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Wastewater biosolids are a promising feedstock for production of value-added renewable chemicals. Methane-producing archaea (methanogens) are already used to produce renewable biogas via the anaerobic treatment of wastewater. The ability of methanogens to efficiently convert dissolved organic carbon into methane makes them an appealing potential platform for biorefining using metabolic engineering. We have engineered a strain of the methanogen Methanosarcina acetivorans to produce the volatile hemiterpene isoprene in addition to methane. The engineered strain was adapted to grow in municipal wastewater through cultivation in a synthetic wastewater medium. When introduced to municipal wastewater the engineered methanogens were able to compete with the indigenous microorganisms and produce 0.97 mM of isoprene (65.9 ± 21.3 g per m3 of effluent). The production of isoprene in wastewater appears to be dependent on the quantity of available methanogenic substrate produced during upstream digestion by heterotrophic fermenters. This shows that with minimal adaptation it is possible to drop-in engineered methanogens to existing wastewater environments and attain value-added products in addition to the processing of wastewater. This shows the potential for utilizing methanogens as a platform for low-cost production of renewable materials without expensive feedstocks or the need to build or adapt existing facilities. 

ABSTRACT Isoprene is a valuable petrochemical used for a wide variety of consumer goods, such as adhesives and synthetic rubber. We were able to achieve a high yield of renewable isoprene by taking advantage of the naturally high-flux mevalonate lipid synthesis pathway in anaerobic methane-producing archaea (methanogens). Our study illustrates that by genetically manipulating Methanosarcina species methanogens, it is possible to create organisms that grow by producing the hemiterpene isoprene. Mass balance measurements show that engineered methanogens direct up to 4% of total carbon flux to isoprene, demonstrating that methanogens produce higher isoprene yields than engineered yeast, bacteria, or cyanobacteria, and from inexpensive feedstocks. Expression of isoprene synthase resulted in increased biomass and changes in gene expression that indicate that isoprene synthesis depletes membrane precursors and redirects electron flux, enabling isoprene to be a major metabolic product. Our results demonstrate that methanogens are a promising engineering chassis for renewable isoprene synthesis. IMPORTANCE A significant barrier to implementing renewable chemical technologies is high production costs relative to those for petroleum-derived products. Existing technologies using engineered organisms have difficulty competing with petroleum-derived chemicals due to the cost of feedstocks (such as glucose), product extraction, and purification. The hemiterpene monomer isoprene is one such chemical that cannot currently be produced using cost-competitive renewable biotechnologies. To reduce the cost of renewable isoprene, we have engineered methanogens to synthesize it from inexpensive feedstocks such as methane, methanol, acetate, and carbon dioxide. The “isoprenogen” strains we developed have potential to be used for industrial production of inexpensive renewable isoprene.