Produce natural and non-natural compounds using multi-species microbiomes.

Microbiome Engineering

Produce natural and non-natural compounds using multi-species microbiomes.

Current State-of-the-Art

Current efforts to use microbiomes for biosynthesis have focused on using consortia to improve production efficiency or to produce compounds that are difficult to synthesize using a single strain. Different strains or species have been co-cultured to optimize substrate utilization. Multiple species that are naturally well-suited to convert the molecule of interest can be co-cultured to identify optimal species compositions to achieve high titers. Mutualistic interactions can be engineered in synthetic multi-species consortia to increase fermentation efficiency.¹^,²^,³ Co-cultures of different species can also be used to convert multiple substrates into the desired products through different metabolic routes. This strategy was used to demonstrate complete conversion of glucose and xylose to ethanol.⁴ Similarly, when multiple sugars are available for fermentation, mixtures of “specialist” strains that ferment only one of each type of sugar can outperform individual strains that ferment multiple sugars.⁵ Microbial co-cultures have been demonstrated to use carbon dioxide and synthesis gas (syngas) to produce alcohols and other metabolites, while single cultures would only be capable of producing acetate.⁶^,⁷^,⁸

Another approach relies on consortia where biosynthetic reactions are distributed among naturally-occurring or engineered specialist organisms that are best-suited to perform select reactions within a metabolic pathway. Consortia have been engineered where engineered strains remove the metabolic byproducts generated by a primary producer strain.⁹ There are also several examples of production pathways split between different strains. Using this strategy, compounds previously too burdensome or toxic for using a single strain could be produced.¹⁰^,¹¹^,¹² Quorum sensing regulation and division of labor have also been reported to engineer time-controlled expression of portions of the pathway and improve production.¹³

Most current work has used a limited selection of model organisms (e.g., Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis) with well-established genetic tools. Significant advances in microbiome engineering will require expanding these models to include microbes from across the tree of life, including Archaea.

Breakthrough Capabilities & Milestones

Distributed compound biosynthesis using multiple microbial species.

Identify and engineer transporters, sensors, and/or extracellular enzymes that facilitate the movement of intermediate compounds within a microbiome.

Engineer species to convert common biological intermediates or electron carriers (e.g., acetate, formate, butyrate, ethanol) into higher value precursor molecules for further transformations.

Bottleneck/Challenge: Intermediates may be toxic to cells and inhibit microbial growth or production.¹⁴

Potential Solution: Engineer microbiome metabolism to decrease toxicity of specific compounds (e.g., acetogenic consumption of methanol to bypass formaldehyde production).

Potential Solution: Engineer microbiomes to simultaneously consume multiple intermediates for production, so they produce practical amounts of compounds without risk of cellular toxicity.

Produce additional compounds (e.g., addition of multiple functional groups) using a different microbial species to catalyze the reaction.

Engineer multi-step synthetic pathways spanning multiple organisms, to reduce accumulation of toxic intermediates.

Bottleneck/Challenge: Many bioactive compounds are the result of long metabolic pathways, so differentiating between toxicity and general cell stress (due to large gene clusters) may be challenging.

Potential Solution: Improve existing metabolomics models to better distinguish between rate-limiting catalysis, metabolite toxicity, or additional factors.

Potential Solution: Design biosensors to detect metabolic bottlenecks and indicate which parts of a pathway should be moved to a different organism.

Bottleneck/Challenge: In natural environments, exported compounds may be metabolized by other non-engineered microbes.

Potential Solution: Engineer microbes that self-associate into higher density clusters to outcompete non-engineered microbes.

Spatially organize communities with distinct catalysis environments (e.g., reducing or oxidizing) to facilitate more efficient and specific biosynthesis.

Bottleneck/Challenge: Microbiomes must be able to resist dispersive forces or reorganize a disrupted structure in natural environments.

Potential Solution: Engineer exosomes/liposomes that can incorporate proteins from multiple organisms (i.e., multiple vesicles fuse together and all cargo intermingles) to create spatially separated reaction compartments.

