{ "types" : { "Bookmark" : { "pluralLabel" : "Bookmarks" }, "Publication" : { "pluralLabel" : "Publications" }, "GoldStandardPublication" : { "pluralLabel" : "GoldStandardPublications" }, "GoldStandardBookmark" : { "pluralLabel" : "GoldStandardBookmarks" }, "Tag" : { "pluralLabel" : "Tags" }, "User" : { "pluralLabel" : "Users" }, "Group" : { "pluralLabel" : "Groups" }, "Sphere" : { "pluralLabel" : "Spheres" } }, "properties" : { "count" : { "valueType" : "number" }, "date" : { "valueType" : "date" }, "changeDate" : { "valueType" : "date" }, "url" : { "valueType" : "url" }, "id" : { "valueType" : "url" }, "tags" : { "valueType" : "item" }, "user" : { "valueType" : "item" } }, "items" : [ { "type" : "Publication", "id" : "https://puma.ub.uni-stuttgart.de/bibtex/2d2fbd13b8243050d76f6784383286149/bastian", "tags" : [ "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" ], "intraHash" : "d2fbd13b8243050d76f6784383286149", "interHash" : "dd4270d58d31233ae25eebd4b8cf903e", "label" : "Stereospecificity of Corynebacterium glutamicum 2,3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2,3-butanediol", "user" : "bastian", "description" : "", "date" : "2018-02-09 13:18:17", "changeDate" : "2018-02-09 12:18:56", "count" : 1, "pub-type": "article", "journal": "Appl. Microbiol. Biotechnol.", "year": "2016", "url": "", "author": [ "Dušica Radoš","David L. Turner","Teresa Catarino","Eugenia Hoffart","Ana Rute Neves","Bernhard J. Eikmanns","Bastian Blombach","Helena Santos" ], "authors": [ {"first" : "Dušica", "last" : "Radoš"}, {"first" : "David L.", "last" : "Turner"}, {"first" : "Teresa", "last" : "Catarino"}, {"first" : "Eugenia", "last" : "Hoffart"}, {"first" : "Ana Rute", "last" : "Neves"}, {"first" : "Bernhard J.", "last" : "Eikmanns"}, {"first" : "Bastian", "last" : "Blombach"}, {"first" : "Helena", "last" : "Santos"} ], "volume": "100","number": "24","pages": "10573--10583","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.", "pmid" : "27687994", "issn" : "1432-0614", "language" : "eng", "doi" : "10.1007/s00253-016-7860-6", "bibtexKey": "rados_stereospecificity_2016" } , { "type" : "Publication", "id" : "https://puma.ub.uni-stuttgart.de/bibtex/29ce34124eaffe2a799aade38deec413d/bastian", "tags" : [ "Bacterial","Bioreactors,","Butylene","Complex,","Corynebacterium","Dehydrogenase","Dehydrogenase,","Engineering","Family,","Glucose,","Glycols,","L-Lactate","Lactococcus","Metabolic","Multigene","Oxygen,","Proteins,","Pyruvate","glutamicum,","lactis,","myown" ], "intraHash" : "9ce34124eaffe2a799aade38deec413d", "interHash" : "518ec5750d920964df1a659788edff11", "label" : "Engineering Corynebacterium glutamicum for the production of 2,3-butanediol", "user" : "bastian", "description" : "", "date" : "2018-02-09 13:18:17", "changeDate" : "2018-02-09 12:18:56", "count" : 1, "pub-type": "article", "journal": "Microb. Cell Fact.", "year": "2015", "url": "", "author": [ "Dušica Radoš","Ana Lúcia Carvalho","Stefan Wieschalka","Ana Rute Neves","Bastian Blombach","Bernhard J. Eikmanns","Helena Santos" ], "authors": [ {"first" : "Dušica", "last" : "Radoš"}, {"first" : "Ana Lúcia", "last" : "Carvalho"}, {"first" : "Stefan", "last" : "Wieschalka"}, {"first" : "Ana Rute", "last" : "Neves"}, {"first" : "Bastian", "last" : "Blombach"}, {"first" : "Bernhard J.", "last" : "Eikmanns"}, {"first" : "Helena", "last" : "Santos"} ], "volume": "14","pages": "171","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.", "pmid" : "26511723", "issn" : "1475-2859", "pmcid" : "PMC4625470", "language" : "eng", "doi" : "10.1186/s12934-015-0362-x", "bibtexKey": "rados_engineering_2015" } ] }