Model-Guided Design of Synthetic Microbial Consortia for Next-Generation Biofuel Production
Deepan Thiruppathy1* (firstname.lastname@example.org), Neal N. Hengge2, Lizzette Moreno García2, Gustavo Lastiri-Pancardo1, Violeta Sànchez i Nogué2, Jeffrey G. Linger2, Michael T. Guarnieri2, and Karsten Zengler1
1University of California–San Diego; 2National Renewable Energy Laboratory
The team propose to lay the foundation for bioproduction using multifaceted microbial communities. Researchers will build metabolic community models of increasing complexity by integrating multiomics datasets. These models will guide engineering designs for optimized production of biofuels from lignocellulosic biomass. Furthermore, researchers will use innovative approaches to augment existing communities for improved bioproduction and complete conversion of different biomass feedstock. Overall, these strategies will provide knowledge of the functional metabolic exchanges driving interspecies interactions in microbial communities, thus providing insights into fundamental biological processes. Lessons learned here would be crucial for designing stable microbial communities for various biotechnology applications in the future.
Microbial communities are everywhere, and their influence on the environment is gaining recognition for their industrial potential, such as bioenergy production. The multiplicity of intertwined, interspecies metabolite interactions within these communities regulates their ultimate functional organization and assembly. This allows them to perform complex functional tasks unreachable by axenic systems, such as the breakdown of hardy lignocellulosic materials into high-energy volatile fatty acids (VFAs).
Bioproduction of one such fatty acid, butyrate (BA), from sustainable lignocellulosic sources has gained attention owing to butyrate’s versatile applications as a precursor for jet-fuel, polymers, fibers, and even cosmetics. However, current industrial processes have been forced to rely on monoculture setups requiring expensive enzymatic raw-material preprocessing. Thus, there is a need to rationally design reproducible, tunable consortia that can replicate the collective capabilities of natural communities, thereby negating the need for the expensive preprocessing steps and significantly intensifying the economic benefit of using cheap feedstocks.
Here, team members characterized the metabolic interactions of the mutualistic co-culture of Clostridium thermocellum and Clostridium thermobutyricum, recently shown to be effective in converting lignocellulosic biomass to butyrate (Chi et al. 2018), and identified bottlenecks that could be relieved by augmentation with additional microbes to increase biomass conversion performance and efficiency. The approach is two-pronged. Researchers first used high quality and manually curated genome-scale metabolic models (GEMs) for both species to unravel the metabolic exchange network of the co-culture, compartmentalized as a community model containing 1,777 reactions, 1,679 metabolites, and 1,569 genes. This allowed the team to computationally identify metabolic bottlenecks responsible for the co-culture’s limited butyrate production efficiency. Simultaneously, team members experimentally identified substrate inefficiencies in the co-culture setup by measuring solids deconstruction percentages and characterizing the monomeric and oligomeric sugars in the substrate left unused. Researchers tested the co-culture on raw corn-stover and deacetylated-milled corn stover (DMR) and determined highest butyrate production from DMR (without enzymatic pre-processing), with a solid deconstruction of 83.1%. The largest percentage of leftover unused sugar moieties were xylose oligomers along with some arabinose and glucose oligomers.
To identify candidate isolates that could augment the co-culture to improve carbohydrate utilization, researchers collected various soil samples and enriched them on raw corn stover, bagasse, switchgrass, poplar, and pine substrates as well as on supernatants from the DMR/co-culture experiments. The enrichments were carried out under anoxic conditions at 55°C, identical to those used with the Clostridia co-culture. They were passaged multiple times into fresh media, keeping the raw lignocellulosic plant materials as their sole carbon source, to ensure the selected members are producing butyrate, which was validated by HPLC.
Following isolation, the isolates will be tested for compatibility with the co-culture using a high- throughput community design and construction method (Coker et al. 2022). These constructed consortia will then be further engineered to optimize production of butyrate. Engineering strategies will be guided by community metabolic models, ensuring the collective capability of the designed community is reproducible and optimized toward bioproduction. This study will lay the foundation for advanced bioproduction using multifaceted microbial communities inspired from nature and will expand knowledge on intra-community microbial interactions.
Chi, X., et al. 2018. “Hyper-production of Butyric Acid from Delignified Rice Straw by a Novel Consolidated Bioprocess,” Bioresour. Technol. 254, 115–120.
Coker, J., et al. 2022. “A Reproducible and Tunable Synthetic Soil Microbial Community Provides New Insights into Microbial Ecology,” mSystems 7, e00951-22.
This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award DE- SC0022137.