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Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis.

ABSTRACT: Solventogenic clostridia are an important class of microorganisms that can produce various biofuels. One of the bottlenecks in engineering clostridia stems from the fact that central metabolic pathways remain poorly understood. Here, we utilized the power of (13) C-based isotopomer analysis to re-examine central metabolic pathways of Clostridium acetobutylicum ATCC 824. We demonstrate using [1,2-(13) C]glucose, MS analysis of intracellular metabolites, and enzymatic assays that C. acetobutylicum has a split TCA cycle where only Re-citrate synthase (CS) contributes to the production of ?-ketoglutarate via citrate. Furthermore, we show that there is no carbon exchange between ?-ketoglutarate and fumarate and that the oxidative pentose-phosphate pathway (oxPPP) is inactive. Dynamic gene expression analysis of the putative Re-CS gene (CAC0970), its operon, and all glycolysis, pentose-phosphate pathway, and TCA cycle genes identify genes and their degree of involvement in these core pathways that support the powerful primary metabolism of this industrial organism.

SUBMITTER: Crown SB

PROVIDER: S-EPMC6468983 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis.

Crown Scott B SB Indurthi Dinesh C DC Ahn Woo Suk WS Choi Jungik J Papoutsakis Eleftherios T ET Antoniewicz Maciek R MR

Biotechnology journal 20101104 3

Solventogenic clostridia are an important class of microorganisms that can produce various biofuels. One of the bottlenecks in engineering clostridia stems from the fact that central metabolic pathways remain poorly understood. Here, we utilized the power of (13) C-based isotopomer analysis to re-examine central metabolic pathways of Clostridium acetobutylicum ATCC 824. We demonstrate using [1,2-(13) C]glucose, MS analysis of intracellular metabolites, and enzymatic assays that C. acetobutylicum ...[more]

PMID: 21370473

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Project description:Bacterial fermentation of carbohydrates from sustainable lignocellulosic biomass into commodity chemicals by the anaerobic bacterium Clostridium acetobutylicum is a promising alternative source to fossil fuel-derived chemicals. Recently, it was demonstrated that xylose is not appreciably fermented in the presence of arabinose, revealing a hierarchy of pentose utilization in this organism (L. Aristilde, I. A. Lewis, J. O. Park, and J. D. Rabinowitz, Appl Environ Microbiol 81:1452-1462, 2015, https://doi.org/10.1128/AEM.03199-14). The goal of the current study is to characterize the transcriptional regulation that occurs and perhaps drives this pentose hierarchy. Carbohydrate consumption rates showed that arabinose, like glucose, actively represses xylose utilization in cultures fermenting xylose. Further, arabinose addition to xylose cultures led to increased acetate-to-butyrate ratios, which indicated a transition of pentose catabolism from the pentose phosphate pathway to the phosphoketolase pathway. Transcriptome sequencing (RNA-Seq) confirmed that arabinose addition to cells actively growing on xylose resulted in increased phosphoketolase (CA_C1343) mRNA levels, providing additional evidence that arabinose induces this metabolic switch. A significant overlap in differentially regulated genes after addition of arabinose or glucose suggested a common regulation mechanism. A putative open reading frame (ORF) encoding a potential catabolite repression phosphocarrier histidine protein (Crh) was identified that likely participates in the observed transcriptional regulation. These results substantiate the claim that arabinose is utilized preferentially over xylose in C. acetobutylicum and suggest that arabinose can activate carbon catabolite repression via Crh. Furthermore, they provide valuable insights into potential mechanisms for altering pentose utilization to modulate fermentation products for chemical production. IMPORTANCE Clostridium acetobutylicum can ferment a wide variety of carbohydrates to the commodity chemicals acetone, butanol, and ethanol. Recent advances in genetic engineering have expanded the chemical production repertoire of C. acetobutylicum using synthetic biology. Due to its natural properties and genetic engineering potential, this organism is a promising candidate for converting biomass-derived feedstocks containing carbohydrate mixtures to commodity chemicals via natural or engineered pathways. Understanding how this organism regulates its metabolism during growth on carbohydrate mixtures is imperative to enable control of synthetic gene circuits in order to optimize chemical production. The work presented here unveils a novel mechanism via transcriptional regulation by a predicted Crh that controls the hierarchy of carbohydrate utilization and is essential for guiding robust genetic engineering strategies for chemical production.

Dataset Information

Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis.

Publications

Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis.

Similar Datasets

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