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.
Figure 2.
Phylogenetic analysis of type 1a and type 1b fatty acids synthases.
a) A phylogenetic tree that shows FAS type 1a is found in Actinobacteria, Stramenopiles, and Fungi. b) A phylogenetic tree of MAS-family type 1b proteins. This gene family is expanded in the genera Mycobacterium, Frankia, and Streptomyces but present in single copy in Rhodococcus belonging to branch containing the PKS12 from M. tuberculosis NP_216564 that also contains similar protein domain architecture.
Figure 3.
Analysis of genes and gene families implicated in the TAGs cycle.
a) The number of genes in the TribeMCL gene-clusters implicated by metabolic reconstruction for each reaction of the TAGs cycle are displayed in a heat map with species clustered according to their TAGs cycle genetic profile. The number of genes corresponding to TribeMCL gene-clusters implicated for each biochemical reaction (EC number) is color coded from 0 to >15. b) The sum of unique genes for each bacterial species implicated for the TAGs cycle by metabolic reconstruction and TribeMCL analysis.
Figure 4.
Identification and purification of odd-carbon straight-chain fatty acids generated by Rhodococcus.
a and b) GC-FID analysis of FAMEs synthesized during fermentation. Freeze-dried whole cells fermented on glucose of R. opacus PD630 (a) and R. jostii RHA1 (b). c and d) GC-FID analysis of FAMEs derived from TLC-purified TAGs of R. opacus PD630 (c) and R. jostii RHA1 (d) grown on glucose e) odd lipids C15∶0, C17∶0, and C17∶1 increase during fermentation of both R. opacus PD630 and R. jostii RHA1. f) Rhodococcus grown on propionate generates mostly C15∶0, C17∶0, and C17∶1 fatty acids that were used for purification and confirmation by mass spectrometry and structural 1H-NMR. These analyses demonstrated that the purified Rhodococcus odd-carbon fatty acids were straight-chain.
Figure 5.
Screens of four bacterial species for growth on carbohydrates and alcohols.
a) Compounds were clustered according to how R. opacus PD630, R. jostii RHA1, C. glutamicum 13032, and R. eutropha H16 were able to grow on oligosaccharides from 2–4 days. Yellow indicates evidence of growth. b) alcohol and monosaccharide compounds were clustered according to bacterial growth as in a. c) Comparison of three Rhodococcus chromosomes revealed that R. opacus B4 and R. opacus PD630 shared two divergent operons dedicated to galactose and oligogalactoside metabolism but R. jostii RHA1 only had a small piece of this chromosomal region containing the GalK and GalT genes omitting α- and β-galactosidases, Solute Binding Protein (SBP), two Solute Binding Protein Transporter (SBPT) proteins, and a DeoR family transcriptional regulatory protein.
Figure 6.
Screens of four bacterial species for growth on carboxylate compounds (organic acids).
Compounds were clustered as in Figure 5a.