Efficient anaerobic whole cell stereoselective bioreduction with recombinant saccharomyces cerevisiae

Abstract

In this study we investigate the NADPH-dependent stereoselective reduction of the bicyclic diketone bicyclo[2.2.2]octane-2,6-dione (BCO2,6D) to the chiral ketoalcohol (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one (BCO2one6ol). Our aim was to develop a whole cell batch process for reduction of carbonyl substrates with (i) a high cosubstrate yield (formed product/consumed cosubstrate) and (ii) a high conversion rate under anaerobic conditions with Saccharomyces cerevisiae as biocatalyst and glucose as cosubstrate. Five open reading frames (ORFs), YMR226c, YDR368w, YOR120w, YGL157w, and YGL039w, encoding reductases involved in the conversion of BCO2,6D were identified using cell-free extract from strains belonging to the ExClone collection (yeast ORF expression clones; ResGen, Invitrogen Corp., UK). We report the one-step purification and characterization of three major BCO2,6D reductases, YMR226cp, YDR368wp (YPR1p), and YOR120wp (GCY1p). The reductases were overexpressed under a strong constitutive promoter and the impact on cosubstrate yield, conversion time, glucose consumption rate, and reduction rate was investigated when reductases were overexpressed either alone or in combination with low phosphoglucose isomerase activity (encoded by YBR196c). Combining overexpression of BCO2,6D reductase with reduced glycolytic rate (low phosphoglucose isomerase activity) offers a fast whole cell stereoselective bioreduction system useful for facilitated anaerobic batch conversions.

A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion

“…The reaction was started by adding 20 μl of the protein sample to the mix at 25°C for 3 min. Whole cell bioreduction was carried in deep 96-well plates using procedures described previously (Katz et al, 2003a;Hwang et al, 2009). …”

Asymmetric synthesis of (R)-1,3-butanediol from 4-hydroxy-2-butanone by a newly isolated strain Candida krusei ZJB-09162

“…Nevertheless, the change in temperature did not remarkably affect ee. The stereoselective bioreduction system in whole cells is composed of two parallel reactions, the asymmetric reduction of carbonyl compounds catalyzed by reductase under the concomitant oxidation of reduced cofactors and the regeneration of the reduced cofactor through the dissimilatory metabolism of a cosubstrate (Katz et al 2003). Therefore, cosubstrate plays a crucial role in cofactor recycling and is essential to the continuous and smooth proceeding of the biocatalytic reduction (Yang et al 2008).…”

Enhancement of substrate concentration in microbial stereoinversion through one-pot oxidation and reduction by aqueous two-phase system

“…Microbial stereoinversion is a new and high-efficiency one-pot process for the production of chiral alcohols with theoretical yield of 100% [1][2][3], in which one enantiomer was selectively inter-converted into its mirror-image counterpart by a microorganism [4]. Although the number of stereoinversion processes has been increasing rapidly in the recent years, one severe drawback of low substrate concentration hindered its industrial scale application.…”