Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation

Kirsten Schroer, Klaus Peter Luef, Franz Stefan Hartner, Anton Glieder, Beate Pscheidt

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)8-17
Number of pages10
JournalMetabolic engineering
Volume12
Issue number1
DOIs
Publication statusPublished - 2010

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glutathione-independent formaldehyde dehydrogenase
Pichia
Biotransformation
NAD
Methanol
Regeneration
Oxidation
butanediol dehydrogenase
alcohol oxidase
Formaldehyde
Kinetics
Formate Dehydrogenases
Butylene Glycols
Recycling
Saccharomyces cerevisiae
Oxidoreductases
Alcohols
Pharmaceutical Preparations

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Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation. / Schroer, Kirsten; Luef, Klaus Peter; Hartner, Franz Stefan; Glieder, Anton; Pscheidt, Beate.

In: Metabolic engineering, Vol. 12, No. 1, 2010, p. 8-17.

Research output: Contribution to journalArticleResearchpeer-review

Schroer, Kirsten ; Luef, Klaus Peter ; Hartner, Franz Stefan ; Glieder, Anton ; Pscheidt, Beate. / Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation. In: Metabolic engineering. 2010 ; Vol. 12, No. 1. pp. 8-17.
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AB - 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.

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