A Novel Methanol Dehydrogenase Enzyme and its Engineered Variants from Cupriavidus

Tech ID: 29433 / UC Case 2016-104-0


UCLA researchers in the Department of Chemical and Biomolecular Engineering have engineered a Cupriavidus necator N-1 NAD-dependent methanol dehydrogenase (Mdh) variant with much improved catalytic efficiency and specificity toward methanol, compared with the existing NAD-dependent Mdhs with or without endogenous activator protein (ACT) activation.


Methanol is becoming an attractive substrate for bioconversion to chemical commodities due to the abundance of methane. Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Synthetic methylotrophy can be achieved by overexpressing heterologous enzymes for methanol oxidation and engineering a formaldehyde assimilation pathway to produce central metabolites for growth.

Enzymes that are capable of methanol oxidation are categorized into three groups based on their terminal electron acceptors: pyrroloquinoline quinone-dependent methanol dehydrogenases (Mdhs), methanol oxidases, and NAD-dependent Mdhs. Among the three types of methanol-oxidizing enzymes, NAD-dependent Mdhs are the favorable option for synthetic methylotrophy due to their applicability in both aerobic and anaerobic conditions. Furthermore, electrons derived from methanol oxidation are stored in NADH, which can be used to drive production of target metabolites without sacrificing additional carbons. Among a limited number of NAD-dependent Mdhs been reported, the Bacillus methanolicus Mdh exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). For the purpose of metabolic engineering, it would be useful to identify a Mdh with high activity without the need for ACT.


Researchers at UCLA have characterized andengineered a NAD-dependent alcohol dehydrogenase (Mdh) from Cupriavidus necator N-1. This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Unlike previously reported Mdhs, this Mdh2 enzyme does not require activation by known activators such as Bacillus methanolicus ACT, Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibits higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. An engineered variant of Mdh2 (CT4-1) has demonstrated improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.


Microbial production of chemicals involving NAD-dependent methanol oxidation:

  • Bioconversion of methanol to amino acids 
  • Synthesis of biofuel and related alcohol products


  • Enzymatic activity is independent of activator protein 
  • Greatly improved enzyme catalytic efficiency and substrate specificity in a wide range of temperatures

State Of Development

Enzyme activity has been tested in lab.

Patent Status

Patent Pending

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  • Liao, James C.

Other Information


methanol dehydrogenase, Mdh, enzyme, chemical production, alcohol, biofuel, methanol bioconversion

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