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12 Publications visible to you, out of a total of 12
Quantitative analysis of amino acid metabolism in liver cancer links glutamate excretion to nucleotide synthesis

Abstract (Expand)

Many cancer cells consume glutamine at high rates; counterintuitively, they simultaneously excrete glutamate, the first intermediate in glutamine metabolism. Glutamine consumption has been linked to … replenishment of tricarboxylic acid cycle (TCA) intermediates and synthesis of adenosine triphosphate (ATP), but the reason for glutamate excretion is unclear. Here, we dynamically profile the uptake and excretion fluxes of a liver cancer cell line (HepG2) and use genome-scale metabolic modeling for in-depth analysis. We find that up to 30% of the glutamine is metabolized in the cytosol, primarily for nucleotide synthesis, producing cytosolic glutamate. We hypothesize that excreting glutamate helps the cell to increase the nucleotide synthesis rate to sustain growth. Indeed, we show experimentally that partial inhibition of glutamate excretion reduces cell growth. Our integrative approach thus links glutamine addiction to glutamate excretion in cancer and points toward potential drug targets.

Authors: Avlant Nilsson, Jurgen R. Haanstra, Martin Engqvist, Albert Gerding, Barbara M. Bakker, Ursula Klingmüller, Bas Teusink, Jens Nielsen

Date Published: 27th Apr 2020

Publication Type: Journal

Genome-scale Model Constrained by Proteomics Reveals Metabolic Changes in Streptomyces coelicolor M1152 Compared to M145

Abstract (Expand)

Streptomyces coelicolor M1152 is a widely used host strain for the heterologous production of novel small molecule natural products, genetically engineered for this purpose through e.g. deletion of … four of its native biosynthetic gene clusters (BGCs) for improved precursor supply. Regardless of its potential, a systems understanding of its tight regulatory network and the effects of the significant genomic changes in M1152 is missing. In this study, we compare M1152 to its ancestor M145, thereby connecting observed phenotypic differences to changes on transcription and translation. Measured protein levels are connected to predicted metabolic fluxes, facilitated by an enzyme-constrained genome-scale model (GEM), that by itself is a consensus result of a community effort. This approach connects observed differences in growth rate and glucose consumption to changes in central carbon metabolism, accompanied by differential expression of important regulons. Results suggest that precursors supply is not limiting secondary metabolism, informing that alternative strategies will be beneficial for further development of S. coelicolor for heterologous production of novel compounds.

Authors: Snorre Sulheim, Tjaša Kumelj, Dino van Dissel, Ali Salehzadeh-Yazdi, Chao Du, Gilles P. van Wezel, Kay Nieselt, Eivind Almaas, Alexander Wentzel, Eduard J Kerkhoven

Date Published: 8th Oct 2019

Publication Type: Unpublished

Characterization of the Weimberg Pathway in Caulobacter crescentus

Abstract (Expand)

Caulobacter crescentus is a gram-negative bacterium that can utilize xylose as a substrate using the Weimberg pathway, which converts xylose to α-ketoglutarate in five steps without carbon loss. This … is an interesting pathway for heterologous expression in other organisms in order to enable xylose utilization in biorefinery processes. C. crescentus was grown on xylose, arabinose and glucose, and maximum specific growth rates determined for the three substrates were 0.11 h−1, 0.05 h−1, and 0.15 h−1 respectively. Growth was found to be significantly inhibited at sugar concentration of 20 g L−1, shown primarily by an increased lag phase. Enzyme activity assays showed that the Weimberg pathway was active in cells grown, not only on xylose but also on arabinose. No activity was found for growth on glucose. Furthermore, substantial amounts of α-ketoglutarate—up to a yield of 0.4 g g−1—was excreted during growth on xylose, but no other extracellular intermediates in the Weimberg pathway were detected during growth on xylose. Apparently, C. crescentus is not well adapted for efficient growth on high xylose levels, and responds by an extended lag phase and secretion of α-ketoglutarate.

