{"d2fbd13b8243050d76f6784383286149bastian":{"DOI":"10.1007/s00253-016-7860-6","ISBN":"","ISSN":"1432-0614","URL":"","abstract":"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 butA Cg 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. V max values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 μmol min-1 mg protein-1, and K m 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,butA Cg ). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butA Cg (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.","annote":"","author":[{"family":"Radoš","given":"Dušica"},{"family":"Turner","given":"David L."},{"family":"Catarino","given":"Teresa"},{"family":"Hoffart","given":"Eugenia"},{"family":"Neves","given":"Ana Rute"},{"family":"Eikmanns","given":"Bernhard J."},{"family":"Blombach","given":"Bastian"},{"family":"Santos","given":"Helena"}],"citation-label":"rados_stereospecificity_2016","collection-editor":[],"collection-title":"","container-author":[],"container-title":"Appl. Microbiol. Biotechnol.","documents":[],"edition":"","editor":[],"event-date":{"date-parts":[["2016","dec"]],"literal":"2016"},"event-place":"","id":"d2fbd13b8243050d76f6784383286149bastian","interhash":"dd4270d58d31233ae25eebd4b8cf903e","intrahash":"d2fbd13b8243050d76f6784383286149","issue":"24","issued":{"date-parts":[["2016","dec"]],"literal":"2016"},"keyword":"2,3-Butanediol, Acetoin, Acetolactate Alcohol Butanediol Butylene Carboxy-Lyases, Corynebacterium Engineering Escherichia Glycols, Lactococcus Magnetic Metabolic Oxidoreductases, Proteins, Recombinant Resonance Specificity, Spectroscopy, Stereospecificity, Substrate Synthase, coli, dehydrogenase, glutamicum, lactis, myown","misc":{"pmid":"27687994","issn":"1432-0614","language":"eng","doi":"10.1007/s00253-016-7860-6"},"note":"","number":"24","number-of-pages":"10","page":"10573--10583","page-first":"10573","publisher":"","publisher-place":"","status":"","title":"Stereospecificity of Corynebacterium glutamicum 2,3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2,3-butanediol","type":"article-journal","username":"bastian","version":"","volume":"100"},"9ce34124eaffe2a799aade38deec413dbastian":{"DOI":"10.1186/s12934-015-0362-x","ISBN":"","ISSN":"1475-2859","URL":"","abstract":"BACKGROUND: 2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions.\nRESULTS: A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1).\nCONCLUSIONS: We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.","annote":"","author":[{"family":"Radoš","given":"Dušica"},{"family":"Carvalho","given":"Ana Lúcia"},{"family":"Wieschalka","given":"Stefan"},{"family":"Neves","given":"Ana Rute"},{"family":"Blombach","given":"Bastian"},{"family":"Eikmanns","given":"Bernhard J."},{"family":"Santos","given":"Helena"}],"citation-label":"rados_engineering_2015","collection-editor":[],"collection-title":"","container-author":[],"container-title":"Microb. Cell Fact.","documents":[],"edition":"","editor":[],"event-date":{"date-parts":[["2015","oct"]],"literal":"2015"},"event-place":"","id":"9ce34124eaffe2a799aade38deec413dbastian","interhash":"518ec5750d920964df1a659788edff11","intrahash":"9ce34124eaffe2a799aade38deec413d","issue":"","issued":{"date-parts":[["2015","oct"]],"literal":"2015"},"keyword":"Bacterial Bioreactors, Butylene Complex, Corynebacterium Dehydrogenase Dehydrogenase, Engineering Family, Glucose, Glycols, L-Lactate Lactococcus Metabolic Multigene Oxygen, Proteins, Pyruvate glutamicum, lactis, myown","misc":{"pmid":"26511723","issn":"1475-2859","pmcid":"PMC4625470","language":"eng","doi":"10.1186/s12934-015-0362-x"},"note":"","number":"","page":"171","page-first":"171","publisher":"","publisher-place":"","status":"","title":"Engineering Corynebacterium glutamicum for the production of 2,3-butanediol","type":"article-journal","username":"bastian","version":"","volume":"14"},"a0a5fcfba6017eb6a61292baef557af7bastian":{"DOI":"10.1128/AEM.02972-10","ISBN":"","ISSN":"1098-5336","URL":"","abstract":"We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y(P/S)) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y(P/S) to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y(P/S), indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH + H+ to NADPH + H+. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y(P/S) of about 0.48 mol per mol of glucose in the production phase.","annote":"","author":[{"family":"Blombach","given":"Bastian"},{"family":"Riester","given":"Tanja"},{"family":"Wieschalka","given":"Stefan"},{"family":"Ziert","given":"Christian"},{"family":"Youn","given":"Jung-Won"},{"family":"Wendisch","given":"Volker F."},{"family":"Eikmanns","given":"Bernhard J."}],"citation-label":"blombach_corynebacterium_2011","collection-editor":[],"collection-title":"","container-author":[],"container-title":"Appl. Environ. Microbiol.","documents":[],"edition":"","editor":[],"event-date":{"date-parts":[["2011","may"]],"literal":"2011"},"event-place":"","id":"a0a5fcfba6017eb6a61292baef557af7bastian","interhash":"0ff226fcd2ff0614810a1890f8f46f8e","intrahash":"a0a5fcfba6017eb6a61292baef557af7","issue":"10","issued":{"date-parts":[["2011","may"]],"literal":"2011"},"keyword":"Anaerobiosis, Bacterial Bacterial, Butanols, Chromosomes, Corynebacterium Escherichia Fungal Glucose, Lactococcus Metabolic Networks Pathways, Plasmids, Proteins Proteins, Recombinant Saccharomyces and cerevisiae, coli, glutamicum, lactis, myown","misc":{"pmid":"21441331","issn":"1098-5336","pmcid":"PMC3126470","language":"eng","doi":"10.1128/AEM.02972-10"},"note":"","number":"10","number-of-pages":"10","page":"3300--3310","page-first":"3300","publisher":"","publisher-place":"","status":"","title":"Corynebacterium glutamicum tailored for efficient isobutanol production","type":"article-journal","username":"bastian","version":"","volume":"77"}}