Isoprene is a valuable petrochemical used for a wide variety of consumer goods such as adhesives and synthetic rubber. We were able to achieve 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 spp. methanogens it is possible to create organisms that grow by producing the hemiterpene isoprene. Mass balance measurements show engineered methanogens direct up to 4% total carbon flux to isoprene, demonstrating methanogens produce higher isoprene yields than engineered yeast, bacteria, or cyanobacteria from inexpensive feedstocks. Expression of isoprene synthase resulted in increased biomass and changes in gene expression that indicate isoprene synthesis depletes membrane precursors and redirects electron flux enabling isoprene to be a major metabolic product. Our results demonstrate methanogens are a promising engineering chassis for renewable isoprene synthesis. IMPORTANCE A significant barrier to implementing renewable chemical technologies is high production costs versus 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.
The human gut functions through a complex system of interactions between the host human tissue and the microbes which inhabit it. These diverse interactions are difficult to model or examine under controlled laboratory conditions.
Microbial metabolism and trophic interactions between microbes give rise to complex multispecies communities in microbe-host systems. Bacteroides thetaiotaomicron (B. theta) is a human gut symbiont thought to play an important role in maintaining host health. Untargeted nuclear magnetic resonance metabolomics revealed B. theta secretes specific organic acids and amino acids in defined minimal medium. Physiological concentrations of acetate and formate found in the human intestinal tract were shown to cause dose-dependent changes in secretion of metabolites known to play roles in host nutrition and pathogenesis. While secretion fluxes varied, biomass yield was unchanged, suggesting feedback inhibition does not affect metabolic bioenergetics but instead redirects carbon and energy to CO2 and H2. Flux balance analysis modeling showed increased flux through CO2-producing reactions under glucose-limiting growth conditions. The metabolic dynamics observed for B. theta, a keystone symbiont organism, underscores the need for metabolic modeling to complement genomic predictions of microbial metabolism to infer mechanisms of microbe-microbe and microbe-host interactions. IMPORTANCE Bacteroides is a highly abundant taxon in the human gut, and Bacteroides thetaiotaomicron (B. theta) is a ubiquitous human symbiont that colonizes the host early in development and persists throughout its life span. The phenotypic plasticity of keystone organisms such as B. theta is important to understand in order to predict phenotype(s) and metabolic interactions under changing nutrient conditions such as those that occur in complex gut communities. Our study shows B. theta prioritizes energy conservation and suppresses secretion of “overflow metabolites” such as organic acids and amino acids when concentrations of acetate are high. Secreted metabolites, especially amino acids, can be a source of nutrients or signals for the host or other microbes in the community. Our study suggests that when metabolically stressed by acetate, B. theta stops sharing with its ecological partners.
Methanogens are anaerobic archaea which conserve energy by producing methane. Found in nearly every anaerobic environment on earth, methanogens serve important roles in ecology as key organisms of the global carbon cycle, and in industry as a source of renewable biofuels. Environmentally, methanogenic archaea play an essential role in the reintroducing unavailable carbon to the carbon cycle by anaerobically converting low-energy, terminal metabolic degradation products such as one and two-carbon molecules into methane which then returns to the aerobic portion of the carbon cycle. In industry, methanogens are commonly used as an inexpensive source of renewable biofuels as well as serving as a vital component in the treatment of wastewater though this is only the tip of the iceberg with respect to their metabolic potential. In this review we will discuss how the efficient central metabolism of methanoarchaea could be harnessed for future biotechnology applications.
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.
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