The D494G Point Mutation in the Bifunctional Alcohol and Aldehyde Dehydrogenase (adhE) of Clostridium Thermocellum Leads to Improved Ethanol Production

Previously we have engineered Clostridium thermocellum for increased ethanol production by eliminating lactate and acetate production.  The resulting strain did not grow well and carbon flux was diverted to amino acid production instead of ethanol.  The strain was evolved by serial transfer and ethanol production increased.  To understand the reason for this change, the strain was resequenced and compared to the unevolved strain.  A SNP in the bifunctional alcohol/aldehyde dehydrogenase adhE was found that creates a D494G substitution in the amino acid sequence.  The mutation appears to alter the cofactor specificity for the alcohol dehydrogenase reaction and may explain the reason for the additional ethanol production.