Authors: Henrik Almqvist, Sara Jonsdottir Glaser, Celina Tufvegren, Lisa Wasserstrom, Gunnar Lidén

Date Published: 1st Jun 2018

Publication Type: Not specified

Multi-omics Reveals the Lifestyle of the Acidophilic, Mineral-Oxidizing Model Species Leptospirillum ferriphilum T

Abstract (Expand)

Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well … as biomining operations. Despite its perceived importance, no complete genome sequence of this model species' type strain is available, limiting the possibilities to investigate the strategies and adaptations Leptospirillum ferriphilumT applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT DSM 14647 obtained by PacBio SMRT long read sequencing for use as a high quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as substrate and bioleaching cultures containing chalcopyrite (CuFeS2). Leptospirillum ferriphilumT adaptations to growth on chalcopyrite included a possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, expression and translation of genes responsible for chemotaxis and motility were enhanced.

Authors: Stephan Christel, Malte Herold, Sören Bellenberg, Mohamed El Hajjami, Antoine Buetti-Dinh, Igor V. Pivkin, Wolfgang Sand, Paul Wilmes, Ansgar Poetsch, Mark Dopson

Date Published: 1st Feb 2018

Publication Type: Not specified

Modeling metabolic networks including gene expression and uncertainties

Abstract (Expand)

Constraint based methods, such as the Flux Balance Analysis, are widely used to model cellular growth processes without relying on extensive information on the regulatory features. The regulation is … instead substituted by an optimization problem usually aiming at maximal biomass accumulation. A recent extension to these methods called the dynamic enzyme-cost Flux Balance Analysis (deFBA) is a fully dynamic modeling method allowing for the prediction of necessary enzyme levels under changing environmental conditions. However, this method was designed for deterministic settings in which all dynamics, parameters, etc. are exactly known. In this work, we present a theoretical framework extending the deFBA to handle uncertainties and provide a robust solution. We use the ideas from multi-stage nonlinear Model Predictive Control (MPC) and its feature to represent the evolution of uncertainties by an exponentially growing scenario tree. While this representation is able to construct a deterministic optimization problem in the presence of uncertainties, the computational cost also increases exponentially. We counter this by using a receding prediction horizon and reshape the standard deFBA to the short-time deFBA (sdeFBA). This leads us, along with further simplification of the scenario tree, to the robust deFBA (rdeFBA). This framework is capable of handling the uncertainties in the model itself as well as uncertainties experienced by the modeled system. We applied these algorithms to two case-studies: a minimal enzymatic nutrient uptake network, and the abstraction of the core metabolic process in bacteria.

Authors: Henning Lindhorst, Sergio Lucia, Rolf Findeisen, Steffen Waldherr

Date Published: 13th Jun 2017

Publication Type: Not specified

Miniaturized and automated adaptive laboratory evolution: Evolving Corynebacterium glutamicum towards an improved d-xylose utilization.

Abstract (Expand)

Adaptive Laboratory Evolution (ALE) is increasingly being used as a technique for untargeted strain optimization. This work aimed at developing an automated and miniaturized ALE approach based on … repetitive batch cultivations in microtiter plates. The new method is applied to the recently published strain Corynebacterium glutamicum pEKEx3-xylXABCDCc, which is capable of utilizing d-xylose via the Weimberg (WMB) pathway. As a result, the significantly improved strain WMB2evo was obtained, showing a specific growth rate of 0.26h-1 on d-xylose as sole carbon and energy source. WMB2evo grows stable during lab-scale bioreactor operation, demonstrating the high potential of this strain for future biorefinery applications. Genome sequencing of cell samples from two different ALE processes revealed potential key mutations, e.g. in the gene cg0196 (encoding for the transcriptional regulator IolR of the myo-inositol metabolism). These findings open up new perspectives for the rational engineering of C. glutamicum towards improved d-xylose utilization.

Authors: A. Radek, N. Tenhaef, M. F. Muller, C. Brusseler, W. Wiechert, J. Marienhagen, T. Polen, S. Noack

Date Published: 30th May 2017

Publication Type: Not specified

Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.

Abstract (Expand)

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a … hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Authors: Z. Xu, Y. Wang, K. F. Chater, H. Y. Ou, H. H. Xu, Z. Deng, M. Tao

Date Published: 8th Jan 2017

Publication Type: Not specified

Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains

Abstract (Expand)

Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been … pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization.

