Man-made photovoltaic device is relatively efficient in converting sunlight to electricity, but the electrical energy generated is difficult to store. Current methods, such as chemical batteries, hydraulic pumping, and water splitting, suffer from low energy density or incompatibility with current transportation infrastructure. The biological systems, on the other hand, can store sunlight in high energy-density carbon-carbon bonds. However, the photosynthesis has low solar energy harvesting efficiency, for which no near-term improvements are in sight. One way to solve both problems is to combine man-made solar cells to biological Carbon dioxide (CO2) fixation and fuel production. Therefore, a microbial process to produce biofuels from CO2 and electricity is needed. In this work, we first used metabolic engineering methods to achieve the production of isobutanol, n-butanol, and 3-methyl-1-butanol (3MB) in a lithoautotrophic bacterium Ralstonia eutropha H16, which can utilize formate as the sole carbon and energy source. We then demonstrated electrochemical production of formate from CO2 and finally integrated the electrochemical reactions with the microbial fuel production. Besides achieving the goals in engineering, this work explored important design principles of metabolic pathway and developed new tools in synthetic biology.
To produce higher alcoholos, two different carbon chain building routes were constructed. First, Coenzyme A (CoA)-dependent pathway was used to synthesize isobutanol and n-butanol in R. eutropha H16. We demonstrated for the first time the production of isobutanol, a branched-chain alcohol, using the CoA-dependent pathway in recombinant R. eutropha H16. The isobutanol production pathway deviates from the CoA-dependent n-butanol production pathway in that it contains an extra carbon chain rearrangement step catalyzed by the isobutyryl-CoA mutase in R. eutropha, which has not been characterized previously. Metabolic engineering methods such as heterologous gene expression, codon optimization, and promoter strength altering were applied to first achieve the production of ~200mg/L n-butanol from fructose or ~30mg/L from formate by engineered R. eutropha. The isobutyryl-CoA mutase was then added to the pathway to achieve the production of ~30mg/L isobutanol from fructose. The carbon skeleton rearrangement chemistry explored here might be used to expand the repertoire of the chemicals accessible with the CoA-dependent pathway.
Next, keto acid-dependent pathway was used to synthesize isobutanol and 3MB in R. eutropha H16. We integrated the set of genes for isobutanol and 3MB production into R. eutropha H16 genome, namely alsS from Bacillus subtilis, and ilvC and ilvD from Escherichia coli. The genes kivd from Lactococcus lactis and yqhD from E.coli were then introduced using a plasmid. R. entropha uses poly[R-(-)-3-hydroxybutyrate] (PHB) as a storage compound and as the metabolic sink for carbon and reducing equivalents. We disrupted the PHB synthesis and used the synthetic isobutanol and 3MB production pathway as the new metabolic sink. In a pH-coupled formic acid feeding fermentor, the engineered strain LH74D produced fuels with the final titer of over 1.4 g/l (~846mg/l isobutanol and ~570mg/l 3MB) and peak productivity of 25 mg/l/h.
Most natural metabolic pathways are regulated on transcriptional level or on protein level by allosteric effectors. In this work, non-native regulatory mechanisms were introduced to the synthetic pathways. In particular, on transcriptional level, a synthetic anhydrotetracycline (aTc)-controllable gene expression system in R. eutropha H16 was developed and applied in regulating the biofuel production gene. The system is composed of a controllable promoter containing the operator tetO, the repressor tetR, and the inducer aTc. The active hybrids between the tetO operators and the native PrrsC were first identified and shown to be repressable by tetR. Next, two mutants of the native PphaC1 promoter were obtained from a high-throughput screening of 300 candidates to tune the tetR expression. The optimized system, which contains the PrrsC-O1-O1 hybrid promoter and the PphaC1-G3::tetR cassette, has decreased leaky expression level and can be regulated gradually by different aTc concentration with a ~11 fold dynamic range. The system was used to alleviate cellular toxicity caused by AlsS overexpression, which impeded our metabolic engineering work on isobutanol production in R. eutropha H16. The system reported in this study may be a useful tool for future research and engineering work in this organism.
Finally, we combined the electrochemical formate production with the microbial fuel synthesis by the engineered host in an integrated process. The challenge was that the growth of the microbial host was inhibited by the electrochemical reaction in a transient manner, suggesting that unstable compounds such as reactive oxygen or nitrogen species might be responsible for the growth inhibition. Reporter assays in R. eutropha H16 showed that sodC and norA promoters, which drives the genes for O2− and NO defense pathway, respectively, were induced when electricity was on. To circumvent this toxicity problem, a porous ceramic cup was used to shield the anode. This inexpensive shield provides a tortuous diffusion path for chemicals. Therefore, the reactive compounds produced by the anode may be quenched before reaching the cells growing outside the cup. Using this approach, healthy growth of Ralstonia strains and production of over 140mg/l biofuels were achieved with the electricity and CO2 as the sole source of energy and carbon, respectively.