Efficient anaerobic whole cell stereoselective bioreduction with recombinant Saccharomyces cerevisiae.
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.
Department of Applied Microbiology, Lund University, P.O. Box 124, 221 00 Lund, Sweden.
Michael
M
Katz
Torbjörn
T
Frejd
Bärbel
B
Hahn-Hägerdal
Marie F
MF
Gorwa-Grauslund
D000693:Anaerobiosis / Q000502:physiology; D001711:Biotransformation; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D002455:Cell Division; D004789:Enzyme Activation; D000431:Ethanol / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D013237:Stereoisomerism
C487950:6-hydroxybicyclo(2.2.2)octane-2-one; D001643:Bridged Bicyclo Compounds; D011994:Recombinant Proteins; C487951:bicyclo(2.2.2)octane-2,6-dione; D000431:Ethanol; D010088:Oxidoreductases; D005947:Glucose