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Results for “Microbial metabolic pathways”

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PubMed2002

Metabolic pathway analysis in trypanosomes and malaria parasites.

Identification of novel drug targets is required for the development of new classes of drugs to overcome drug resistance and replace less efficacious treatments. In theory, knowledge of the entire genome of a pathogen identifies every potential drug target in any given microbe. In practice, the sheer complexity and the inadequate or inaccurate annotation of genomic information makes target identification and selection somewhat more difficult. Analysis of metabolic pathways provides a useful conceptual framework for the identification of potential drug targets and also for improving our understanding of microbial responses to nutritional, chemical and other environmental stresses. A number of metabolic databases are available as tools for such analyses. The strengths and weaknesses of this approach are discussed.

Animals↗

PubMed2006

Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae.

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.

Alkaloids↗

PubMed1998

Directed evolution of new enzymes and pathways for environmental biocatalysis.

Biocatalysis is important in both natural and engineered environments. The major global reactions in the biospheric cycling of carbon, nitrogen, and other elements are catalyzed by microorganisms. The global carbon cycle includes millions of organic compounds that are made by plants, microorganisms, and organic chemists. Most of those compounds are transformed by microbial enzymes. Degradative metabolism is known as catabolism and yields principally carbon dioxide, methane, or biomass. Microbial catabolic enzymes are a great resource for biotechnology. They are the building blocks for engineering novel metabolic pathways and evolving improved enzymes in the laboratory. Two multicomponent bacterial oxygeneases, cytochrome P450cam and toluene dioxygenase, catalyze the dechlorination of polyhalogenated C2 compounds. Seven genes encoding those functional enzyme complexes were coexpressed in a Pseudomonas and shown to metabolize pentachloreothane to nonhalogenated organic acids that were metabolized further to carbon dioxide. In another example, the enzyme catalyzing the dechlorination of the herbicide atrazine was subjected to iterative DNA shuffling to produce mutations. By using a plate screening assay, mutated atrazine chlorohydrolase that catalyzed a more rapid dechlorination of atrazine was obtained. The mutant genes were sequences and found to encode up to 11 amino acid changes. Atrazine chlorohydrolase is currently being used in a model municipal water treatment system to test the feasibility of using enzymes for atrazine decontamination. These data suggest that the natural diversity of bacterial catabolic enzymes provides the starting point for improved biocatalytic systems that meet the needs of commercial applications.

Biodegradation, Environmental↗

PubMed1997

The effect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils.

The effects of several bioremediation stimulants, including potential metabolism pathway inducers, inorganic/organic nutrients, and surfactants on the metabolism of phenanthrene and pyrene, as well as the population dynamics of PAH degrading microorganisms was examined in five soils with differing background PAH concentrations, exposure histories and physical properties. Most of the supplements either had no significant effect or decreased the mineralization of [14C]-phenanthrene and [14C]-pyrene in soil slurry microcosms. The effect of a particular supplement, however, was often not uniform within or across soils. Decreased mineralization of [14C]-phenanthrene and [14C]-pyrene was usually due to either preferential use of the supplement as carbon source and/or stimulation of non-PAH degrading microorganisms. Many of the supplements increased populations of heterotrophic microorganisms, as measured by plate counts, but did not increase populations of phenanthrene degrading microorganisms, as measured by the [14C]-PAH mineralization MPN analysis or cellular incorporation of [14C]-PAH. These results suggest that the PAH degrading community at each site may be unique in their response to materials added in an attempt to stimulate PAH degradation. The characteristics of the site, including exposure history, soil type, and temporal variation may all influence their response.

Biodegradation, Environmental↗

PubMed2000

Microbial consortia involved in the anaerobic degradation of hydrocarbons.

