Cloning and Overexpression of theExiguobacteriumsp. F42 Gene Encoding a New Short Chain Dehydrogenase, Which Catalyzes the Stereoselective Reduction of Ethyl 3-Oxo-3-(2-thienyl)propanoate to Ethyl (S)-3-Hydroxy-3-(2-thienyl)propanoate

Abstract

Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.

Highly enantioselective bioreduction of N-methyl-3-oxo-3-(thiophen-2-yl) propanamide for the production of (S)-duloxetine

“…Accordingly, the preparative-scale transformation was terminated after 48 h and (S)-MHTPA was obtained at a yield of 90% with[99.5% ee (96% HPLC yield). The reaction afforded much higher substrate tolerance than previous reports (Breuer 2008;Soni and Banerjee 2005;Wada et al 2004). In addition, the stability of both substrate and product in water enhanced the reproductivity and facilitated product purification.…”

“…1a), was achieved with an ee of 98% by using isolated dehydrogenases from Exiguobacterium sp. (Wada et al 2004) and Thermoanaerobacter sp. (Breuer 2008), respectively, with 5-10 mM substrate in the presence of NADPH.…”

Bioreductive production of enantiopure (S)-duloxetine intermediates catalyzed with ketoreductase ChKRED15

“…8). The reaction afforded much higher substrate tolerance than previously reported bioreduction of duloxetine precursors [16,21,37,38]. For the same substrate, previous study used high concentration of whole cells of R. glutinis (42 g CDW/l), which yielded compromised stereoselectivity when substrate concentration exceeded 30 g/l [16].…”

“…It has been reported that intermediates (S)-2b and (S)-6b can be achieved with >99% ee using the whole cells of Candida tropicalis [20] and Rhodotorula glutinis [16], respectively, and (S)-3b and (S)-5b with 98% ee using isolated ketoreductases from Exiguobacterium sp. [21] and Thermoanaerobacter sp. [22], respectively.…”

“…For substrate 3a, a substrate concentration of 50 mM (9.9 g/l) has been reported using the Exiguobacterium sp. F42 ketoreductase expressed in Escherichia coli to achieve 80% product yield (>98% ee) in 6 h [21].…”