Researchers at the US Department of Energy Joint BioEnergy Institute (JBEI) and Lawrence Berkeley National Laboratory (Berkeley Lab) have discovered a new enzyme that will enable microbial production of a renewable alternative to petroleum-based toluene, a widely used octane booster in gasoline that has a global market of 29 million tons per year.
A major focus of research at JBEI, and in the broader community of biofuel researchers, is the production of industrially and commercially relevant fuels and chemicals from renewable resources, such as lignocellulosic biomass, rather than from petroleum. The enzyme discovered in this study will enable the first-time microbial production of bio-based toluene, and indeed, the first microbial production of any aromatic hydrocarbon biofuel. A paper on the study is published in the journal Nature Chemical Biology.
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.
The enzyme discovery resulted from the intensive study of two very different microbial communities that produced toluene. One community contained microbes from lake sediment, and the other from sewage sludge.
The process of enzyme discovery for this project was both challenging and unconventional. The researchers first started working with a bacterial species reported to make toluene, but when those reports appeared to be irreproducible, the scientists turned to the environment for toluene-producing cultures—specifically to municipal sewage and anoxic lake sediment.
All enzyme discovery projects are challenging. But moving from discovery in a single bacterial species, to discovery in a complex microbial community from sewage sludge or lake sediments, was more difficult by orders of magnitude. This study became a needle-in-a-haystack search for the toluene-producing enzyme in a candidate pool of hundreds of thousands of enzymes.
Metagenome analyses revealed that these microbial communities each contained more than 300,000 genes—the equivalent of more than 50 bacterial genomes. Another challenge was that the anaerobic microbial communities and many of their enzymes were sensitive to oxygen, forcing the scientists to manipulate cultures and enzymes under strictly anaerobic conditions.
JBEI is a DOE Bioenergy Research Center funded by DOE's Office of Science.