Dataset Information

Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

ABSTRACT: The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ?aceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ?aceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

SUBMITTER: Mustafi N

PROVIDER: S-EPMC3895001 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

Mustafi Nurije N Grünberger Alexander A Mahr Regina R Helfrich Stefan S Nöh Katharina K Blombach Bastian B Kohlheyer Dietrich D Frunzke Julia J

PloS one 20140117 1

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the p ...[more]

PMID: 24465669

Similar Datasets

Project description:The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2 The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3- pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3- levels, such as external addition of HCO3-, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3- pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3- pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains.IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3- pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3- pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3- levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3- pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.

Project description:Carotenoid biosynthesis in Corynebacteriumglutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (PcrtE) and of its own gene (PcrtR). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacteriumcallunae, Acidipropionibacteriumjensenii, Paenarthrobacternicotinovorans, Micrococcusluteus and Pseudarthrobacterchlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a PcrtE_gfpuv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the PcrtE_gfpuv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations.

Project description:For many bacterial proteins, specific localizations within the cell have been demonstrated, but enzymes involved in central metabolism are usually considered to be homogenously distributed within the cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex found in actinobacteria, the hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH). In non-actinobacterial cells, PDH and ODH form separate multienzyme complexes of megadalton size composed of three different subunits, E1, E2, and E3. The actinobacterial PDH-ODH complex is composed of four subunits, AceE (E1p), AceF (E2p), Lpd (E3), and OdhA (E1oE2o). Using fluorescence microscopy, we observed that in Corynebacterium glutamicum, all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots are present at mid-cell. These results further confirm the existence of the hybrid complex. The unphosporylated OdhI protein, which binds to OdhA and inhibits ODH activity, was co-localized with OdhA at the poles, whereas phosphorylated OdhI, which does not bind OdhA, was distributed in the entire cytoplasm. Isocitrate dehydrogenase and glutamate dehydrogenase, both metabolically linked to ODH, were evenly distributed in the cytoplasm. Based on the available structural data for individual PDH-ODH subunits, a novel supramolecular architecture of the hybrid complex differing from classical PDH and ODH complexes has to be postulated. Our results suggest that localization at the poles or at mid-cell is most likely caused by nucleoid exclusion and results in a spatially organized metabolism in actinobacteria, with consequences yet to be studied. IMPORTANCE Enzymes involved in the central metabolism of bacteria are usually considered to be distributed within the entire cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex of actinobacteria, the hybrid pyruvate dehydrogenase/2-oxoglutarate dehydrogenase (PDH-ODH) complex composed of four different subunits. Using fusions with mVenus or mCherry and fluorescence microscopy, we show that all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots were observed at mid-cell. These results clearly support the presence of the hybrid PDH-ODH complex and suggest a similar localization in other actinobacteria. The observation of a defined spatial localization of an enzyme complex catalyzing two key reactions of central metabolism poses questions regarding possible consequences for the availability of substrates and products within the cell and other bacterial enzyme complexes showing similar behavior.

Project description:The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

Dataset Information

Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

Publications

Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets