Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities.

BACKGROUND: Microbial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology. Different strategies for their uncovering such as bioprospecting, which relies mainly on metagenomic approaches in combination with sequence-based bioinformatic analyses, have emerged; yet accurate function prediction of their proteomes and deciphering the in vivo activity of an enzyme remains challenging. RESULTS: Here, we present environmental activity-based protein profiling (eABPP), a multi-omics approach that extends genome-resolved metagenomics with mass spectrometry-based ABPP. This combination allows direct profiling of environmental community samples in their native habitat and the identification of active enzymes based on their function, even without sequence or structural homologies to annotated enzyme families. eABPP thus bridges the gap between environmental genomics, correct function annotation, and in vivo enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases from eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. CONCLUSIONS: By reporting enzyme activities within an ecosystem in their native state, we anticipate that eABPP will not only advance current methodological approaches to sequence homology-guided enzyme discovery from environmental ecosystems for subsequent biocatalyst development but also contributes to the ecological investigation of microbial community interactions by dissecting their underlying molecular mechanisms.

Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus.

The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Šresolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Šresolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications.

Application of the endogenous CRISPR-Cas type I-D system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems are widely distributed among bacteria and archaea. In this study, we demonstrate the successful utilization of the type I-D CRISPR-Cas system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Given its extreme growth conditions characterized by a temperature of 75°C and pH 3, an uracil auxotrophic selection system was previously established, providing a basis for our investigations. We developed a novel plasmid specifically designed for genome editing, which incorporates a mini-CRISPR array that can be induced using xylose, resulting in targeted DNA cleavage. Additionally, we integrated a gene encoding the ß-galactosidase of Saccharolobus solfataricus into the plasmid, enabling blue-white screening and facilitating the mutant screening process. Through the introduction of donor DNA containing genomic modifications into the plasmid, we successfully generated deletion mutants and point mutations in the genome of S. acidocaldarius. Exploiting the PAM (protospacer adjacent motif) dependence of type I systems, we experimentally confirmed the functionality of three different PAMs (CCA, GTA, and TCA) through a self-targeting assessment assay and the gene deletion of upsE. Our findings elucidate the application of the endogenous Type I-D CRISPR-Cas system for genetic engineering in S. acidocaldarius, thus expanding its genetic toolbox.

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling.

Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications so far mainly in biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from the thermophilic and lignocellulose-degrading white rot fungus Phanerochaete chrysosporium. By employing a comparative ABPP-based functional screen, including a direct profiling of wood substrate-bound enzymes, we identify those lignocellulose-degrading carbohydrate esterase (CE1 and CE15) and glycoside hydrolase (GH3, GH5, GH16, GH17, GH18, GH25, GH30, GH74 and GH79) enzymes specifically active in presence of the substrate. As expression of fungal enzymes remains challenging, our ABPP-mediated approach represents a preselection procedure for focusing experimental efforts on the most promising biocatalysts. Furthermore, this approach may also allow the functional annotation of domains-of-unknown functions (DUFs). The ABPP-based biocatalyst screening described here may thus allow the identification of active enzymes in a process of interest and the elucidation of novel biocatalysts that share no sequence similarity to known counterparts.

Exopolysaccharide composition and size in Sulfolobus acidocaldarius biofilms.

Extracellular polymeric substances (EPS) comprise mainly carbohydrates, proteins and extracellular DNA (eDNA) in biofilms formed by the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius. However, detailed information on the carbohydrates in the S. acidocaldarius biofilm EPS, i.e., the exopolysaccharides (PS), in terms of identity, composition and size were missing. In this study, a set of methods was developed and applied to study the PS in S. acidocaldarius biofilms. It was initially shown that addition of sugars, most significantly of glucose, to the basal N-Z-amine-based growth medium enhanced biofilm formation. For the generation of sufficient amounts of biomass suitable for chemical analyses, biofilm growth was established and optimized on the surface of membrane filters. EPS were isolated and the contents of carbohydrates, proteins and eDNA were determined. PS purification was achieved by enzymatic digestion of other EPS components (nucleic acids and proteins). After trifluoroacetic acid-mediated hydrolysis of the PS fraction, the monosaccharide composition was analyzed by reversed-phase liquid chromatography (RP-LC) coupled to mass spectrometry (MS). Main sugar constituents detected were mannose, glucose and ribose, as well as minor proportions of rhamnose, N-acetylglucosamine, glucosamine and galactosamine. Size exclusion chromatography (SEC) revealed the presence of one single PS fraction with a molecular mass of 4-9 × 104 Da. This study provides detailed information on the PS composition and size of S. acidocaldarius MW001 biofilms and methodological tools for future studies on PS biosynthesis and secretion.

