26]. We propose the FAS 1a acyl-CoA products become accessible to the other synthases once released from within the b barrel structure of the FAS hexamer by the palmitoyl transferase activity of the MPT domain. Acetyl/acyl transferase (AT) domains in type 1 fatty acid synthases display remarkable substrate diversity. In the type 1a synthase, the AT domain loads acetyl–CoA on the FAS-ACP domain then is subsequently transferred to a cysteine in the keto synthase (KS) domain. The second substrate for condensation is the elongation substrate malonyl-CoA that is transfered by the FAS-MPT domain to the FAS-ACP domain then delivered to the PLoS Genetics | www.plosgenetics.org 12 September 2011 | Volume 7 | Issue 9 | e1002219