An engineered microbe transforms carbon dioxide into a prospective liquid fuel in tandem with electrical power rather than light.

Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype–phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde.

Methanol is an important intermediate in the utilization of natural gas for synthesizing other feedstock chemicals. Typically, chemical approaches for building C-C bonds from methanol require high temperature and pressure. Biological conversion of methanol to longer carbon chain compounds is feasible; however, the natural biological pathways for methanol utilization involve carbon dioxide loss or ATP expenditure. Here we demonstrated a biocatalytic pathway, termed the methanol condensation cycle (MCC), by combining the nonoxidative glycolysis with the ribulose monophosphate pathway to convert methanol to higher-chain alcohols or other acetyl-CoA derivatives using enzymatic reactions in a carbonconserved and ATP-independent system. We investigated the robustness of MCC and identified operational regions. We confirmed that the pathway forms a catalytic cycle through 13 C-carbon labeling. With a cell-free system, we demonstrated the conversion of methanol to ethanol or n-butanol. The high carbon efficiency and low operating temperature are attractive for transforming natural gas-derived methanol to longer-chain liquid fuels and other chemical derivatives.ethanol is industrially produced from synthetic gas-derived olefins and alkanes (1-7). These reactions typically involve high temperatures and pressures that require large capital investment (8, 9). The condensation of methanol to higher-chain alcohols such as ethanol or n-butanol is thermodynamically favorable (ΔG°′ = −68 and −182 kJ/mol, respectively), but the direct condensation of methanol to higher-chain alcohols has been quite challenging. Using the Guerbet reaction, methanol can upgrade short alcohols (such as n-propanol) to longer alcohols; however, methanol cannot self-couple (10). Metal acetylides can convert methanol to isobutanol, although this process was demonstrated to be noncatalytic (11).Nature has evolved several distinct ways to assimilate methanol to form metabolites necessary for growth. In principle, metabolites resulting from these methylotrophic pathways can be used to form higher-chain alcohols, although inherent pathway limitations prevent complete carbon conservation (Fig. S1). In the ribulose monophosphate pathway (RuMP), three formaldehydes condense to pyruvate, which is decarboxylated to form acetyl-CoA and CO 2 , reducing the carbon efficiency to 67%. The serine pathway requires an external supply of ATP to drive otherwise unfavorable reactions. Similarly, oxidation of methanol to CO 2 followed by CO 2 fixation using the Calvin-Benson-Bassham (CBB) cycle also requires additional ATP. To generate the required ATP input, extra carbon must be spent to drive oxidative phosphorylation. To our knowledge, natural methylotrophs are not capable of using the reductive acetyl-CoA pathway, which can produce acetyl-CoA without carbon loss or ATP requirement through carbon reassimilation after complete oxidation of methanol. This route is extremely oxygen sensitive and difficult to engineer due to the complex cofactors involved, and achieving carbo...