(228j) Identifying NAD-Dependent Methanol Dehydrogenases for Synthetic Methylotrophy
AIChE Annual Meeting
Monday, November 14, 2016 - 3:15pm to 5:45pm
Methanol oxidoreductase enzymes are classified by the cofactor associated with methanol oxidation. Generally, methylotrophic yeasts utilize methanol oxidases, Gram-negative methylotrophic bacteria utilize pyrroloquinoline quinone-dependent MDHs and Gram-positive methylotrophic bacteria utilize NAD-dependent MDHs. NAD-dependent MDH enzymes offer the best selection towards synthetic methylotrophy since methanol oxidation supplies reducing equivalents in the form of NAD(P)H to drive downstream metabolite-production pathways. 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 with these MDH enzymes, including unfavorable thermodynamics of methanol oxidation and broad substrate selectivity. We report a high-throughput screening technique that we developed to identify improved MDH enzymes through 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.