Authors: A. Radek, M. F. Muller, J. Gatgens, L. Eggeling, K. Krumbach, J. Marienhagen, S. Noack

Date Published: 15th Jun 2016

Publication Type: Not specified

Identification of HNRNPK as regulator of hepatitis C virus particle production

Abstract (Expand)

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, … detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses.

Authors: M. Poenisch, P. Metz, H. Blankenburg, A. Ruggieri, J. Y. Lee, D. Rupp, I. Rebhan, K. Diederich, L. Kaderali, F. S. Domingues, M. Albrecht, V. Lohmann, H. Erfle, R. Bartenschlager

Date Published: 8th Jan 2015

Publication Type: Not specified

Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of D-xylose containing substrates

Abstract (Expand)

Biomass-derived d-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. The industrially important platform organism Corynebacterium … glutamicum has already been engineered to grow on this pentose as sole carbon and energy source. However, all currently described C. glutamicum strains utilize d-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of CO2, in particular, when aiming for the synthesis of alpha-ketoglutarate and its derivatives (e.g. l-glutamate). Driven by the motivation to engineer a more carbon-efficient C. glutamicum strain, we functionally integrated the Weimberg pathway from Caulobacter crescentus in C. glutamicum. This five-step pathway, encoded by the xylXABCD-operon, enabled a recombinant C. glutamicum strain to utilize d-xylose in d-xylose/d-glucose mixtures. Interestingly, this strain exhibited a tri-phasic growth behavior and transiently accumulated d-xylonate during d-xylose utilization in the second growth phase. However, this intermediate of the implemented oxidative pathway was re-consumed in the third growth phase leading to more biomass formation. Furthermore, C. glutamicum pEKEx3-xylXABCDCc was also able to grow on d-xylose as sole carbon and energy source with a maximum growth rate of mumax=0.07+/-0.01h(-1). These results render C. glutamicum pEKEx3-xylXABCDCc a promising starting point for the engineering of efficient production strains, exhibiting only minimal carbon loss on d-xylose containing substrates.

Authors: A. Radek, K. Krumbach, J. Gatgens, V. F. Wendisch, W. Wiechert, M. Bott, S. Noack, J. Marienhagen

Date Published: 12th Oct 2014

Publication Type: Not specified

Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters

Abstract (Expand)

We have constructed derivatives of Streptomyces coelicolor M145 as hosts for the heterologous expression of secondary metabolite gene clusters. To remove potentially competitive sinks of carbon and … nitrogen, and to provide a host devoid of antibiotic activity, we deleted four endogenous secondary metabolite gene clusters from S. coelicolor M145--those for actinorhodin, prodiginine, CPK and CDA biosynthesis. We then introduced point mutations into rpoB and rpsL to pleiotropically increase the level of secondary metabolite production. Introduction of the native actinorhodin gene cluster and of gene clusters for the heterologous production of chloramphenicol and congocidine revealed dramatic increases in antibiotic production compared with the parental strain. In addition to lacking antibacterial activity, the engineered strains possess relatively simple extracellular metabolite profiles. When combined with liquid chromatography and mass spectrometry, we believe that these genetically engineered strains will markedly facilitate the discovery of new compounds by heterologous expression of cloned gene clusters, particularly the numerous cryptic secondary metabolic gene clusters that are prevalent within actinomycete genome sequences.

Authors: Juan Pablo Gomez-Escribano, Mervyn J. Bibb

Date Published: 1st Mar 2011

Publication Type: Not specified

Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment

Abstract (Expand)

Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome … and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIalpha), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIalpha product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIalpha was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIalpha and stimulate its kinase activity. The absence of PI4KIIIalpha activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

Authors: S. Reiss, I. Rebhan, P. Backes, I. Romero-Brey, H. Erfle, P. Matula, L. Kaderali, M. Poenisch, H. Blankenburg, M. S. Hiet, T. Longerich, S. Diehl, F. Ramirez, T. Balla, K. Rohr, A. Kaul, S. Buhler, R. Pepperkok, T. Lengauer, M. Albrecht, R. Eils, P. Schirmacher, V. Lohmann, R. Bartenschlager

Date Published: 18th Jan 2011

Publication Type: Not specified

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