Dataset Information

Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

ABSTRACT: 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 (~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.

SUBMITTER: Rados D

PROVIDER: S-EPMC4018904 | biostudies-other | 2014 May

REPOSITORIES: biostudies-other

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Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

Radoš Dušica D Turner David L DL Fonseca Luís L LL Carvalho Ana Lúcia AL Blombach Bastian B Eikmanns Bernhard J BJ Neves Ana Rute AR Santos Helena H

Applied and environmental microbiology 20140307 10

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 a ...[more]

PMID: 24610842

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Project description:In the heart, detection of hyperpolarized [(13)C]bicarbonate and (13)CO(2) by magnetic resonance (MR) after administration of hyperpolarized [1-(13)C]pyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by [1-(13)C]pyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized [1-(13)C]pyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized [1-(13)C]pyruvate was rapidly converted to [1-(13)C]lactate, [1-(13)C]alanine, [1-(13)C]malate, [4-(13)C]malate, [1-(13)C]aspartate, [4-(13)C]aspartate, and [(13)C]bicarbonate. Livers from fasted animals had increased lactate:alanine, consistent with elevated NADH:NAD(+). The appearance of asymmetrically enriched malate and aspartate indicated high rates of anaplerotic pyruvate carboxylase activity and incomplete equilibration with fumarate. Hyperpolarized [(13)C]bicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of [4-(13)C]oxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of [4-(13)C]oxaloacetate to generate (13)CO(2) at isocitrate dehydrogenase, or decarboxylation of [1-(13)C]pyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from [4-(13)C]malate and [4-(13)C]aspartate was markedly blunted and signal from [(13)C]bicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized [1-(13)C]pyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of [(13)C]bicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.

Project description:Background:Succinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study. Results:In this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (?ldhA?pta?pqo?catPsod-ppcPsod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose-xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L-1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L-1 h-1 during the anaerobic stage. Conclusion:This work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.

Dataset Information

Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

Publications

Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

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