Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production.

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 ±â€¯0.006 (h-1) 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. 13C tracing showed that 43% and 71% of ethanol and 1-butanol produced had labeled carbon derived from methanol, respectively.

Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

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.

Building carbon-carbon bonds using a biocatalytic methanol condensation cycle.

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 carbon-conserved 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.

Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq.

The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP) organism due to its ability to produce the biofuel products, hydrogen, and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP) produced 30 and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than two-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR) analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(P)H hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism.

Synthetic non-oxidative glycolysis enables complete carbon conservation.

Glycolysis, or its variations, is a fundamental metabolic pathway in life that functions in almost all organisms to decompose external or intracellular sugars. The pathway involves the partial oxidation and splitting of sugars to pyruvate, which in turn is decarboxylated to produce acetyl-coenzyme A (CoA) for various biosynthetic purposes. The decarboxylation of pyruvate loses a carbon equivalent, and limits the theoretical carbon yield to only two moles of two-carbon (C2) metabolites per mole of hexose. This native route is a major source of carbon loss in biorefining and microbial carbon metabolism. Here we design and construct a non-oxidative, cyclic pathway that allows the production of stoichiometric amounts of C2 metabolites from hexose, pentose and triose phosphates without carbon loss. We tested this pathway, termed non-oxidative glycolysis (NOG), in vitro and in vivo in Escherichia coli. NOG enables complete carbon conservation in sugar catabolism to acetyl-CoA, and can be used in conjunction with CO2 fixation and other one-carbon (C1) assimilation pathways to achieve a 100% carbon yield to desirable fuels and chemicals.