KS domain for decarboxylating condensation with acetyl-CoA resulting in acetoacetyl-ACP. The type 1b synthases that inititiate fatty acid biosynthesis, like the mammalian FAS, lack the MPT domain thus use their AT domain to load both acetyl-CoA and malonyl-CoA. MAS-family type 1b enzymes display significant substrate diversity by varying both the acyl substrates (acetyl-CoA, acyl-CoAs) as well as elongation substrates (malonyl-CoA, methylmalonyl-CoA). We found seven Actinomycetales genera that contained the MAS-family type 1b synthases compared to 4 genera that encode the type 1a synthase. Six out of seven Actinomycetales genera that encode type 1b synthases also encode at least one storage enzyme of the DGAT/WE family. We conclude the relatively widespread taxonomic representation of the type 1b synthases and the diversity of substrates these enzymes react on suggests that there are lipid variants yet to be identified. The large number of genes dedicated to lipid metabolism in Actinomycetales is the result of gene duplications, multifunctional FAS type 1a and MAS-family type 1b gene emergence, horizontal gene transfer, and emergence of the DGAT/WE enzymes that catalyze the transesterification of fatty acyl-CoAs with diacylgly- cerol. We performed phylogenetic analysis on genes identified by metabolic reconstruction in an attempt to predict the most likely activities for each enzyme encoded in the R. opacus PD630 genome. We used the TribeMCL algorithm to describe the size of the gene family and all of the family members for a set of related Actinomycetales. Phylogenetic tree formation with these related species was used to resolve the orthologous from paralogous enzymes, thus facilitating the extrapolation of functional data for related proteins to our in silico model of Rhodococcus Opacuscyc14.5. The broad catabolism and elaborate lipid biosynthesis described in Rhodococcus indicates that there is substantial enzymatic activity on chemically-related compounds that could be explained by expansion in gene-family sizes and genetic drift to encode enzymes with slightly variant substrate recognitions from ancestrally-related enzymes but still catalyze similar chemical reactions. The 8632 genes in R. opacus PD630 provide a large arsenal of metabolic enzymes for an oleaginous lifestyle within soil. We found protein domains contained in transport proteins to be the most abundant class of domains encoded in Rhodococcus as well as a distinctive profile of transporter types that are distinguishing for Rhodococcus (Figure S13). Identification of structural genes and operons that play a role in the lipid body assembly process have already begun to benefit from genomic sequence [63]. Chemoheterotrophic organisms capable of fermenting sugars and a broad spectrum of organic compounds derived from cellulosic and other natural resource biomasses through biocon- version provides an industrial process to convert agricultural side-, natural resource-, and industrial waste-streams into fungible fuels. R. opacus PD630 differs from R. jostii RHA1 in its ability to catabolize the cellulosic sugar galactose and oligogalactosides. Both Rhodococcus species degraded the cellulosic sugars glucose and rhamnose; however, none of the species tested were able to degrade the disaccharide cellobiose containing b1–4 linked glucose. Cellulose catabolism requires hydrolysis of b1–4 linked glucose, indicating that these species do not encode for these hydrolases. The most abundant component of hemicellulose is xylan that is broken down to xylose. Consistent with the cellobiose deficiency these species were unable to catabolize xylose. Genetic complementation of cellulosic degradation pathways in Rhodococcus provides a streamlined approach for cellulosic biomass conversion into oil-based fuels. Rhodococcus species catabolized a diverse array of carboxylic acids that corresponded to an expansion in the acyl- CoA ligase gene family that link organic acids with biosynthesis and catabolism. Organic acids are produced in the mixed-acid Genomics of Rhodococcus for Biofuels fermentations of cellulose degrading organisms thus indicating a potential next-generation strategy for 2-phase fermentations of cellulosic biomass to TAGs that could be done without genetic modification. Our working model of the R. opacus PD630 metabolism began with genome sequence that allowed phylogenetic comparisons to be made with related species that have been studied in far greater molecular detail. Phenotypic information about catabolism in Rhodococcus provided a powerful multigenic test that guides the metabolic reconstruction towards completion through phenotype- directed pathway curation. Literature-based pathway curation united the reported biochemical reactions of M. phylei FAS type 1a protein with metabolic products that we purified and character- ized during metabolic model refinement of R. opacus PD630. The improvements to our genetic model of R. opacus PD630 metabolism provides a template for further refinement with the integration of data from genetics, biochemistry, metabolomics, lipidomics, and transcriptomics that will be the focus of future work. Materials and Methods Bacterial Species Used in This Study R. opacus PD630 was obtained from the DSMZ strain 44193. R. eutropha H16 ATCC17699 and C. glutamicum ATCC13032 were from ATCC. R. jostii RHA1 was a gift from Lindsay Eltis at the University of British Columbia. Rhodococcus opacus PD630 DNA Sequencing and Genome Annotation Genomic DNA was extracted from R. opacus PD630 as in [64] without the addition of mutanolysin. Genomic DNA was sequenced at 454 Life Sciences (A Roche Company) according to manufacturer protocol for shotgun (Rapid Library) and 3 kb paired-end reads using GS FLX Titanium sequencing chemistry. The resulting DNA sequence was assembled using the GS De Novo Assembler software version 2.0. Open Reading Frames (ORFs) were predicted from assembled genome sequence using a combination of in silico ORF predictions and gene mapping from the annotated R. jostii RHA1 genome. In silico ORFs were predicted using GeneMark [65] and Glimmer3 [66]. For synteny based gene-mapping Nucmer [67] was used to find local alignments between every contiguous sequence read (contig) in the R. jostii RHA1 and every contig in the R. opacus PD630. A whole-genome synteny map was then built from these local alignments by chaining together collinear hits, allowing up to 10,000 bases of undetectable similarity between anchors, then filtering out chains that overlap a larger chain on either sequence by more than 90% of their length. The remaining chains, corresponding to syntenic regions of DNA, were globally aligned using LAGAN [68]. Each transcript in the R. jostii RHA1 genome was mapped onto the R. opacus PD630 by attempting two mapping techniques and then selecting the transcript with the higher- scoring global alignment to the reference transcript’s coding sequence. The first method projects the boundaries of each gene onto the target genome using the coordinates in the raw whole- genome alignment. The second method uses the whole-genome alignment to define a target region containing the reference transcript, and then uses GeneWise to build a gene model by aligning the reference protein to the target region. Final ORFs were defined by comparison of in silico ORFs and mapped ORFs with hits to Pfam [69] and the top blast hits against the non- redundant protein database. ORFs with overlap to non-coding RNA features (see below) were reviewed and removed when PLoS Genetics | www.plosgenetics.org 13 September 2011 | Volume 7 | Issue 9 | e1002219