Widespread head-to-head hydrocarbon biosynthesis in bacteria and role of OleA

Abstract

Previous studies identified the oleABCD genes involved in head-to-head olefinic hydrocarbon biosynthesis. The present study more fully defined the OleABCD protein families within the thiolase, alpha/beta-hydrolase, AMP-dependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively. Only 0.1 to 1% of each superfamily represents likely Ole proteins. Sequence analysis based on structural alignments and gene context was used to identify highly likely ole genes. Selected microorganisms from the phyla Verucomicrobia, Planctomyces, Chloroflexi, Proteobacteria, and Actinobacteria were tested experimentally and shown to produce long-chain olefinic hydrocarbons. However, different species from the same genera sometimes lack the ole genes and fail to produce olefinic hydrocarbons. Overall, only 1.9% of 3,558 genomes analyzed showed clear evidence for containing ole genes. The type of olefins produced by different bacteria differed greatly with respect to the number of carbon-carbon double bonds. The greatest number of organisms surveyed biosynthesized a single long-chain olefin, 3,6,9,12,15,19,22,25,28-hentriacontanonaene, that contains nine double bonds. Xanthomonas campestris produced the greatest number of distinct olefin products, 15 compounds ranging in length from C(28) to C(31) and containing one to three double bonds. The type of long-chain product formed was shown to be dependent on the oleA gene in experiments with Shewanella oneidensis MR-1 ole gene deletion mutants containing native or heterologous oleA genes expressed in trans. A strain deleted in oleABCD and containing oleA in trans produced only ketones. Based on these observations, it was proposed that OleA catalyzes a nondecarboxylative thiolytic condensation of fatty acyl chains to generate a beta-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.

FIG. 1.

Structure-based amino acid sequence alignments of OleA, OleB, OleC, and OleD from S. oneidensis MR-1 (denoted MR1 in the figure) with highly conserved regions of proteins catalyzing divergent reactions from each respective superfamily. Accession numbers or PDB identifiers of the proteins used are as follows. OleA thiolase superfamily: OleA from Shewanella oneidensis MR-1 (gi 24373309), β-ketoacyl-acyl carrier protein synthase III (FabH) from Escherichia coli (1EBL), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG_CoA) from Staphylococcus aureus (1XPK), and chalcone synthase (Chalcone) from Medicago sativa (1CGZ); blue residues indicate the glutamate that abstracts a proton to produce a carbanion for the nondecarboxylative condensation reaction, red indicates the absolutely conserved cysteine of the superfamily that forms a covalent bond with the substrate, and green residues are involved in formation of an oxyanion hole. OleB α/β-hydrolase superfamily: OleB from Shewanella oneidensis MR-1 (gi 24373310), haloalkane dehalogenase (HAD) from Xanthobacter autotrophicus GJ10 (1B6G), epoxide hydrolase (EH) from Agrobacterium radiobacter AD1 (1EHY), and prolyloligopeptidase (prolyl) from porcine brain (1H2W); red residues indicate the catalytic nucleotide (Ser, Asp, or Cys in the whole superfamily), green indicates the general acid, and blue indicates the conserved histidine that activates water. OleC AMP-dependent ligase/synthetase superfamily: OleC from Shewanella oneidensis MR-1 (gi 24373311), gramicidin synthetase (gramicidin) from Brevibacillus brevis (1AMU), acetyl-CoA synthetase (AcCoA) from Saccharomyces cervisiae (1RY2), and luciferase from the Japanese firefly (2D1Q); red indicates absolute conservation in the three consensus regions identified by Conti et al. ([STG]-[STG]-G-[ST]-[TSE]-[GS]-x-[PALIVM]-K, [YFW]-[GASW]-x-[TSA]-E, [STA]-[GRK]-D) (15); blue and green indicate Thr/Ser residues thought to be involved in binding the phosphoryl group in ATP and AMP. OleD short chain dehydrogenase/reductase superfamily: OleD from Shewanella oneidensis MR-1 (gi 24373312), UDP-galactose-4-epimerase (Udp-gal-4 epim) from humans (1EK6), 7-α-hydroxysteroid dehydrogenase (7a-HOstroid DH) from Escherichia coli (1AHH), and D-3-hydroxybutyrate dehydrogenase (D-3-HObut DH) from Pseudomonas fragi (3ZTL); blue identifies the tyrosine anion that abstracts the proton from the substrate, red is a lysine that stabilizes the tyrosine anion, green is a glycine-rich region involved in cofactor NAD(P)⁺ binding, and pink is the serine that orients the substrate or stabilizes intermediates.

