Discovery of enzymes for toluene synthesis from anoxic microbial communities

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum

Anaerobic Initial Reaction of n-Alkanes in a Denitrifying Bacterium: Evidence for (1-Methylpentyl)succinate as Initial Product and for Involvement of an Organic Radical in n-Hexane Metabolism

A novel type of denitrifying bacterium (strain HxN1) with the capacity to oxidize n-alkanes anaerobically with nitrate as the electron acceptor to CO(2) formed (1-methylpentyl)succinate (MPS) during growth on n-hexane as the only organic substrate under strict exclusion of air. Identification of MPS by gas chromatography-mass spectrometry was based on comparison with a synthetic standard. MPS was not formed during anaerobic growth on n-hexanoate. Anaerobic growth with [1-(13)C]n-hexane or d(14)-n-hexane led to a 1-methylpentyl side chain in MPS with one (13)C atom or 13 deuterium atoms, respectively. This indicates that the 1-methylpentyl side chain originates directly from n-hexane. Electron paramagnetic resonance spectroscopy revealed the presence of an organic radical in n-hexane-grown cells but not in n-hexanoate-grown cells. Results point at a mechanistic similarity between the anaerobic initial reaction of n-hexane and that of toluene, even though n-hexane is much less reactive; the described initial reaction of toluene in anaerobic bacteria is an addition to fumarate via a radical mechanism yielding benzylsuccinate. We conclude that n-hexane is activated at its second carbon atom by a radical reaction and presumably added to fumarate as a cosubstrate, yielding MPS as the first stable product. When 2,3-d(2)-fumarate was added to cultures growing on unlabeled n-hexane, 3-d(1)-MPS rather than 2,3-d(2)-MPS was detected, indicating loss of one deuterium atom by an as yet unknown mechanism