Simplified Enzymatic Synthesis of 2-Keto-3-Deoxy-D-Gluconate from D-Gluconate Using the Gluconate Dehydratase from Thermoproteus tenax.

Many research areas, e.g., basic research but also applied fields of biotechnology, biomedicine, and diagnostics often suffer from the unavailability of metabolic compounds. This is mostly due to missing easy and efficient synthesis procedures. We herein describe the biocatalytic/enzymatic production of 2-keto-3-deoxy-D-gluconate, an intermediate of central metabolic pathways in all three domains of life and also of bacterial polysaccharides, lipopolysaccharides, and cell wall components. The method is based on the gluconate dehydratase from the hyperthermophilic crenarchaeon Thermoproteus tenax, which can be easily recombinantly overproduced in Escherichia coli and-due to its intrinsic thermostability-rapidly be purified by two precipitation steps. The enzyme completely converts D-gluconate to solely stereochemically pure KDG, taking benefits from the enol-keto-tautomerism of the primary reaction product. The final product can then easily be separated from the protein by ultrafiltration. The simple one-step procedure, which is suitable at least for the lab-scale/gram-scale production of KDG, replaces lengthy multi-step reactions and is easily scalable. This approach also illustrates the great application potential of Archaea with their unusual metabolic pathways and enzymes for the synthesis of added value products.

Thermostable and O2-Insensitive Pyruvate Decarboxylases from Thermoacidophilic Archaea Catalyzing the Production of Acetaldehyde.

Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO2. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen.

Workflows for optimization of enzyme cascades and whole cell catalysis based on enzyme kinetic characterization and pathway modelling.

To move towards a circular bioeconomy, sustainable strategies for the utilization of renewable, non-food biomass wastes such as lignocellulose, are needed. To this end, an efficient bioconversion of d-xylose - after d-glucose the most abundant sugar in lignocellulose - is highly desirable. Most standard organisms used in biotechnology are limited in metabolising d-xylose, and also in vitro enzymatic strategies for its conversion have not been very successful. We herein discuss that bioconversion of d-xylose is mostly hampered by missing knowledge on the kinetic properties of the enzymes involved in its metabolism. We propose a combination of classical enzyme characterizations and mathematical modelling approaches as a workflow for rational, model-based design to optimize enzyme cascades and/or whole cell biocatalysts for efficient d-xylose metabolism.

Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress.

Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80°C and pH 2 to 3. Due to its unique physiological properties, allowing life at environmental extremes, and the recent availability of genetic tools, this extremophile has received increasing interest for biotechnological applications. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius toward the industrially relevant organic solvent 1-butanol. In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred at up to 1.5% (vol/vol) 1-butanol, while planktonic growth was observed at up to 1% (vol/vol) 1-butanol. Confocal laser-scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy, three different cell morphotypes were observed in response to 1-butanol. Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in the absence and presence of 1-butanol. In response to 1% (vol/vol) 1-butanol, transcript levels of genes encoding motility and cell envelope structures, as well as membrane proteins, were reduced. Cell division and/or vesicle formation were upregulated. Furthermore, changes in immune and defense systems, as well as metabolism and general stress responses, were observed. Our findings show that the extreme lifestyle of S.acidocaldarius coincided with a high tolerance to organic solvents. This study provides what may be the first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldariusIMPORTANCEArchaea are unique in terms of metabolic and cellular processes, as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic, and polyomics studies has provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius, which is adapted to the two extremes of low pH and high temperature, to study its tolerance and robustness as well as its global cellular response toward organic solvents, as exemplified by 1-butanol. We were able to identify biofilm formation as a primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes, such as cell division and cellular defense systems, thus suggesting a global response for the protection at the population level.

The biology of thermoacidophilic archaea from the order Sulfolobales.

Thermoacidophilic archaea belonging to the order Sulfolobales thrive in extreme biotopes, such as sulfuric hot springs and ore deposits. These microorganisms have been model systems for understanding life in extreme environments, as well as for probing the evolution of both molecular genetic processes and central metabolic pathways. Thermoacidophiles, such as the Sulfolobales, use typical microbial responses to persist in hot acid (e.g. motility, stress response, biofilm formation), albeit with some unusual twists. They also exhibit unique physiological features, including iron and sulfur chemolithoautotrophy, that differentiate them from much of the microbial world. Although first discovered >50 years ago, it was not until recently that genome sequence data and facile genetic tools have been developed for species in the Sulfolobales. These advances have not only opened up ways to further probe novel features of these microbes but also paved the way for their potential biotechnological applications. Discussed here are the nuances of the thermoacidophilic lifestyle of the Sulfolobales, including their evolutionary placement, cell biology, survival strategies, genetic tools, metabolic processes and physiological attributes together with how these characteristics make thermoacidophiles ideal platforms for specialized industrial processes.

Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E.

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6-7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or ß-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous ß-glycosidase (EGIDFPOO_00532) with ß-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.

Salt Stress Response of Sulfolobus acidocaldarius Involves Complex Trehalose Metabolism Utilizing a Novel Trehalose-6-Phosphate Synthase (TPS)/Trehalose-6-Phosphate Phosphatase (TPP) Pathway.

The crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway, and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of strains with single and double deletions of ΔtreY and ΔtreT in S. acidocaldarius revealed that in addition to these two pathways, a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family, establishing a new function within this group of enzymes. This novel GT4-like TPS was found to be present mainly in the Sulfolobales The ΔtreY ΔtreT Δtps triple mutant of S. acidocaldarius, which lacks the ability to synthesize trehalose, showed no altered phenotype under standard conditions or heat stress but was unable to grow under salt stress. Accordingly, in the wild-type strain, a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress-mediated induction of all three trehalose-synthesizing pathways. This demonstrates that in Archaea, trehalose plays an essential role for growth under high-salt conditions.IMPORTANCE The metabolism and function of trehalose as a compatible solute in Archaea was not well understood. This combined genetic and enzymatic approach at the interface of microbiology, physiology, and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea Here, we unraveled the complexity of trehalose metabolism, and we present a comprehensive study on trehalose function in stress response in S. acidocaldarius This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea, involving many novel biocatalysts, such as glycosyltransferases, with great potential in biotechnology.

Corrigendum: Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus.

[This corrects the article DOI: 10.3389/fbioe.2020.00185.].

Identification of XylR, the Activator of Arabinose/Xylose Inducible Regulon in Sulfolobus acidocaldarius and Its Application for Homologous Protein Expression.

The thermophilic archaeon Sulfolobus acidocaldarius can use different carbon sources for growth, including the pentoses D-xylose and L-arabinose. In this study, we identified the activator XylR (saci_2116) responsible for the transcriptional regulation of the pentose transporter and pentose metabolizing genes in S. acidocaldarius. A xylR deletion mutant showed growth retardation on D-xylose/L-arabinose containing media and the lack of transcription of the respective ABC transporter. In contrast to so far used promoters for expression in S. acidocaldarius, the xylR responsive promoters have a very low background activity. Finally, two XylR dependent promoters next to the long-established maltose inducible promotor were used to construct a high-throughput expression vector system for S. acidocaldarius to efficiently clone and express proteins in S. acidocaldarius.

Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals.

2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator (CnL-KdpD) and D-KdpD from Pseudomonas putida (PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on CnL-KdpD, whereas increased activity and stability were observed for PpD-KdpD in the presence of Mg2+. Furthermore, CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas PpD-KdpD was inhibited with D-xylonate (I50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while PpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points • L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively. • Mg2+affected activity and stabilities of D-KdpDs, but not of L-KdpDs. • CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield. • Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.

Phosphorylation of the acyl-CoA binding pocket of the FadR transcription regulator in Sulfolobus acidocaldarius.

The archaeal model organism Sulfolobus acidocaldarius possesses a TetR-like transcription factor that represses a 30-kb gene cluster encoding fatty acid metabolism enzymes. Interaction of this regulator, FadRSa, with acyl-CoA molecules causes a DNA dissociation, which may lead to a derepression of the gene cluster. Previously, a phosphoproteome analysis revealed the phosphorylation of three consecutive amino acids in the acyl-CoA ligand binding pocket. Here, we study this phosphorylation event and show that ArnC, a Hanks-type protein kinase, targets a threonine within the phosphoacceptor motif in vitro. Electrophoretic mobility shift assays using a phosphomimetic mutant of FadRSa demonstrate that the presence of negatively charged groups on the phosphoacceptor motif causes an inhibition of the ligand binding that desensitizes the responsiveness of the regulator to acyl-CoA molecules. Based on these observations, we propose a model in which phosphorylation of FadRSa in its ligand-binding pocket acts as an additional regulatory layer silencing acyl-CoA responsive derepression of fatty acid and lipid degradation. Moreover, given the recently discovered interplay between FadRSa and the chromosome structuring protein coalescin, FadRSa phosphorylation could also influence local chromosome conformation under specific cellular conditions.

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus.

The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a Vmax of 61.6 U mg-1 and a KM of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses.

N-Myc-induced metabolic rewiring creates novel therapeutic vulnerabilities in neuroblastoma.

N-Myc is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of Myc family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of Myc proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of N-Myc overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of N-Myc induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of N-Myc. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of N-Myc overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered N-Myc induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.

A combined experimental and modelling approach for the Weimberg pathway optimisation.

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.