The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876–1878, 2001). To resolve this discrepancy, the enzyme encoded by butACg was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by 1H-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (∼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. Vmax values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 μmol min−1 mg protein−1, and Km values were 0.23 ± 0.02 and 1.49 ± 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butACg). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butACg (from 0.30 ± 0.03 to 0.004 ± 0.001 μmol min−1 mg protein−1), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.
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Technical assistance by Sara Rebelo is gratefully acknowledged. The generous advice provided by Dr. Nuno Borges was very important to the development of this work.
Compliance with ethical standards
This work was supported by Fundação para a Ciência e a Tecnologia, Portugal, project MOSTMICRO UID/CBQ/04612/2013. The NMR spectrometers are part of the National NMR Facility, supported by Fundação para a Ciência e a Tecnologia (RECI/BBB-BQB/0230/2012).
Conflict of interest
The authors declare that they have no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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