In this review, we examine the energetics of well-characterized biodegradation pathways and explore the possibilities for these to support growth of multiple organisms interacting in consortia. The relevant phenotypic and/or phylogenetic characteristics of isolates and consortia mediating hydrocarbon degradation coupled with different terminal electron-accepting processes (TEAP) are also reviewed. While the information on metabolic pathways has been gained from the analysis of individual isolates, the energetic framework presented here demonstrates that microbial consortia could be readily postulated for hydrocarbon degradation coupled to any TEAP. Several specialized reactions occur within these pathways, and the organisms mediating these are likely to play a key role in defining the hydrocarbon degradation characteristics of the community under a given TEAP. Comparing these processes within and between TEAPs reveals biological unity in that divergent phylotypes display similar degradation mechanisms and biological diversity in that hydrocarbon-degraders closely related as phylotypes differ in the type and variety of hydrocarbon degradation pathways they possess. Analysis of microcosms and of field samples suggests that we have only begun to reveal the diversity of organisms mediating anaerobic hydrocarbon degradation. Advancements in the understanding of how hydrocarbon-degrading communities function will be significantly affected by the extent to which organisms mediating specialized reactions can be identified, and tools developed to allow their study in situ.

Bacteria, Anaerobic↗

PubMed2006

Xenobiotic reductase A in the degradation of quinoline by Pseudomonas putida 86: physiological function, structure and mechanism of 8-hydroxycoumarin reduction.

A continuous evolutionary pressure of the biotic and abiotic world has led to the development of a diversity of microbial pathways to degrade and biomineralize aromatic and heteroaromatic compounds. The heterogeneity of compounds metabolized by bacteria like Pseudomonas putida indicates the large variety of enzymes necessary to catalyse the required reactions. Quinoline, a N-heterocyclic aromatic compound, can be degraded by microbes along different pathways. For P. putida 86 quinoline degradation by the 8-hydroxycoumarin pathway has been described and several intermediates were identified. To select enzymes catalysing the later stages of the 8-hydroxycoumarin pathway P. putida 86 was grown with quinoline. The FMN-containing enzyme xenobiotic reductase A (XenA) was isolated and analysed for its reactivity with intermediates of the 8-hydroxycoumarin pathway. XenA catalyses the NADPH-dependent reduction of 8-hydroxycoumarin and coumarin to produce 8-hydroxy-3,4-dihydrocoumarin and 3,4-dihydrocoumarin, respectively. Crystallographic analysis of XenA alone and in complex with the two substrates revealed insights into the mechanism. XenA shows a dimeric arrangement of two (beta/alpha)(8) barrel domains each binding one FMN cofactor. High resolution crystal structures of complexes with 8-hydroxycoumarin and coumarin show different modes of binding for these molecules in the active site. While coumarin is ideally positioned for hydride transfer from N-5 of the isoalloxazine ring to C-4 of coumarin, 8-hydroxycoumarin forms a non-productive complex with oxidised XenA. Orientation of 8-hydroxycoumarin in the active site appears to be dependent on the electronic state of the flavin. We postulate that XenA catalyses the last step of the 8-hydroxycoumarin pathway before the heterocyclic ring is hydrolysed to yield 3-(2,3-dihydroxyphenyl)-propionic acid. As XenA is also found in P. putida strains unable to degrade quinoline, it appears to have more than one physiological function and is an example of how enzymes with low substrate specificity can help to explain the variety of degradation pathways possible.

Bacterial Proteins↗

PubMed1990

The metabolism of CGP-291: the use of microorganisms as models of mammalian metabolism.

CGP-291 is an investigational antiprotozoal agent with unknown metabolism. Microbial systems were utilized, as a model of mammalian metabolism, to predict the oxidative metabolic pathway of this nitroimidazole. Large-scale fermentation of CGP-291 with Beauvaria bassiana produced two major metabolites, IV and V. Structures of both were elucidated by comparing spectral data of metabolites to that of the starting material. The presence of two minor monohydroxylated metabolites was verified using LC-MS.

Chromatography, Thin Layer↗

PubMed2005

Reactive oxygen species and development in microbial eukaryotes.

Reactive oxygen species (ROS) have been regarded as inevitable harmful by-products of aerobic metabolism. Growing evidence, however, suggests that ROS play important physiological roles. This raises questions about the pathways that different groups of organisms use to produce and sense ROS. In microbial eukaryotes, recent data show (i) increased ROS levels during cell differentiation, (ii) the existence of ROS-producing enzymes, such as NADPH oxidases (NOX), (iii) the involvement of NOX in developmental processes, and (iv) a conservation in the signal-transduction mechanisms used to detect ROS. This shows that manipulation of reactive species, as strategy to regulate cell differentiation, is ubiquitous in eukaryotes and suggests that such strategy was selected early in evolution.