Potential Solution: Engineer cell surface proteins that facilitate specific flocculation properties so sub-groups of microbes naturally reassociate when disrupted.

Compound biosynthesis using community members that support a primary production strain.

Engineer microbiomes to remove inhibitory compounds (e.g., hydrogen or sulfur-containing compounds) to stimulate catalysis in a primary species.

Bottleneck/Challenge: Anaerobic microbial communities frequently produce hydrogen as an electron sink for metabolism, but high levels of hydrogen inhibit enzyme function and reduce substrate to product conversion.

Potential Solution: Design microbiomes containing hydrogenotrophic microbes alongside hydrogen-producing microbes, so that the hydrogenotrophs can detoxify excess concentrations of hydrogen.

Engineer microbiomes to maintain homeostasis in the local environment to ensure highest efficiency of the primary producer strain in situ.

Use existing microbial partnerships to synthesize value-added compounds (e.g., anaerobic fungi convert plant biomass into ethanol, acids, and hydrogen gas. Methanogens and other hydrogenotrophs use the hydrogen gas as an electron donor to make something valuable, like methane).

Bottleneck/Challenge: Mechanisms of extracellular transfer of reducing power (e.g., secretion of reduced compounds such as hydrogen, electrode-microbe interactions) may not be compatible between electron-donating and accepting organisms.¹⁵

Potential Solution: Heterologous expression of pathways for direct extracellular electron transfer or uptake to enhance efficiency of syntrophic partnerships.

Design communities of organisms consuming multiple different carbon sources, all of which transfer their reducing power to a universal carrier (e.g., microbial species, extracellular protein, small molecule) that can be used by one or more producer organisms for biosynthesis.

Bottleneck/Challenge: Identifying a universal carrier that is compatible with all organisms in the microbiome may be difficult.

Potential Solution: Explore use of compounds that can pass through membranes such as neutral red and anthraquinone-2,6-disulfonate (AQDS).

Bottleneck/Challenge: Electron transfer may be inhibited or quenched by compounds found in the local environment and/or may react to form toxic radicals.

Potential Solution: Engineer constituents of the community to scavenge compounds created by rogue electron transfer events such as superoxide and hydroxyl radical.

Potential Solution: Design community to include small numbers to respire or remove oxygen that may intrude into the system.

Potential Solution: Identify reactive compounds in the local environment and engineer strains able to quench or transform these compounds.

Rapid design of engineered microbiomes that functionally complement existing natural microbiomes.

Develop sampling and analysis methods that can be adapted to diverse environmental niches.

Identify ‘available’ environmental niches based on the metagenomic, metatranscriptomic and metabolomic profiles of an existing natural community.

Bottleneck/Challenge: Environmental niches are frequently colonized stochastically by the first microbes adapted to that environment. However, other microbes may exist that could thrive in those niches and improve community function.

Potential Solution: Computational models that can predict community output (e.g., metabolites consumed/produced, essential cofactors required) as a function of microbiota composition, metabolic function, and microbial competition.

Design a microbiome capable of invading a natural community and producing a specific metabolite in the environment of interest.

Bottleneck/Challenge: Establishing new microbial members to introduce a new function that outcompetes the existing community is hard. Existing communities are frequently robust, and resist introgression of other species.

Potential Solution: Engineer a microbe that temporarily induces dysbiosis (e.g., secretes an antimicrobial compound) to destabilize the community and create a niche for the introduced species.

Generate a modular toolbox of microbes with defined function to support the desired environmental niche (e.g., provide resistance to compounds, generate specific products or byproducts).

