Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

Abstract

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.

FIG. 1.

Partial amino acid alignments of three translated M. luteus genes with homology to condensing enzymes involved in fatty acid biosynthesis: (A) Mlut_09290 (FabF), (B) Mlut_09310 (FabH), and (C) Mlut_13230. Alignments include the most similar sequences from E. coli and close relatives Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1. Three conserved active-site residues (see text) are highlighted: Cys-His-His (panel A) or Cys-His-Asn (panels B and C). Gray areas indicate sequence identity. (A) GenBank accession numbers: E. coli, NP_415613; strain TC1, YP_948166; strain FB24, YP_831948. (B) GenBank accession numbers: E. coli, NP_415609; strain TC1, YP_948164; strain FB24, YP_831946. (C) GenBank accession numbers: E. coli, NP_415609; strain TC1, YP_947743; strain FB24, YP_832433.

FIG. 2.

(A) TIC of diazomethane-derivatized extracts of fatty acid-overproducing E. coli expressing Mlut_13230 (strain EGS180; blue) or no M. luteus genes (strain EGS084; black). Long-chain ketones (27:2, 27:1, 29:2, 29:1; blue fill) were observed when Mlut_13230 was expressed and were not observed in the control. (B) TIC of underivatized extracts of fatty acid-overproducing E. coli expressing Mlut_13230-13250 (strain EGS145; red), Mlut_13230 (strain EGS180; blue), or no M. luteus genes (strain EGS084; black). Long-chain alkenes (27:3, 27:2, 29:3, 29:2; red fill) were observed only when Mlut_13230-13250 were present and were not observed with Mlut_13230 alone or in the negative control. There are peaks from strain EGS180 that coelute with 29:3 and 29:2 alkenes; however, inspection of extracted ion profiles for the molecular ions of these alkenes (m/z 402 and 404) demonstrates that the alkenes are not present in strain EGS180 (insets).

FIG. 3.

(A) EI mass spectra (70 eV) of the two unsaturated C₂₇ monoketones (labeled 27:2 and 27:1) in Fig. 2A. (B) EI mass spectra (70 eV) of the two C₂₇ alkenes (labeled 27:3 and 27:2) in Fig. 2B.

FIG. 4.

Extracted ion chromatograms (m/z 291) of extracts from in vitro studies with purified Mlut_13230 protein. Duplicate results are shown for assays including Mlut_13230, tetradecanoyl-CoA, and crude lysate from wild-type E. coli DH1 (red); controls without DH1 lysate (blue); and controls without Mlut_13230 protein (black). The peak has the same retention time as the 27:2 monoketone observed during in vivo studies with Mlut_13230 (Fig. 2A), and m/z 291 is characteristic of that compound (Fig. 3A).

FIG. 5.

Alkene production (A) and expression of alkene biosynthesis genes (B) through different growth stages of M. luteus. All variables are plotted as a percentage of their maximum values, and duplicate results are shown. Alkenes 1 and 2 (A) are 29:1 alkenes (see text); inset chromatograms showing the relative enhancement of alkene 2 over time are shown. In panel B, results of RT-qPCR analysis of Mlut_13230, Mlut_13240, and Mlut_13250 over time are shown.

FIG. 6.

Proposed pathway for alkene biosynthesis from condensation of fatty acids. Compounds shown as CoA thioesters may, in fact, be ACP thioesters. The unsaturated monoketones observed in this study (Fig. 2, 3, and 4) correspond to the metabolite following the first dehydratase reaction. In M. luteus, the starting compounds are likely iso- and anteiso-branched C₁₅ fatty acids and the predominant products are iso- and anteiso-branched C₂₉ monoalkenes.