Characterization of a Stereospecific Diacetyl/acetoin Reductase from Klebsiella oxytoca M1 and Its Application for Production of (2S,3S)-2,3-butanediol
- Characterization of a Stereospecific Diacetyl/acetoin Reductase from Klebsiella oxytoca M1 and Its Application for Production of (2S,3S)-2,3-butanediol
- 조숙형; 우한민; 엄영순
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- Klebsiella oxytoca M1 produces a mixture of 2,3-butanediol (BDO) isomers (meso- and (2S,3S)-2,3-BDO). To characterize a stereo-specificity of diacetyl/acetoin reductase (DAR) in K. oxytoca M1, the dar gene was cloned, overexpressed in Escherichia coli and purified by Ni-affinity chromatography. The optimal conditions of DAR for diacetyl reduction were pH 6.0 and 40 ˚C, whereas those for (2S,3S)-2,3-BDO oxidation were pH 7.0 and 40 ˚C. There was no significant oxidation activity towards meso-2,3-BDO and (2R,3R)-2,3-BDO. These results suggest that DAR in K. oxytoca M1 has a strict stereospecificity in not only reduction of diacetyl to (2S,3S)-2,3-BDO via S-acetoin but also oxidation of (2S,3S)-2,3-BDO. To produce pure (2S,3S)-2,3-BDO, the budB gene encoding acetolactate synthase and dar gene from K. oxytoca M1 were introduced into E. coil BW25113 using medium copy plasmid. As a result, (2S,3S)-2,3-BDO was produced by E. coil BW25113 (pBbA5c-budB-dar) with trace amount of meso-2,3-BDO without supplemented diacetyl from glucose. Therefore, this biotechnological route may have potential for use in the industrial production of (2S,3S)-2,3-BDO.
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