Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

Abstract

BackgroundEthyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile chiral precursor for many pharmaceuticals. Although several biosynthesis strategies have been documented to convert ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE, the catalytic efficiency and stereoselectivity are still too low to be scaled up for industrial applications. Due to the increasing demand of (R)-CHBE, it is essential to explore more robust biocatalyst capable of preparing (R)-CHBE efficiently.ResultsA stereoselective carbonyl reductase toolbox was constructed and employed into the asymmetric reduction of COBE to (R)-CHBE. A robust enzyme designed as BgADH3 from Burkholderia gladioli CCTCC M 2012379 exhibited excellent activity and enantioselectivity, and was further characterized and investigated in the asymmetric synthesis of (R)-CHBE. An economical and satisfactory enzyme-coupled cofactor recycling system was created using recombinant Escherichia coli cells co-expressing BgADH3 and glucose dehydrogenase genes to regenerate NADPH in situ. In an aqueous/octanol biphasic system, as much as 1200 mmol COBE was completely converted by using substrate fed-batch strategy to afford (R)-CHBE with 99.9 % ee at a space-time yield per gram of biomass of 4.47 mmol∙L−1∙h−1∙g DCW−1.ConclusionsThese data demonstrate the promising of BgADH3 in practical synthesis of (R)-CHBE as a valuable chiral synthon. This study allows for the further application of BgADH3 in the biosynthesis of chiral alcohols, and establishes a preparative scale process for producing (R)-CHBE with excellent enantiopurity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0301-x) contains supplementary material, which is available to authorized users.

Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

“…For instance, methyl ( R )‐3‐hydroxybutanoate is a precursor of antibiotic candidate (−)‐albocycline; [17] whereas methyl ( S )‐3‐hydroxybutanoate is an important synthon for the preparation of chaetoviridin A [18] . Ethyl ( R )‐4‐chloro‐3‐hydroxybutyrate serves as a versatile precursor for compounds with pharmacological activity, such as l ‐carnitine, ( R )‐4‐amino‐3‐hydroxybutyric acid, and ( R )‐4‐hydroxy‐pyrrolidone; [19] whereas its ( S )‐stereoisomer is a key intermediate to synthesize the cholesterol‐lowering blockbuster drug atorvastatin [20] . The demand for each stereoisomer of chiral β‐hydroxy esters inevitably leads to the demand for the specific enzymes.…”

Characterization of a Carbonyl Reductase from Rhodococcus erythropolis WZ010 and Its Variant Y54F for Asymmetric Synthesis of (S)-N-Boc-3-Hydroxypiperidine

“…Furthermore, attempts with biphasic catalysis in the presence of water-immiscible organic solvents have demonstrated an intriguing potential for overcoming the inhibition from substrate/co-substrate, increasing the solubility of substrates, easy product removal, decreasing the spontaneous hydrolysis of substrate/product, and avoiding unfavorable equilibria [16,17,18,19]. In an aqueous/octanol biphasic system, the biosynthesis process of ethyl ( R )-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli was established, in which 1.2 M ethyl 4-chloro-3-oxobutanoate was completely converted to afford ethyl ( R )-4-chloro-3-hydroxybutyrate through the substrate fed-batch strategy [20]. In addition, the integration of protein engineering and medium engineering can further improve the effectiveness of asymmetric reduction at a high substrate load [20,21,22].…”

“…In an aqueous/octanol biphasic system, the biosynthesis process of ethyl ( R )-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli was established, in which 1.2 M ethyl 4-chloro-3-oxobutanoate was completely converted to afford ethyl ( R )-4-chloro-3-hydroxybutyrate through the substrate fed-batch strategy [20]. In addition, the integration of protein engineering and medium engineering can further improve the effectiveness of asymmetric reduction at a high substrate load [20,21,22].…”

Converting the 3‐quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4‐chloroacetoacetate reductase by site‐directed mutagenesis

“…1,2 (R)-CHBE is the precursor for the synthesis of l-carnitine and R-γ-amino-βhydroxybutyric acid. [3][4][5] And (S)-CHBE is a key chiral intermediate for the synthesis of statins which are one of the most effective drugs for cardiovascular diseases. For example, atorvastatin, as an inhibitor of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase, can decrease low-density lipoprotein cholesterol and associate the decline in coronary heart disease mortality 6 (S)-CHBE can also be used to synthesize Slagenins B and C and 1,4dihydropyridine type β-blockers.…”