Footnotes

Mee, M. T., Collins, J. J., Church, G. M., & Wang, H. H. (2014). Syntrophic exchange in synthetic microbial communities. Proceedings of the National Academy of Sciences, 111(20), E2149–E2156. https://doi.org/10.1073/pnas.1405641111
Wang, E.-X., Ding, M.-Z., Ma, Q., Dong, X.-T., & Yuan, Y.-J. (2016). Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation. Microbial Cell Factories, 15(1), 21. https://doi.org/10.1186/s12934-016-0418-6
Wang, E.-X., Liu, Y., Ma, Q., Dong, X.-T., Ding, M.-Z., & Yuan, Y.-J. (2019). Synthetic cell–cell communication in a three-species consortium for one-step vitamin C fermentation. Biotechnology Letters, 41(8–9), 951–961. https://doi.org/10.1007/s10529-019-02705-2
Fu, N., Peiris, P., Markham, J., & Bavor, J. (2009). A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures. Enzyme and Microbial Technology, 45(3), 210–217. https://doi.org/10.1016/j.enzmictec.2009.04.006
Eiteman, M. A., Lee, S. A., & Altman, E. (2008). A co-fermentation strategy to consume sugar mixtures effectively. Journal of Biological Engineering, 2(1), 3. https://doi.org/10.1186/1754-1611-2-3
Haas, T., Krause, R., Weber, R., Demler, M., & Schmid, G. (2018). Technical photosynthesis involving CO2 electrolysis and fermentation. Nature Catalysis, 1(1), 32–39. https://doi.org/10.1038/s41929-017-0005-1
Molitor, B., Mishra, A., & Angenent, L. T. (2019). Power-to-protein: Converting renewable electric power and carbon dioxide into single cell protein with a two-stage bioprocess. Energy & Environmental Science, 12(12), 3515–3521. https://doi.org/10.1039/C9EE02381J
Richter, H., Molitor, B., Diender, M., Sousa, D. Z., & Angenent, L. T. (2016). A Narrow pH Range Supports Butanol, Hexanol, and Octanol Production from Syngas in a Continuous Co-culture of Clostridium ljungdahlii and Clostridium kluyveri with In-Line Product Extraction. Frontiers in Microbiology, 7. https://doi.org/10.3389/fmicb.2016.01773
Bernstein, H. C., Paulson, S. D., & Carlson, R. P. (2012). Synthetic Escherichia coli consortia engineered for syntrophy demonstrate enhanced biomass productivity. Journal of Biotechnology, 157(1), 159–166. https://doi.org/10.1016/j.jbiotec.2011.10.001
Bernstein, H. C., Paulson, S. D., & Carlson, R. P. (2012). Synthetic Escherichia coli consortia engineered for syntrophy demonstrate enhanced biomass productivity. Journal of Biotechnology, 157(1), 159–166. https://doi.org/10.1016/j.jbiotec.2011.10.001
Jones, J. A., Vernacchio, V. R., Collins, S. M., Shirke, A. N., Xiu, Y., Englaender, J. A., Cress, B. F., McCutcheon, C. C., Linhardt, R. J., Gross, R. A., & Koffas, M. A. G. (2017). Complete Biosynthesis of Anthocyanins Using E. coli Polycultures. MBio, 8(3), mBio.00621-17, e00621-17. https://doi.org/10.1128/mBio.00621-17
Zhou, K., Qiao, K., Edgar, S., & Stephanopoulos, G. (2015). Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nature Biotechnology, 33(4), 377–383. https://doi.org/10.1038/nbt.3095
Jones, J. A., Vernacchio, V. R., Sinkoe, A. L., Collins, S. M., Ibrahim, M. H. A., Lachance, D. M., Hahn, J., & Koffas, M. A. G. (2016). Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids. Metabolic Engineering, 35, 55–63. https://doi.org/10.1016/j.ymben.2016.01.006
Claassens, N. J., Cotton, C. A. R., Kopljar, D., & Bar-Even, A. (2019). Making quantitative sense of electromicrobial production. Nature Catalysis, 2(5), 437–447. https://doi.org/10.1038/s41929-019-0272-0
Kracke, F., Vassilev, I., & Kömer, J. O. (2015). Microbial electron transport and energy conservation – the foundation for optimizing bioelectrochemical systems. Frontiers in Microbiology, 6. https://doi.org/10.3389/fmicb.2015.00575