PUMA publications for /user/bastian/magnetichttps://puma.ub.uni-stuttgart.de/user/bastian/magneticPUMA RSS feed for /user/bastian/magnetic2024-03-29T08:12:50+01:00Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicumhttps://puma.ub.uni-stuttgart.de/bibtex/22e74e76dec66d069069ac7fbe5bedf94/bastianbastian2018-02-09T13:18:17+01:00Acid, Anaerobiosis, Carbon Corynebacterium Dioxide Glucose, Isotope Isotopes, Labeling, Magnetic Resonance Spectroscopy, Succinic glutamicum, myown <span data-person-type="author" class="authorEditorList "><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Dušica Radoš" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/0"><span itemprop="name">D. Radoš</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="David L. Turner" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/1"><span itemprop="name">D. Turner</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Luís L. Fonseca" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/2"><span itemprop="name">L. Fonseca</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Ana Lúcia Carvalho" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/3"><span itemprop="name">A. Carvalho</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Bastian Blombach" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/4"><span itemprop="name">B. Blombach</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Bernhard J. Eikmanns" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/5"><span itemprop="name">B. Eikmanns</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Ana Rute Neves" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/6"><span itemprop="name">A. Neves</span></a></span>, </span> and <span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Helena Santos" itemprop="url" href="/person/138e7d0611f298c2eedc8bd9086a28955/author/7"><span itemprop="name">H. Santos</span></a></span></span>. </span><span class="additional-entrytype-information"><span itemtype="http://schema.org/PublicationIssue" itemscope="itemscope" itemprop="isPartOf"><em><span itemprop="journal">Appl. Environ. Microbiol.</span>, </em> <em><span itemtype="http://schema.org/PublicationVolume" itemscope="itemscope" itemprop="isPartOf"><span itemprop="volumeNumber">80 </span></span>(<span itemprop="issueNumber">10</span>):
<span itemprop="pagination">3015--3024</span></em> </span>(<em><span>May 2014<meta content="May 2014" itemprop="datePublished"/></span></em>)</span>Fri Feb 09 13:18:17 CET 2018Appl. Environ. Microbiol.may103015--3024Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of {CO}2 on anaerobic succinate production by {Corynebacterium} glutamicum802014Acid, Anaerobiosis, Carbon Corynebacterium Dioxide Glucose, Isotope Isotopes, Labeling, Magnetic Resonance Spectroscopy, Succinic glutamicum, myown Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using (13)C-labeled glucose and (13)C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97\% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low ({\textasciitilde}5\%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H(+):organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.Stereospecificity of Corynebacterium glutamicum 2,3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2,3-butanediolhttps://puma.ub.uni-stuttgart.de/bibtex/2d2fbd13b8243050d76f6784383286149/bastianbastian2018-02-09T13:18:17+01:002,3-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 <span data-person-type="author" class="authorEditorList "><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Dušica Radoš" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/0"><span itemprop="name">D. Radoš</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="David L. Turner" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/1"><span itemprop="name">D. Turner</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Teresa Catarino" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/2"><span itemprop="name">T. Catarino</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Eugenia Hoffart" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/3"><span itemprop="name">E. Hoffart</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Ana Rute Neves" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/4"><span itemprop="name">A. Neves</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Bernhard J. Eikmanns" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/5"><span itemprop="name">B. Eikmanns</span></a></span>, </span><span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Bastian Blombach" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/6"><span itemprop="name">B. Blombach</span></a></span>, </span> and <span><span itemtype="http://schema.org/Person" itemscope="itemscope" itemprop="author"><a title="Helena Santos" itemprop="url" href="/person/1dd4270d58d31233ae25eebd4b8cf903e/author/7"><span itemprop="name">H. Santos</span></a></span></span>. </span><span class="additional-entrytype-information"><span itemtype="http://schema.org/PublicationIssue" itemscope="itemscope" itemprop="isPartOf"><em><span itemprop="journal">Appl. Microbiol. Biotechnol.</span>, </em> <em><span itemtype="http://schema.org/PublicationVolume" itemscope="itemscope" itemprop="isPartOf"><span itemprop="volumeNumber">100 </span></span>(<span itemprop="issueNumber">24</span>):
<span itemprop="pagination">10573--10583</span></em> </span>(<em><span>December 2016<meta content="December 2016" itemprop="datePublished"/></span></em>)</span>Fri Feb 09 13:18:17 CET 2018Appl. Microbiol. Biotechnol.dec2410573--10583Stereospecificity of {Corynebacterium} glutamicum 2,3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2,3-butanediol10020162,3-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 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.