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.

三十多年前在缺氧的湖泊沉积物中报告了微生物甲苯的生物合成，但是催化这种生化挑战性反应的酶从未被发现。在这里，我们报告产生甲苯的酶PhdB（一种细菌来源的糖基自由基酶，催化苯乙酸酯的脱羧反应）及其同源活化酶PhdA（一种自由基S）-腺苷甲硫氨酸酶，在两个不同的产生甲苯的缺氧微生物群落中发现。从复杂的微生物群落（> 300,000个基因）而不是从微生物分离物中发现酶的非常规过程涉及宏基因组学和元蛋白质组学支持的生物化学，以及体外重组酶活性的确认。这项工作扩大了已知的糖基自由基酶（以前仅表征了7种反应类型）和产生芳烃的酶的催化范围，并将使首次利用可再生资源（例如木质纤维素生物质）生化合成芳族燃料碳氢化合物成为可能。 ，而不是石油。