Genomics of Rhodococcus for Biofuels Figure 1. Phylogenetic and metabolic pathway features of lipid biosynthesis in Rhodococcus. a) A phylogenetic representation of oleaginous genes in Actinomycetales. An AMPHORA-based tree of related genera (left) provides context to the copy number of unique oleaginous genes within each genera presented in three columns to the right. Remarkable oleaginous genes in Actinobacteria are fatty acid synthase (FAS) type 1a, MAS-family type 1b, and diacylglycerol acyl-transferases (DGATs). Oleaginous gene family members in each genus were counted and color-coded from 0 to .10. A phylogenetic tree of the FAS type 1a gene is presented to the right of the FAS column. b) Fatty acids biosynthesis in Rhodococcus. Acetyl-CoA is the product of many biochemical reactions and a limiting substrate in lipid biosynthesis. Acetyl-CoA can also be generated from acetic acid found in the environment by ligation to –CoA. ATP-hydrolysis enables bicarbonate coupling to acetyl-CoA forming malonyl-CoA, the substrate used for elongation. The lipid biosynthesis -CoA substrates are incorporated in Rhodococcus by three fatty acid biosynthesis systems that begins with multifunctional FAS type 1a. This synthase contains the following enzyme activities: malonyl palmityl transferase (MPT), acetyl transferase (AT), keto synthase (KS), keto reductase (KR), dehydratase (DH), enoyl reductase (ER), acyl carrier protein (ACP) catalyzing decarboxylating condensation of malonyl-ACP with acetyl-CoA, propionoyl-CoA, or acyl-CoAs to generate straight-chain fatty acyl-CoAs that range in size (C16–C26) and include odd- carbon fatty acids. Phosphopantotheinyl transferase (PPT) converts the ACP domain of FAS into the active form via attachment of phosphopantotheinate to a serine residue and is encoded by the down stream gene in the Rhodococcus bicistronic FAS operon. During inititiation malonyl-ACP is condensed with either acetyl-CoA or propionoyl-CoA to form a C4 or C5 straight-chain intermediate, respectively. Further elongation cycles consume malonyl-ACP for C2 additions with concomitant release of CO2 for each round of Claisen-type condensation reaction. The fatty acyl- CoA products of FAS ,C20 are attached to glycerol catalyzing production of phospholipids and triacylglycerols (TAGs) or processed by number of other routes. Fatty acyl-CoAs can be further elongated by the FAS II system or MAS-family proteins. Wax esters (WEs) are generated by transesterification of fatty acyl-CoAs with fatty alcohols. TAGs, WEs, phospholipds, FAS II and MAS products come together to form compartments within Rhodococcus. Lipid bodies (yellow, LB) house the stored lipids surrounded by cytoplasm (blue, CP). The Plasma Membrane (orange, PM) is surrounded by cell wall (green, CW). The mycolic acid layer (black, MA) on the outside of Rhodococcus cells contains the long chain lipids. 1c) Comparison of biochemical activities of type II proteins and type I protein domains. Colors of FAS II genes correspond to related function in type 1 protein domains. Notable differences between synthase type 1 domains and type 2 proteins include: the MPT domain is dual functional in FAS type 1a thus releasing –CoA products through palmitoyl-CoA activity. The AT domain in type 1a provides acetyl-CoA and propionoyl-CoA substrates for condensation. In type 1b synthase the AT domain provides long chain acyl-CoAs for condensation on the KS domains. 1d) Methyl-branched lipid biosynthesis model for type 1b synthase activity from related mycobacterial protein PKS12. The C3 organic salt propionate can be ligated to CoA generating propionoyl-CoA, a substrate for Rhodococus lipid biosynthesis that is also produced in several metabolic degradation pathways. Propionoyl-CoA can be used directly by FAS during initiation or carboxylated to form methylmalonyl-CoA making it a substrate for a second multifunctional fatty acid synthase with a type 1b protein domain architecture present in Rhodococcus OPAG_06239. The type 1b synthase belongs to the mycocerosic acid synthase (MAS)-family of proteins that in Mycobacterium were shown to incorporate methylmalonyl-CoA into growing fatty acid chains creating methyl-branched lipids. doi:10.1371/journal.pgen.1002219.g001 have emerged in Actinobacteria (Figure 1a) and was horizontally transferred to only those eukaryotic branches containing fungi and stramenopiles (Figure 2a). Our genome-based metabolic reconstruction revealed close metabolic pathway relationships when we compared the lipid metabolism enzymes of the phylogenetically related genera Mycobacterium, Nocardia, and Rhodococcus. These Actinomycetales contain multiple pathways for lipid biosynthesis utilizing a unique combination of multifunctional fatty acid synthases (FAS), a type 1a synthase and a related MAS-family type 1b synthase that generate linear- and branched-fatty acids respectively (Figure 1D). Diacyl glycerol acyl transferase (DGAT) enzymes convert fatty and other carboxylic acids into TAGs [29]. DGAT genes are also of limited genus representation in eubacteria and interestingly are present in six out of seven genera that also contain at least one gene of the MAS-family type 1b synthases (Figure 1a). Closely related genera to Rhodococcus share expanded gene-families of DGATs [29,30]. A phylogenetic tree based on 701 shared genes present in a single copy and general genomic features of representative species used for comparisons with Rhodococcus are presented in Figure S3. The phylogenetic distance between the bacterial taxa that contain the FAS type 1a suggest this gene has likely been horizontally transferred within Actinomycetales. The FAS gene was duplicated in Corynebacterium and horizontally transferred between Bifidobacterium and Corynebacterium. The FAS type 1a protein from Rhodococcus is highly related, ranging between 60– 65% amino acid identity, to the enzyme in mycobacteria. The FAS protein is comprised of 3128 amino acids with 7 domains (Figure 1b), six of which catalyze distinct biochemical reactions (Figure 1c). Multiple sequence alignment of Actinomycetales FAS type 1a proteins reveals conservation across all domains (Dataset S1 and Dataset S2). The order of type 1a synthase protein domains, high sequence similarity, and presence of all three key lipid biosynthesis systems (FAS type 1a, MAS-family type 1b, and FAS II) suggest that the substrates and products are similar for the shared enzymatic network of lipid synthases within the suborder Nocardiacae and Mycobacterium. A working model for carbon flow in Rhodococcus lipid biosynthesis is presented in Figure 1b. Enzymes from Expanded Gene Families Generate Lipid Biosynthesis Primers Acetyl-CoA is the product of many catabolic reactions and a key substrate in lipid biosynthesis (http://tinyurl.com/4fgl6zo). Ace- tate and longer chain carboxylic acids captured from the environment can be converted to -CoA derivatives by CoA synthetases (EC 6.2.1.1) thus feeding these organic acids into lipid metabolism (Figure 1b). The first committed step in lipid biosynthesis is catalyzed by acetyl-CoA carboxylases that function as a/b complexes in Rhodococcus and related bacteria of the suborder Corynebacterineae to generate malonyl-CoA that is utilized by FAS type 1a, MAS-family type 1b, and FAS II enzymes for fatty acid biosynthesis (Figure 1b and 1d). Malonyl-CoA is generated by ATP-hydrolysis dependent carboxylation of acetyl- CoA in reaction EC 6.4.1.2 by an expanded family of AccA (a) and AccD (b) enzymes in Rhodococcus (Figure 3a). Homologous Rhodococcus AccA and AccD proteins were analyzed phylogenet- ically with those from related species (Figure S4). Some paralogous enzymes from these gene families were reported to recognize the – CoA derivatives of distinct carbon chain length organic acids [31,32,33,34] suggesting that a diverse pool of organic acids could be carboxylated and incorporated into cellular lipids by Rhodo- coccus. The pool of propionoyl-CoA in Rhodococcus can be converted into methylmalonyl-CoA via ATP dependent carboxylation by propionoyl-CoA carboxylase in reaction EC 6.4.1.3 (Figure 1d). In Mycobacterium and Streptomyces methylmalonyl-CoA is a lipid biosynthetic substrate that is incorporated into methyl-branched lipids by the type 1b fatty acid synthases, a member of the mycocerosic acid synthase MAS-family of proteins [35]. OPAG_06239 is homologous (41% identity over 3527 amino acids) to a protein within the MAS-family with all of the same PLoS Genetics | www.plosgenetics.org 4 September 2011 | Volume 7 | Issue 9 | e1002219