Antioxidants↗

PubMed2005

Bioinformatics in microbial biotechnology--a mini review.

The revolutionary growth in the computation speed and memory storage capability has fueled a new era in the analysis of biological data. Hundreds of microbial genomes and many eukaryotic genomes including a cleaner draft of human genome have been sequenced raising the expectation of better control of microorganisms. The goals are as lofty as the development of rational drugs and antimicrobial agents, development of new enhanced bacterial strains for bioremediation and pollution control, development of better and easy to administer vaccines, the development of protein biomarkers for various bacterial diseases, and better understanding of host-bacteria interaction to prevent bacterial infections. In the last decade the development of many new bioinformatics techniques and integrated databases has facilitated the realization of these goals. Current research in bioinformatics can be classified into: (i) genomics--sequencing and comparative study of genomes to identify gene and genome functionality, (ii) proteomics--identification and characterization of protein related properties and reconstruction of metabolic and regulatory pathways, (iii) cell visualization and simulation to study and model cell behavior, and (iv) application to the development of drugs and anti-microbial agents. In this article, we will focus on the techniques and their limitations in genomics and proteomics. Bioinformatics research can be classified under three major approaches: (1) analysis based upon the available experimental wet-lab data, (2) the use of mathematical modeling to derive new information, and (3) an integrated approach that integrates search techniques with mathematical modeling. The major impact of bioinformatics research has been to automate the genome sequencing, automated development of integrated genomics and proteomics databases, automated genome comparisons to identify the genome function, automated derivation of metabolic pathways, gene expression analysis to derive regulatory pathways, the development of statistical techniques, clustering techniques and data mining techniques to derive protein-protein and protein-DNA interactions, and modeling of 3D structure of proteins and 3D docking between proteins and biochemicals for rational drug design, difference analysis between pathogenic and non-pathogenic strains to identify candidate genes for vaccines and anti-microbial agents, and the whole genome comparison to understand the microbial evolution. The development of bioinformatics techniques has enhanced the pace of biological discovery by automated analysis of large number of microbial genomes. We are on the verge of using all this knowledge to understand cellular mechanisms at the systemic level. The developed bioinformatics techniques have potential to facilitate (i) the discovery of causes of diseases, (ii) vaccine and rational drug design, and (iii) improved cost effective agents for bioremediation by pruning out the dead ends. Despite the fast paced global effort, the current analysis is limited by the lack of available gene-functionality from the wet-lab data, the lack of computer algorithms to explore vast amount of data with unknown functionality, limited availability of protein-protein and protein-DNA interactions, and the lack of knowledge of temporal and transient behavior of genes and pathways.

Journal Article↗

PubMed2001

Flux analysis: a basic tool of microbial physiology.

Flux analysis (FA) is a means of organizing data to show flux through the central metabolic pathways (CMPs). It quantifies flux from uptake of carbon to the outputs of the CMPs, which are the precursors used for biosynthesis, acetate excretion and CO2. Fluxes to precursors reflect the commands of the genome and acetate excretion balances fluxes to precursor supply when uptake exceeds the capacity of the CMPs to allocate carbon in exactly the correct amount to each precursor. No other products have been detected in 11 phenotypes of Escherichia coli ML308. FA of each of these 11 phenotypes (with some additional variations in culture conditions, some selected mutations and one genetic construct) are shown as flux (mol (kg dry weight biomass)-1 h-1) and are the starting point for further exploration of the physiology of E. coli: FAs suggest the possibility of four strategies to reduce acetate excretion and these have been tested in two of the phenotypes (glucose and pyruvate). All are successful to some degree but results are not always what were expected. FA of such interventions suggest that some 'global' control mechanisms operate in E. coli ML308 independent of carbon source. There is a division in the CMPs between those pathways that use phosphorylated intermediates and those that do not and these, in turn, are divided into the Krebs cycle and the C2 and C3 monocarboxylic acids. Altogether, there are four 'compartments' and each contains intermediates that are also precursors.

