초록
<P>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 <I>Pichia pastoris</I> for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (<I>DAS1</I> and <I>DAS2</I>), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting <I>Δdas1 Δdas2</I> 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 <I>P. pastoris Δdas1 Δdas2</I> 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 CO<SUB>2</SUB> 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.</P>