A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

Abstract

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.

Biosynthesis of (S)-4-chloro-3-hydroxybutanoate ethyl using Escherichia coli co-expressing a novel NADH-dependent carbonyl reductase and a glucose dehydrogenase

“…The amplified DNA fragment was double-digested with NdeI and BamHI and inserted into the expression vector pET-22b. Expression in E. coli transformants was as described previously (Ye et al, 2009a).…”

“…Several enzymes in the SDR family that produce (S)-CHBE with 99% e.e. have been found, including PsCR I from P. stipitis (Ye et al, 2009a), S1 from Candida magnoliae (Yasohara et al, 2000), and KaCR from Kluyveromyces aestuarii (Yamamoto et al, 2004). Amino acid sequence alignment showed only 54% identity between PsCR II and PsCR I, 53% identity between PsCR II and S1, and 46% between PsCR II and KaCR.…”

“…The reduction of COBE is a more economical way of producing (S)-CHBE because COBE is inexpensive and easily synthesized. P. stipitis, however, produces (S)-CHBE with low enantiomeric excess (Ye et al, 2009a). This non-satisfactory value was obtained mainly because wild strains contain several enzymes with variable or opposing stereospecificities that reduce COBE to products of different chirality (Stewart 2001).…”

“…E. coli Rosetta (DE3) containing the polycistronic plasmid pET-BP II exhibited an activity of 10 U/mg with COBE as a substrate, and 11 U/mg with glucose as a substrate. Since COBE and CHBE were both inhibitory for enzymes and cells in an aqueous mono-phase system (Ye et al, 2009a), biosynthesis of (S)-CHBE using recombinant E. coli harboring pET-BP II was examined using a water/butyl acetate diphasic system for efficient partitioning of COBE and CHBE (Ye et al, 2010a). In this reaction system, NADH was regenerated by coupling glucose oxidation with BmGDH.…”

“…For example, we previously described a novel carbonyl reductase (PsCR I) from Pichia stipitis that produces (S)-CHBE at greater than 99% enantiomeric excess (e.e.) (Ye et al, 2009a). Although several enzymes for the production of (S)-CHBE have been found that required NADPH for catalysis, few studies have addressed reductases that need the cofactor NADH as an electron donor.…”