Sustainable Biochemical Production from C1 Substrates

Single-carbon substrates as feedstocks for bioprocessing

Single-carbon (C1) compounds including carbon monoxide, methanol and formic acid have emerged as promising feedstocks for biofuel and biochemical production by engineered microbes. These substrates can be produced renewably from CO2 and other waste materials, bypassing food security and land conversion concerns raised over traditional biofuel feedstocks.

Acetogens efficiently metabolize C1 compounds

Acetogens like Eubacterium limosum are an ancient, anaerobic group of bacteria that evolved in energy-starved conditions. They grow on C1 compounds, extracting as much energy as possible from these substrates as possible with the highly efficient Wood-Ljungdahl pathway.

Important aspects of acetogen metabolism are still unclear

Without a full, systems-level characterization of acetogen metabolism, it is impossible to rationally engineer these microbes to produce chemicals at commercially viable metrics. For example, the electron donor for the key enzyme methylenetetrahydrofolate reductase in E. limosum is still unknown. One possible scenario is pictured on the left, but experimental evidence is lacking. We are using a range of techniques to probe this hypothesis, as well as other interesting fundamental questions about metabolism and regulation in E. limosum to provide foundational knowledge for metabolic engineering efforts.

Synthetic biology tools for acetogens are underdeveloped

Compared to traditional 'chassis' organisms like E. coli and yeast, acetogens are much more difficult to genetically engineer. We have developed a range of foundational tools for E. limosum, including an expanded range of antibiotics and selection markers, high-efficiency transformation procedures, and characterized promoter libraries. More recently, we have developed advanced workflows for efficient integration of large biosynthetic gene clusters into the chromosome, as well as a CRISPR-recombineering platform for rapidly generating precise point mutations, deletions and small insertions. See our Publications page for more details, and the Resources page for detailed protocols for using these systems and a list of plasmids we will gladly share with the academic community.

Synthetic co-culture for maximizing production from C1 substrates

Because of their anaerobic lifestyle, the range of products that could be made at commercially relevant metrics by acetogens is somewhat constrained. We are pioneering a novel co-culture approach to overcome this problem, which combines the efficiency of anaerobic metabolism with the increased energy available from aerobic respiration in the same reactor to achieve simultaneously high yields and productivities . In this system, C1 metabolism and product formation are split between two different microbes. The acetogen converts C1 compounds to acetate at high yield. A partner aerobic organism is responsible for converting the acetate to a higher-value product, and for rapidly consuming the oxygen in the reactor to establish the microaerobic conditions necessary for acetogen growth. Essentially, a symbiotic relationship is established, in which the aerobe protects the anaerobe from oxygen in exchange for fixed carbon.

Controlling Sulfur Metabolism in the Gut Microbiota

Probiotics engineered to manipulate gut microbial metabolism are a promising therapeutic platform

Dysbiosis in the human gut microbiota is increasingly recognized as an important driver of host health. Correlations have been described between microbial composition and a variety of diseases, including cardiovascular disease, inflammatory bowel disease, obesity, and even some neurological disorders. A primary method by which bacteria exert influence on host health is through their production and breakdown of small molecule metabolites that act as chemical messengers between microbes and host cells. The expanding role of gut microbial metabolites in health and disease has motivated the development of approaches to modulate microbial metabolism for therapeutic outcomes. One promising approach involves engineered probiotics; genetically modified commensal microbes programmed to produce a therapeutic metabolite or consume a toxic one to mitigate its effects. Several companies have developed strains targeting diseases ranging from lung cancer to phenylketonuria (PKU), with demonstrated efficacy in mouse models6 and non-human primates, and several advancing to clinical development

Dynamic control for precision medicine

While engineered strains have been successfully engineered to either reduce the concentration of a toxic metabolite or produce a therapeutic one, strains capable of controlling the level of a metabolite within a narrow window have not been developed. Such ‘smart probiotics’, able to dynamically respond to the environment and either produce or consume a compound in response to the local concentration, would be useful for stabilizing metabolites which play a concentration-dependent role in host health and disease. For example, ulcerative colitis and Crohn’s disease have been linked to microbially produced hydrogen sulfide (H2S), with a growing consensus that low levels of H2S have anti-inflammatory properties and support a healthy epithelium, whereas high concentrations of H2S are genotoxic, inhibit mitochondrial function and butyrate oxidation, and potentially weaken the mucosal barrier. Given that H2S concentrations vary spatially and temporally throughout the mucosa, controlling H2S within a tight range is not possible with current small-molecule sulfide donors, which release sulfide regardless of local concentration. We are developing a new synthetic biology-based approach to controlling microbial metabolites in situ, in which the engineered microbe uses a transcription factor responsive to the metabolite of interest to dynamically balance the expression of metabolic pathways for production and consumption of the metabolite, thus producing a stable, titratable concentration in a manner analogous to a thermostat.