Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

Martina Geier, Christoph Brandner, Gernot A Strohmeier, Mélanie Hall, Franz Stefan Hartner, Anton Glieder

Research output: Contribution to journalArticle

Abstract

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

LanguageEnglish
Pages1741-8
Number of pages8
JournalBeilstein Journal of Organic Chemistry
Volume11
DOIs
StatusPublished - 2015

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Chassis
NAD
Catalysis
Methanol
formaldehyde transketolase
butanediol dehydrogenase
Acetoin
Oxidation
Enzymes
Yeast
Protein Isoforms
Carbon
Genes
Fluxes
Catalysts
Substrates
Costs

Keywords

  • Journal Article

Cite this

Engineering Pichia pastoris for improved NADH regeneration : A novel chassis strain for whole-cell catalysis. / Geier, Martina; Brandner, Christoph; Strohmeier, Gernot A; Hall, Mélanie; Hartner, Franz Stefan; Glieder, Anton.

In: Beilstein Journal of Organic Chemistry , Vol. 11, 2015, p. 1741-8.

Research output: Contribution to journalArticle

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AU - Glieder,Anton

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