Electrical-biological hybrid system for carbon efficient isobutanol production.

We have developed an electrical-biological hybrid system wherein an engineered microorganism consumes electrocatalytically produced formate from CO2 to supplement the bioproduction of isobutanol, a valuable fuel chemical. Biological CO2 sequestration is notoriously slow compared to electrochemical CO2 reduction, while electrochemical methods struggle to generate carbon-carbon bonds which readily form in biological systems. A hybrid system provides a promising method for combining the benefits of both biology and electrochemistry. Previously, Escherichia coli was engineered to assimilate formate and CO2 in central metabolism using the reductive glycine pathway. In this work, we have shown that chemical production in E. coli can benefit from single carbon substrates when equipped with the RGP. By installing the RGP and the isobutanol biosynthetic pathway into E. coli and by further genetic modifications, we have generated a strain of E. coli that can consume formate and produce isobutanol at a yield of >100% of theoretical maximum from glucose. Our results demonstrate that carbon produced from electrocatalytically reduced CO2 can bolster chemical production in E. coli. This study shows that E. coli can be engineered towards carbon efficient methods of chemical production.

Adaptive laboratory evolution for improved tolerance of isobutyl acetate in Escherichia coli.

Previously, Escherichia coli was engineered to produce isobutyl acetate (IBA). Titers greater than the toxicity threshold (3 g/L) were achieved by using layer-assisted production. To avoid this costly and complex method, adaptive laboratory evolution (ALE) was applied to E. coli for improved IBA tolerance. Over 37 rounds of selective pressure, 22 IBA-tolerant mutants were isolated. Remarkably, these mutants not only tolerate high IBA concentrations, they also produce higher IBA titers. Using whole-genome sequencing followed by CRISPR/Cas9 mediated genome editing, the mutations (SNPs in metH, rho and deletion of arcA) that confer improved tolerance and higher titers were elucidated. The improved IBA titers in the evolved mutants were a result of an increased supply of acetyl-CoA and altered transcriptional machinery. Without the use of phase separation, a strain capable of 3.2-fold greater IBA production than the parent strain was constructed by combing select beneficial mutations. These results highlight the impact improved tolerance has on the production capability of a biosynthetic system.

Nonphotosynthetic Biological CO2 Reduction.

Alarming changes in environmental conditions have prompted significant research into producing renewable commodities from sources other than fossil fuels. One such alternative is CO2, a determinate greenhouse gas with historically high atmospheric levels. If sequestered, CO2 could be used as a highly renewable feedstock for industrially relevant products and fuels. The vast majority of atmospheric CO2 fixation is accomplished by photosynthetic organisms, which have unfortunately proven difficult to utilize as chassis for industrial production. Nonphotosynthetic CO2 fixing microorganisms and pathways have recently attracted scientific and commercial interest. This Perspective will review promising alternate CO2 fixation strategies and their potential to supply microbially produced fuels and commodity chemicals, such as higher alcohols. Acetogenic fermentation and microbial electrosynthesis are the primary focuses of this review.

Electrical-biological hybrid system for CO2 reduction.

Here we have developed an electrochemical-biological hybrid system to fix CO2. Natural biological CO2 fixation processes are relatively slow. To increase the speed of fixation we applied electrocatalysts to reduce CO2 to formate. We chose a user-friendly organism, Escherichia coli, as host. Overall, the newly constructed CO2 and formate fixation pathway converts two formate and one CO2 to one pyruvate via glycine and L-serine in E. coli. First, one formate and one CO2 are converted to one glycine. Second, L-serine is produced from one glycine and one formate. Lastly, L-serine is converted to pyruvate. E. coli's genetic tractability allowed us to balance various parameters of the pathway. The carbon flux of the pathway was sufficient to compensate L-serine auxotrophy in the strain. In total, we integrated both electrocatalysis and biological systems into a single pot to support E. coli growth with CO2 and electricity. Results show promise for using this hybrid system for chemical production from CO2 and electricity.

Photomixotrophic chemical production in cyanobacteria.

The current global dependence on fossil fuels for both energy and chemical production has spurred concerns regarding long-term resource security and environmental detriments resulting from increased CO2 levels. Through the installation of exogenous metabolic pathways, engineered cyanobacteria strains can directly fix CO2 into industrially relevant chemicals currently produced from petroleum. This review highlights some of the studies that have successfully implemented photomixotrophic conditions to increase cyanobacterial chemical production. Supplementation with fixed carbon sources provides additional carbon building blocks and energy to enhance production and occasionally aid in growth. Photomixotrophic production has increased titers up to 5-fold over traditional autotrophic conditions, demonstrating promising applications for future commercialization.