Sinskey1,5,6* Abstract Comparative and Functional Genomics of Rhodococcus opacus PD630 for Biofuels Development Jason W. Holder1,2, Jil C. Ulrich1, Anthony C. DeBono 1 , Paul A. Godfrey2, Christopher A. Desjardins2, Jeremy Zucker1,2, Qiandong Zeng 2 , Alex L. B. Leach1, Ion Ghiviriga3, Christine Dancel3, Thomas Abeel2, Dirk Gevers2, Chinnappa D. Kodira4, Brian Desany4, Jason P. Affourtit4, Bruce W. Birren2, Anthony J. 1 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2 The Broad Institute, Cambridge, Massachusetts, United States of America, 3 Department of Chemistry, University of Florida, Gainesville, Florida, United States of America, 4 454 Life Sciences, Branford, Connecticut, United States of America, 5 Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 6 Engineering Systems Division, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy. Citation: Holder JW, Ulrich JC, DeBono AC, Godfrey PA, Desjardins CA, et al. (2011) Comparative and Functional Genomics of Rhodococcus opacus PD630 for Biofuels Development. PLoS Genet 7(9): e1002219. doi:10.1371/journal.pgen.1002219 Editor: Paul M. Richardson, Progentech, United States of America Received March 9, 2011; Accepted June 17, 2011; Published September 8, 2011 Copyright: 2011 Holder et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support was received from MIT Energy Initiative Seed Grant program (http://web.mit.edu/mitei/research/spotlights/recipients.html), Cambridge MIT Partnership Program (http://www.cambridge-mit.org/?tag = cmipp), and Shell oil corporation (http://www.shell.com/). This project has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272200900006C (http://www.niaid.nih.gov/Pages/default.aspx). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: asinskey@mit.edu Introduction Bio-Diesel is an energy-rich portable fuel derived mainly from triacylglycerols (TAGs). Biodiesel and related fuels are extracted from oleaginous organisms, both photosynthetic and non-photo- synthetic, that use available energy sources to fix carbon into high levels of stored lipids. In chemoheterotrophic organisms TAGs are synthesized by bioconversion of organic compounds such as the sugars and organic acids derived from globally-abundant cellulosic biomass. A genetic understanding of the oleaginous metabolism of chemoheterotrophic species like Rhodococcus provides critical insight for biofuels development. The high GC content Gram-positive Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1, a close relative that has a completely sequenced genome [1], were previously shown to accumulate large amounts of TAGs and wax esters (WEs) [2,3,4]. Rhodococcus species present an attractive target for industrial processes due to high substrate tolerances and high density culturing on a rapid time scale as compared to many photosynthetic organisms [5,6]. The oleaginous metabolism of Rhodococcus goes beyond abundant lipid biosynthesis to include diverse hydrocarbon catabolism. R. jostii RHA1 was isolated from soil containing 1,2,3,4,5,6-hexachlorocyclohexane (Lindane) [7], while R. opacus PD630 was enriched on phenyldecane as a sole carbon source after isolation from soil sampled at a gas works plant [2]. Rhodococcus can catabolize and detoxify several aromatic hydrocarbons that contaminate soil from industrial waste products. These toxic substrates include polychlorinated biphe- nyls (PCBs) [8,9,10,11] and other halogenated compounds such as Lindane that was used in large quantity for agricultural practices. Limitation of an essential nutrient stimulates enzymatic conversion of the non-limiting essential nutrients into stored polymers such as phosphorous conversion to poly-phosphate [4], acetyl- and other short acyl-CoAs conversion to polyhydroxyalk- anoates (PHAs) [12,13,14], or the production of TAGs and WEs from these same short chain acyl-CoA primers [2,15]. Most prokaryotes store carbon as polyhydroxyalkanoic acids (PHAs) when other essential nutrients such as reduced nitrogen are limiting. By contrast bacteria in the order Actinomycetales have uniquely developed a storage lipid cycle that leads to accumulation of TAGs and WEs [16]. Abundant TAGs accumulation in Rhodococcus provides a pool of fatty acids for b-oxidation as cellular fuel, components of the plasma membrane, and substrates for the PLoS Genetics | www.plosgenetics.org 1 September 2011 | Volume 7 | Issue 9 | e1002219