Abstract
Due to volatile sugar prices, the food vs fuel debate, and recent increases in the supply of natural gas, methanol has emerged as a promising feedstock for the bio-based economy. However, attempts to engineer Escherichia coli to metabolize methanol have achieved limited success. Here, we provide a rigorous systematic analysis of several potential pathway bottlenecks. We show that regeneration of ribulose 5-phosphate in E. coli is insufficient to sustain methanol assimilation, and overcome this by activating the sedoheptulose bisphosphatase variant of the ribulose monophosphate pathway. By leveraging the kinetic isotope effect associated with deuterated methanol as a chemical probe, we further demonstrate that under these conditions overall pathway flux is kinetically limited by methanol dehydrogenase. Finally, we identify NADH as a potent kinetic inhibitor of this enzyme. These results provide direction for future engineering strategies to improve methanol utilization, and underscore the value of chemical biology methodologies in metabolic engineering.
Highlights
Due to volatile sugar prices, the food vs fuel debate, and recent increases in the supply of natural gas, methanol has emerged as a promising feedstock for the bio-based economy
To examine the ability of E. coli cells engineered for methanol metabolism to metabolize formaldehyde, we used the assay procedure developed by Müller et al.[12] to measure [CH2O] in carbon-starved cells treated with 250 mM methanol
Cells expressing hexulose phosphate synthase (Hps) from B. methanolicus and phosphohexulose isomerase (Phi) from Methylococcus capsulatus produced only slightly less formaldehyde (Fig. 2b), despite the superior kinetics of these enzymes compared to methanol dehydrogenase (Mdh) (Supplementary Table 2)
Summary
Due to volatile sugar prices, the food vs fuel debate, and recent increases in the supply of natural gas, methanol has emerged as a promising feedstock for the bio-based economy. Low activity of the enzyme limits total methanol flux, and the high thermodynamic barrier requires both a high intracellular NAD:NADH ratio and rapid consumption of formaldehyde through the ribulose monophosphate (RuMP) cycle, to keep reaction far from equilibrium and driven in the forward direction. This has led to efforts to find and engineer more active variants of Mdh[17], and a strategy involving the co-localization of Mdh and Hps, the first enzyme of the RuMP pathway, to prevent the build-up of formaldehyde[18]. Such an analysis is a critical component of developing a rational strategy to improve methanol assimilation, because it justifies the considerable time and resource expenditure associated with developing a solution to the proposed bottleneck
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