Identification of sewage sludge bacteria that forms the hydrocarbon, toluene

The metabolic diversity of microorganisms (bacteria and fungi) –– combined with ready access to increasingly efficient and less costly DNA-sequencing technologies –– has led to the challenge of unambiguously defined enzymes that have not yet been associated with specific genes that encode amino acid sequences. This has been referred to as “orphan enzymes.” In 2014, for example, an astonishing 22% of defined Enzyme Commission (EC) numbers comprised orphan enzymes. If these specific enzymes could be better linked to a broad range of chemically diverse reactions, then this would enhance the scope and versatility of biochemical transformations harnessed for biotechnological applications.

One area in which knowledge of enzymes is very limited is the biosynthesis of aromatic hydrocarbons (i.e. chemicals containing only hydrogen & carbon), which could be useful as renewable fuels or chemicals made from non-petroleum feedstocks (raw material that can be used to supply or fuel a machine, or an industrial process). The only aromatic hydrocarbon [of which these authors are aware; see attached article] that can currently be synthesized wholly from known enzymes is styrene –– which can be produced from phenylalanine-derived trans-cinnamic acid by enzymes with phenylacrylate decarboxylase activity, such as FDC1 from Saccharomyces cerevisiae (baker’s, or brewer’s yeast). Authors therefore chose to target the aromatic hydrocarbon toluene for enzyme discovery, because it is an important petrochemical with a global market of 29 million tons per year, which can be used to synthesize other aromatic feedstocks, and as an effective octane booster in gasoline. To clarify for some Readers, these two aromatic hydrocarbon chemical structures are illustrated here: (sorry no pix)


Microbial sources of biogenic toluene were first reported more than three decades ago –– yet, the underlying biochemistry and specific enzymes catalyzing toluene biosynthesis were never elucidated. Biogenic toluene has been observed in lake sediments and municipal sewage sludge lacking oxygen, as well as from two bacteria: Tolumonas auensis and Clostridium aerofoetidum. Unfortunately, these findings could not be corroborated.

Authors [see attached article] report the discovery of 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 –– in two distinct anoxic (the complete lack of oxygen) microbial communities. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved the techniques of metagenomics- and metaproteomics-enabled biochemistry, in addition to in vitro confirmation of activity using recombinant enzymes. This intriguing project expands the known catalytic range of glycyl radical enzymes (only seven reaction types had previously been characterized) and aromatic-hydrocarbon-producing enzymes. These data will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass (plant dry matter), rather than only from petroleum.

Nature Chem Biol (2018)

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