Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol

Abstract

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K(m) values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.

Efficient (3R)-Acetoin Production from meso-2,3-Butanediol Using a New Whole-Cell Biocatalyst with Co-Expression of meso-2,3-Butanediol Dehydrogenase, NADH Oxidase, and Vitreoscilla Hemoglobin

“…Thus, developing a suitable system for optically pure (3R)-AC production with high yield is desirable. Compared with chemical synthesis and microbial fermentation, biocatalytic technologies have exhibited potential in optically pure AC production due to high catalytic efficiency and stereoselectivity [12][13][14][15][16].…”

Peer Review #2 of "Ketoreductase TpdE from Rhodococcus jostii TMP1: characterization and application in the synthesis of chiral alcohols (v0.2)"

“…Moreover, the specificity constant (k cat /K M =4472 s -1 M -1 ) value for this substrate was 1.4-fold higher than that for diacetyl. The specificity constants determined for TpdE were of the same order of magnitude as those of other enzymes active towards vicinal diketones including aldo-keto reductase AKR3C1 from Saccharomyces cerevisiae, oxidoreductase GOX0313 from Gluconobacter oxydans and acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 (Calam et al, 2013;Schweiger et al, 2013;Wang et al, 2014). While the bioconversion of diketones to corresponding mono-or dihydroxy alcohols, catalyzed by different alcohol dehydrogenases from various sources, have already been described (Kreit & Elalami, 2002;Edegger et al, 2006;Calam et al, 2013;Schweiger et al, 2013;Park et al, 2014), the ability of SDRs to reduce α-keto esters has been poorly characterized to date.…”

Metabolic engineering of Escherichia coli for production of (2S,3S)-butane-2,3-diol from glucose

“…dissolvens SDM Diacetyl 26.8 0.67 Glucose 32.8 [ 24 ] E. coli JM109 coexpressing meso -BDH from K. pneumoniae and (2 S ,3 S )-2,3-BDH from Brevibacterium saccharolyticum C-1012 Diacetyl 2.2 0.93 Glucose 23.7 [ 25 ] E. coli JM109 expressing (2 S ,3 S )-2,3-BDH from Br. saccharolyticum Racemic acetoin 3.7 0.37 Glucose 85.7 [ 26 ] E. coli expressing acetoin reductase from Rhodococcus erythropolis WZ010 Diacetyl 5.3 1.03 NADH 21.4 [ 27 ] E. coli BL21 coexpressing (2 R ,3 R )-2,3-BDH from Bacillus subtilis 168 and NADH oxidase from Lactobacillus brevis CICC 6004 2,3-BD 2.4 0.12 13.3 [ 28 ] E. coli BL21 (DE3) coexpressing ALS and meso -BDH from E. cloacae subsp. dissolvens SDM Glucose 2.2 0.08 9.4 This work Cost analyses of substrates were performed as described in Additional file 1 …”