FIG. 2.

Analysis of the gene regions of S. oneidensis MR-1 (A), G. bemidijiensis Bem (B), and G. sulfurreducens PCA (C). Genes denoted oleA and fabH are homologs to the oleA from S. oneidensis MR-1. A predicted oleA gene region is shown for S. oneidensis (A) and G. bemidijiensis Bem (B), clustering with oleBCD genes. The fabH gene, which is an oleA homolog with highest percent identity with G. sulfurreducens PCA, fails to cluster with oleBCD homologs.

FIG. 3.

ole gene regions of different bacteria. The gene region configuration is shown on the left, and the bacteria containing each are listed at the right. The double slashes in panels E and F indicate that the genes on either side are not contiguous. Green, oleA; yellow, oleB; red, oleC; blue, oleD; orange, oleBC fusion; white, other genes not currently identified as being involved in hydrocarbon biosynthesis. The different parts represent the most common contiguous four-gene configuration (A), the three-gene cluster in which the oleB and oleC genes are in a single gene, oleBC fusion (B), and gene organization with various insertions between the identified ole genes (C). The white boxes indicate multiple genes that may be encoded in the same or opposite directions to the ole genes. In particular, various Xanthomonas spp. strains have different numbers of genes identified in the indicated locations. (D) Chloroflexi species that have an oligopeptidase inserted between oleB and oleC. Also, the oleA homolog is located after the other genes. (E) Configuration in which pairs of genes are in different parts of the genome. (F) A configuration in which oleA and oleB are located in different parts of the genome but oleC and oleD are clustered. Note: hydrocarbon production was confirmed in at least one organism in each class, A to F. Identifiers for each of the genes are listed in Table S1 of the supplemental material.

FIG. 4.

Gas chromatograms of extracts from different bacteria containing ole genes as identified by bioinformatics. Bacteria were extracted, and extracts were analyzed by GC-MS as described in Materials and Methods. The major products are labeled with their chemical formulas. No hydrocarbon peaks were identified beyond the elution range shown.

FIG. 5.

Gas chromatograms of extracts from wild-type and mutant S. oneidensis strains with and without the oleA gene from S. maltophilia. Extracts are from the following strains: (A) S. oneidensis MR-1 wild type; (B) S. oneidensis MR-1 wild type with S. maltophilia oleA; (C) S. oneidensis ΔoleA; (D) S. oneidensis ΔoleA with S. maltophilia oleA; (E) S. oneidensis ΔoleABCD with S. maltophilia oleA. Products were identified as described in the text.

FIG. 6.

Network protein sequence clusters for OleA (A), OleB (B), OleC (C), and OleD (D). The nodes represent protein sequences, and the edges represent a BLAST linkage that connects the two proteins with an e-score better than e⁻⁷³. Other methods are described in the text. The nodes are numbered to identify the organism from which each Ole protein was derived. The organism names and number identifiers for each sequence are listed in Materials and Methods. The nodes are colored to reflect the type of hydrocarbon produced by that organism: white, a C₃₁H₄₆ nonaene product; dark gray, diene, triene, or tetraene product; light gray, monoene product. Additional network diagrams that depict divergence of the clusters can be found in Fig. S2 of the supplemental material.

FIG. 7.

Parallel biological reaction sequences. At left is the known reaction sequence leading to ketones in humans. At right is the proposed reaction sequence leading to ketones in bacteria expressing OleA.