title: Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis.
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.
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Petersgasse 14, Graz, 8010, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Geier
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Petersgasse 14, Graz, 8010, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Brandner
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Petersgasse 14, Graz, 8010, Austria ; Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, Graz, 8010, Austria.
authors.forename: Gernot A
authors.initials: GA
authors.lastname: Strohmeier
authors.affiliation: Department of Chemistry, University of Graz, Heinrichstrasse 28, Graz, 8010, Austria.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Hall
authors.affiliation: Sandoz GmbH, Biochemiestrasse 10, 6250, Kundl, Austria ; Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, Graz, 8010, Austria.
authors.forename: Franz S
authors.initials: FS
authors.lastname: Hartner
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, Graz, 8010, Austria.
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
keywords: bioreduction; cofactor regeneration; dihydroxyacetone synthase; methanol utilization pathway; whole-cell biotransformation