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Identifying NAD-Dependent Methanol Dehydrogenases for Synthetic Methylotrophy

Authors: 
Bennett, R. K., University of Delaware

Methylotrophy describes the ability of microorganisms to utilize reduced one carbon compounds, such as methane and methanol, as growth and energy sources.  The recent discovery of large natural gas reserves has prompted considerable interest in utilizing these compounds as substrates or co-substrates with sugars in industrial anaerobic fermentation of fuels and chemicals, as higher biomass and product yields are expected from these more reduced substrates.  Native methylotrophs represent poor industrial microorganisms since many are strict aerobes, produce few metabolites and lack genetic engineering tools.  Therefore, the development of synthetic methylotrophy is of considerable interest.  Herein, we discuss the selection of suitable methanol dehydrogenase (MDH) candidates for engineering nonnative methanol metabolism in Escherichia coli, a well-developed host for anaerobic fermentation. 

Methanol oxidoreductase enzymes are classified by the cofactor associated with methanol oxidation.  Generally, methylotrophic yeast utilize a methanol oxidase, gram-negative methylotrophic bacteria utilize a pyrroloquinoline quinone-dependent MDH and gram-positive methylotrophic bacteria utilize a NAD-dependent MDH.  NAD-dependent MDH enzymes offer the best selection towards synthetic methylotrophy since methanol oxidation supplies reducing equivalents in the form of NADH and PQQ biosynthesis is strictly aerobic and nonnative to E. coli.  Of these NAD-dependent MDH enzymes, those from thermophilic Bacillus methanolicus and B. stearothermophilus have been well characterized in vitro.  Of more importance, however, is heterologous in vivo activity of these enzymes in E. coli, which we demonstrate.  We further discuss how to overcome the challenges involved in using these MDH enzymes, including unfavorable methanol oxidation and selectivity.  Toward this, we identified a separate family of NAD-dependent alcohol dehydrogenase enzymes that exhibit high methanol selectivity. 

We also report a high-throughput screening technique that was developed to identify improved MDH enzymes derived from protein engineering.  This technique takes advantage of the native E. coli formaldehyde-responsive transcription factor/promoter system (frmR-Pfrm), derived from the formaldehyde detoxification frmRAB operon.  We constructed a product-responsive reporter strain to isolate improved MDH variants via fluorescence-activated cell sorting (FACS).  This reporter strain enables in vivo quantification of MDH activity via fluorescence detection upon methanol addition.  We demonstrate that this technique successfully isolates MDH variants based on in vivo methanol oxidation activity.

SUPPORTED by the US DOE ARPA-E agency through contract no. DE-AR0000432.