Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation.
Industrial biocatalytic reduction processes require the efficient regeneration of reduced cofactors for the asymmetric reduction of prochiral compounds to chiral intermediates which are needed for the production of fine chemicals and drugs. Here, we present a new engineering strategy for improved NADH regeneration based on the Pichia pastoris methanol oxidation pathway. Studying the kinetic properties of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) and using the derived kinetic data for subsequent kinetic simulations of NADH formation rates led to the identification of FLD activity to constitute the main bottleneck for efficient NADH recycling via the methanol dissimilation pathway. The simulation results were confirmed constructing a recombinant P. pastoris strain overexpressing P. pastoris FLD and the highly active NADH-dependent butanediol dehydrogenase from S. cerevisiae. Employing the engineered strain, significantly improved butanediol production rates were achieved in whole-cell biotransformations.
Schroer
Kirsten
K
Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria.
Peter Luef
Klaus
K
Stefan Hartner
Franz
F
Glieder
Anton
A
Pscheidt
Beate
B
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D007700:Kinetics; D000432:Methanol / Q000378:metabolism*; D009243:NAD / Q000096:biosynthesis* / Q000235:genetics; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D005656:Fungal Proteins; D009243:NAD; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005560:Formate Dehydrogenases; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; D000432:Methanol