Acetates↗

PubMed2026

Integrated metagenomic and metaproteomic insights into current-carrying-coil magnetic field enhanced synergistic methanogenic system and antibiotic resistance gene reduction in cow manure anaerobic digestion.

Anaerobic digestion (AD) is a sustainable strategy for valorizing cow manure (CM). However, the high ammonia (NH3) concentration and low biodegradability of CM limit hydrolysis and methane production. This study investigated the application of a current-carrying-coil-based magnetic field (CCC-MF) to AD of CM. The CCC-MF digesters showed higher soluble chemical oxygen demand and attained 16.59 % higher ammonium nitrogen reduction, contributing to a 37.50 % higher average methane yield than the control. Further, CCC-MF digesters showed higher enzyme activities (alkaline protease + 30 %, acetate kinase + 22 % and hydrazine dehydrogenase + 26 %) and increased microbial metabolic indices (dehydrogenase activity + 17 % and electron transport system activity + 10 %) than the control. Metagenomics analysis revealed that abundances of the bacterial genera Mesotoga, Aminobacterium, Xiashengella, unclassified Candidatus Cloacimonadota, Advenella, Pseudomonas, and Comamonas increased, whereas the acetoclastic methanogen Methanothrix decreased by 2.58 %, accompanied by 2.07- and 1.64-fold increases in hydrogenotrophic methanogens Methanospirillum and Methanobacterium, respectively, in CCC-MF digesters. The abundance of nitrogen dissimilation and assimilation genes NirK, NorB, NarB, NapA, nmo, and GLT1 were enhanced by 1.14, 1.04, 2.30, 1.32, 1.17, and 1.29-fold in CCC-MF digesters compared to the control. Moreover, metaproteomics revealed higher up-regulated differentially expressed proteins in NH3 reduction-related amino acid metabolism pathways in CCC-MF digester compared to control. Additionally, reduced abundances of bacitracin, polymyxin, sulfonamide, and multidrug antibiotic resistance (MAR) gene types were observed in the CCC-MF digesters. The findings suggest that applying CCC-MF may be associated with higher methane production and ammonium reduction, potentially linked to a more favorable synergistic methanogenic system and nitrogen transformation pathways.

Manure↗

PubMed2025

Unlocking microbial potential: advances in omics and bioinformatics for aromatic hydrocarbon degradation.

Aromatic hydrocarbons (AHs) are persistent environmental pollutants with high toxicity. Bacterial degradation of AHs provides a sustainable and cost-effective approach for the remediation of sites contaminated with both mono- and polycyclic aromatic hydrocarbons. Aerobic degradation of AHs typically involves oxygenases-mediated hydroxylation followed by aromatic ring cleavage. In contrast, anaerobic degradation relies on diverse activation mechanisms that ultimately converge on the central intermediate benzoyl-CoA. Over the past decades, research on bacterial degradation of AHs has grown steadily, supported by advances in omics and bioinformatics. In this review, we summarize the current knowledge on the pathways, enzymes, and microbial diversity involved in AH degradation, highlighting how omics and bioinformatic approaches are advancing our understanding of this process. However, to improve our knowledge of microbial AHs catabolism, it is crucial to prioritize the characterization of novel enzymes and pathways, especially those mediating anaerobic and hybrid degradation strategies. Addressing this gap requires the development of specialized resources that incorporate a broader taxonomic diversity and an expanded inventory of anaerobic genes and enzymes supported by experimental evidence. Equally important is the integration of multi-omics technologies, artificial intelligence, and ecological modeling into unified analytical pipelines. These efforts will be key to fully unlocking microbial metabolic potential and guiding more effective bioremediation and monitoring strategies for AHs.

Biodegradation, Environmental↗

PubMed1997

Co-metabolism and microbial growth in the biodegradation of alkylbenzenesulphonates.

