Methanol is an abundant resource mainly produced from natural gas. Due to the large reserve of natural gas and the growing capacity of methanol production, it has been economically attractive to serve as fermentation raw material. Bioconversions of methanol to amino acids and other chemicals have been demonstrated using methylotrophic bacteria. Despite previous successes, many hurdles remain to be overcome before industrial applications. One of the problems for methanol conversion is the limitation of carbon yield via natural pathways. In natural, methanol can be assimilated by ribulose monophosphate pathway (RuMP), serine pathway, or first being oxidized to CO2 followed by CO2 fixation using Calvin-Benson-Bassham (CBB) pathway. However, the carbon efficiency for production of acetyl-CoA, an important central metabolite as well as a precursor for production of a wide variety of products, is compromised by the decarboxylation step or the need of generating ATP in the pathways mentioned above. To address the problem, we designed a synthetic pathway called methanol condensation cycle (MCC). The MCC is capable of converting methanol to acetyl-CoA with complete carbon conservation and ATP-independence. With optimization of enzymes and reaction conditions, we demonstrated conversion of methanol to ethanol and 1-butanol with carbon yield higher than natural pathways. On the other hand, methanol oxidation by methanol dehydrogenase (Mdh) can be a obstacle to efficient methanol utilization. NAD-dependent Mdhs have several advantages in metabolic engineering compare to their counterparts using other co-factors. However, 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). Here, we characterized other Mdhs with better properties for the purpose of synthetic methylotroph. The best Mdh was further engineered to achieve higher activity and specificity toward methanol.

Carbon-efficient conversion of methanol to chemical commodities via synthetic methanol condensation cycle (MCC)

251st National Meeting (2016)

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