UCLA

Global climate change, caused by the emission of greenhouse gas (GHG) to the atmosphere, is one of the key challenges facing humanity in the coming decades. Carbon dioxide (CO2) is one of the leading sources of overall GHG emission, and it is released to the environment in large quantities by the burning of petroleum or other fossil fuels as liquid fuel. Sustainable alternatives to fossil fuels can be produced from biomass using microbial whole-cell catalysis. Since biomass fixes atmospheric CO2, burning these biomass-derived fuels as energy sources reduces the net emission of carbon to the environment. However, current biofuel processes often struggle to compete economically with fossil fuels. A key contributing factor to this problem is the inefficient utilization of feedstock carbon in biomass. Since microbes use conserved metabolic pathways to catabolize carbohydrate feedstocks, inherent limitations in these pathways often cannot be avoided. Moreover, these pathways evolved in nature to prioritize growth rather than production considerations. A notable example of this is the carbon loss when forming two-carbon (C2) metabolites, such as acetyl-CoA. C2 metabolites are precursors to many industrial products, including important biofuels such as ethanol and butanol. Essentially all organisms degrade sugar to pyruvate, a three-carbon (C3) metabolite using the conserved glycolytic pathways. To produce C2, pyruvate is decarboxylated to produce one C2 equivalent and one CO2, thus wasting a third of input carbon and effectively limiting the maximum carbon yield of C2-derived products to 67%. This carbon loss imposes a significant economic constraint on biorefining. While no natural pathway can bypass pyruvate formation, a synthetic non-oxidative glycolysis (NOG) was recently developed that directly degrades sugar phosphates into stoichiometric amounts of C2 equivalents. Here, we first constructed a strain of E. coli, a model microbial organism, which uses NOG for sugar catabolism rather than glycolysis. This was achieved by a combination of rational design and evolution. By coupling the pathway to growth, we were able to use evolution to develop a robust pathway and fine tune the relative activity of pathway enzymes. The resulting strain grew at a comparable rate to wild-type E. coli and could produce acetate, a C2-derived product, at yield exceeding the theoretical maximum using glycolysis. Since it uses NOG for sugar catabolism, this strain has fundamentally rewired metabolism, as it generates C2 metabolites before C3 metabolites. C3 metabolites are generated from C2 using carbon scavenging pathways. Thus, we believe this strain has considerable potential as a host for the production of C2-derived products. Following evolution of the NOG strain, the chromosome was sequenced to elucidate mutations acquired through evolution. The effect of several of these mutations was also investigated here. We found that a transposon insertion inactivating PtsG, a component of the glucose-specific phosphotransferase system, was essential to the NOG strain’s ability to grow in minimal media. When ptsG is deleted, the catabolic activator protein (CAP) is able to upregulate many genes, which is likely necessary for the NOG-dependent growth phenotype. Another transposon insertion in alternative sigma factor rpoS contributed to growth in the NOG strain, likely through the upregulation of enzymes that benefit NOG growth, such as the TCA cycle. We also evaluated mutations where the cell had fine-tuned the activity of pathway enzymes. A mutation knocking down activity in key NOG enzyme phosphoketolase (F/Xpk) was found to be beneficial for growth by potentially alleviating a kinetic trap in the NOG cycle. However, fixing a promotor truncation that had knocked down activity of Pck, a key gluconeogenic enzyme, actually improved growth rate in glucose media by about 15%. Since the mutation occurred before NOG growth was developed, it was reasoned that changing intracellular conditions after the adaptation to NOG-based growth caused an increase in Pck activity to be beneficial. We also explored using the NOG strain for the production of ethanol, a C2-derived liquid fuel. Since ethanol is more reduced than acetate, it cannot be generated without the supply of external reducing power using NOG. Using formate as the electron donor, we were able to improve ethanol yield from 0.26 to 0.82 through the addition of formate in anaerobic production. Further improvements in ethanol yield were limited by the cells ability to produce ATP to maintain sugar phosphorylation. Since the cell is dependent on acetate production to make ATP anaerobically, ethanol production will need to be integrated with respiration, which can convert excess reducing power into ATP using oxygen as an electron acceptor. Finally, we offer strategies for establishing robust, high yield ethanol production using the NOG strain in microaerobic conditions.