Two organisms, CCMI507 and CCMI852, degrading undecylbenzenesulphonate (LAS) by the ortho- and meta-cleavage pathways were studied in cultures where glucose was used as carbon and energy source. CCMI507 (ortho-pathway) started the degradation of LAS at the beginning of the culture development in parallel with glucose utilization. The degradation followed a steady profile of degradation until 77% of LAS was degraded in the culture containing initially 5 mg l-1 of the compound and 81% in the cultures containing initially 10 and 20 mg l-1 of LAS, after 72 h fermentation. The organism CCMI852 (meta-pathway) started degrading the compound only after 20 h, when 75% of glucose was spent and well within the stationary-phase. After 72 h fermentation the level of degradation by CCMI852 varied from 70% (5 mg l-1 of LAS) to around 75% (10 and 20 mg l-1 of LAS).

Benzenesulfonates↗

PubMed2026

Multi-omics revealed the effects of rumen to blood path on early lactation performance in transition dairy cows.

BACKGROUND: The transition period is vitally important to the life cycle of dairy cows. However, the function of the microbiota during both pre- and post-partum and their relationship with ruminal, plasma, and milk metabolites still require systematic investigation. To address this, the 7 highest- and 7 lowest-performing animals among a cohort of 100 dairy cows were selected based on their postpartum energy-corrected milk yield. Rumen fluid and plasma samples were collected during both pre- and post-partum periods, whereas milk samples were obtained postpartum. Shotgun metagenomics of rumen contents in addition to metabolomics of rumen, plasma, and milk samples were performed to evaluate the associations between ruminal microbes and early lactation performance in transition dairy cows. RESULTS: Compared with prepartum cows, postpartum high-yield cows had greater concentrations of ruminal volatile fatty acids and plasma total bile acid. Moreover, plasma urea nitrogen and most amino acids, peptides, and their derivatives in plasma and milk were increased in postpartum high-yield cows, relative to postpartum low-yield cows. Metagenomic analysis revealed that the relative abundances of several species within the Prevotella, Succinimonas, Succinatimonas, and Methanosphaera increased, while other bacteria belong to Alistipes and Bacteroides, and archaeal Methanobrevibacter species decreased in postpartum cows, particularly in postpartum high-yield cows. Co-occurrence network and correlation analysis suggested that Prevotella and Succinatimonas were negatively correlated to Alistipes, Bacteroides, and Methanobrevibacter, potentially contributing to the nutritionally efficient phenotype of postpartum high-yield cows. A metabolic pathway analysis of our metagenomic data revealed that postpartum high-yield cows possessed more microbial genes involved in starch utilization and amino acid synthesis, while a wide range of microbial genes involved in cellulose utilization, acetogenesis, and amino acid degradation were found in prepartum cows with low-yield in postpartum. A structural equation model analysis showed that the increased relative abundances of Prevotella tf.2-5 and Succinatimonas CAG_777 were related to greater concentrations of plasma chenodeoxycholic acid glycine conjugate, milk 5-Methoxytryptophan, and energy-corrected milk yield. Finally, pan-genomic analysis confirmed that Alistipes, Bacteroides, and Methanobrevibacter possess genetic conservation of both hydrogenases and dehydrogenases, which may contribute to energy loss in the rumen via hydrogen dissipation. CONCLUSION: In summary, our findings provide a fundamental understanding of how microbiome-dependent mechanisms contribute to early lactation performance in dairy cows during the transition period. The increased abundance of Prevotella, Succinimonas, and Succinatimonas in postpartum cows suggest that they are important microbes during the transition period and may help in coping with metabolic challenges, while improving nutrient utilization efficiency during this period. Our study underscores the importance of the ruminal microbiome during the transition period and highlights the need for rumen-based nutritional intervention strategies to improve production efficiency in ruminants. Video Abstract.

Animals↗

PubMed2026

Effects of Fecal Microbiota Transplantation on Intestinal Microbial Characteristics and Clinical Phenotypes in Patients with Parkinson's Disease.

