Stereospecificity of Corynebacterium glutamicum 2,3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2,3-butanediol.
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 butACgwas produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by1H-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. Vmaxvalues for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 μmol min-1mg protein-1, and Kmvalues 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-1mg protein-1), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157, Oeiras, Portugal.
Dušica
D
Radoš
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157, Oeiras, Portugal.
David L
DL
Turner
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157, Oeiras, Portugal.|Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
Teresa
T
Catarino
Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Eugenia
E
Hoffart
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157, Oeiras, Portugal.|CED-Discovery, Chr Hansen A/S, 10-12 Bøge Alle, 2970, Hørsholm, Denmark.
Ana Rute
AR
Neves
Institute of Microbiology and Biotechnology, University of Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Germany.
Bernhard J
BJ
Eikmanns
Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Bastian
B
Blombach
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157, Oeiras, Portugal. santos@itqb.unl.pt.|Lisbon Academy of Sciences, R. Academia das Ciências 19, 1249, Lisbon, Portugal. santos@itqb.unl.pt.
Helena
http://orcid.org/0000-0002-8050-9485
H
Santos
D000093:Acetoin / Q000378:metabolism; D000095:Acetolactate Synthase / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002072:Butylene Glycols / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D060847:Metabolic Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D002072:Butylene Glycols; D011994:Recombinant Proteins; C026978:2,3-butylene glycol; D000093:Acetoin; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase; D000095:Acetolactate Synthase; D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase
2,3-Butanediol; Butanediol dehydrogenase; Corynebacterium glutamicum; Stereospecificity