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...

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

Methanol is a potentially attractive substrate for bioproduction of chemicals because of the abundance of natural gas and biogas-derived methane. To move towards utilizing methanol as a sole carbon source, here we engineer an Escherichia coli strain to couple methanol utilization with growth on five-carbon (C5) sugars. By deleting essential genes in the pentose phosphate pathway for pentose utilization and expressing heterologous enzymes from the ribulose-monophosphate (RuMP) pathway, we constructed a strain that cannot grow on xylose or ribose minimal media unless methanol is utilized, creating a phenotype termed "synthetic methanol auxotrophy". Our best strains were able to utilize methanol for growth at a rate of 0.17 ± 0.006 (h) with methanol and xylose co-assimilation at a molar ratio of approximately 1:1. Genome sequencing and reversion of mutations indicated that mutations on genes encoding for adenylate cyclase (cyaA) and the formaldehyde detoxification operon (frmRAB) were necessary for the growth phenotype. The methanol auxotrophic strain was further engineered to produce ethanol or 1-butanol to final titers of 4.6 g/L and 2.0 g/L, respectively. C tracing showed that 43% and 71% of ethanol and 1-butanol produced had labeled carbon derived from methanol, respectively.

SignificanceWe constructed an Escherichia coli strain that does not use glycolysis for sugar catabolism. Instead, it uses the synthetic nonoxidative glycolysis cycle to directly synthesize stoichiometric amounts of the two-carbon building block (acetyl-CoA), which is then converted to three-carbon metabolites to support growth. The resulting strain grows aerobically in glucose minimal medium and can achieve near-complete carbon conservation in the production of acetyl-CoA–derived products during anaerobic fermentation. This strain improves the theoretical carbon yield from 66.7% to 100% in acetyl-CoA–derived product formation.

A bidirectional isolated full-bridge dc-dc converter with a conversion ratio around nine times, soft start-up, and soft-switching features for battery charging/discharging is proposed in this paper. The converter is equipped with an active flyback and two passive capacitor-diode snubbers, which can reduce voltage and current spikes and reduce voltage and current stresses, while it can achieve near zero-voltage-switching and zero-current-switching soft-switching features. In this paper, the operational principle of the proposed converter is first described, and its analysis and design are then presented. A 1.5-kW prototype with a low-side voltage of 48 V and a high-side voltage of 360 V has been implemented, from which measured results have verified the discussed features.