Alterations in the gut microbiota have been associated with Parkinson's disease (PD), but longitudinal microbial changes after fecal microbiota transplantation (FMT) and their clinical associations remain poorly understood. This single-center retrospective observational study included 6 patients with PD, stratified into high- and low-severity subgroups based on disease duration (>6 years vs ≤6 years). Thirty-six fecal samples were collected before FMT and monthly for five months afterward. Microbial diversity, community structure, taxonomic composition, and predicted functional profiles were assessed using 16S ribosomal RNA gene sequencing. Analyses included alpha and beta diversity, taxonomic abundance, linear discriminant analysis effect size, Tax4Fun2-based functional prediction, and Spearman rank correlations between microbial features and clinical indicators. Descriptive analyses indicated differences in microbial richness, diversity, community structure, and predicted functions between severity subgroups and across post-FMT time points. At baseline, the low-severity subgroup had greater microbial richness and diversity than the high-severity subgroup, with relatively higher abundances of taxa including Bifidobacterium and Lactobacillus. One month after FMT, richness and diversity increased from baseline in the high-severity subgroup, accompanied by changes in taxonomic composition. Both subgroups showed time-associated variation in microbial diversity and predicted Kyoto Encyclopedia of Genes and Genomes pathway enrichment after FMT. Predicted functions included carbohydrate and amino acid metabolism, secondary metabolite biosynthesis, membrane transport, and signal transduction. Several operational taxonomic units correlated with indicators of motor impairment, constipation, sleep quality, functional status, and neuropsychiatric symptoms. FMT was therefore associated with longitudinal changes in gut microbial diversity, composition, and predicted functions, and specific microbial features were associated with motor and non-motor indicators. Given the small retrospective cohort, these findings are preliminary and warrant confirmation in larger controlled studies. Future studies should determine whether these microbial alterations are reproducible, persist beyond five months, reflect donor engraftment, and correspond to measurable clinical improvement after transplantation in PD.

Humans↗

PubMed2006

2,4-Dichlorophenoxyacetic acid (2,4-D) biodegradation in river sediments of Northeast-Scotland and its effect on the microbial communities (PLFA and DGGE).

A bench-scale study was conducted to investigate 2,4-D biodegradation rates at different concentrations (10, 100 and 1000 microg per gram of dry weight) in distinct sediments samples collected on the River Ythan, Northeast-Scotland. Mineralisation of 14C 2,4-D occurred mostly within 30 days for all tested concentrations with a degradation rate ranging from 5 to 750 microg d(-1). Biodegradation rates were affected by the biological and biochemical characteristics of the indigenous microbial community in the studied sediments rather than factors such as compound bioavailability and/or toxicity. PLFA-profiling provided evidences of the effect of 2,4-D amendments on the microbial communities and DGGE-profiling showed changes in the genetic potential of the microbial populations which might affect metabolic characteristics of the sediment. PLFAs biomarkers suggested that the pathway of alpha-ketoglutarate-dependent dioxygenase was the main route of 2,4-D biodegradation. This pathway is commonly found in microorganisms of the beta-subdivision of proteobacteria.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2000

Engineering biosynthetic pathways: new routes to chiral amino acids.

The engineering of microbial biosynthetic pathways is advancing rapidly because of new molecular genetic approaches, sophisticated analysis of metabolic flux and the rapid sequencing of diverse bacterial genomes. The classical methods of mutagenesis/selection, originally applied in the development of amino-acid-over-producing bacteria are now being complemented by an increasingly rational strategy.

Amino Acids↗

PubMed2001

New experimental and theoretical tools for metabolic engineering of micro-organisms.

Presently an increasing gap is developing between our experimental capabilities in metabolic engineering of microbial metabolism and our quantitative theoretical understanding of the kinetic interaction between primary metabolism and product pathways. Such theoretical understanding is absolutely needed for a rational design of said metabolic engineering experiments and targets. To obtain such understanding in-vivo kinetic experiments and in-vivo kinetic models are needed. To this end the following new methods for in-vivo kinetic studies of Saccharomyces cerevisiae have recently been developed. The Bioscope device allows the reliable and repeated perturbation (e.g. glucose pulse, or inhibitor etc.) of steady state biomass outside the fermentor and subsequent sampling and quenching to measure glycolytical intermediates and nucleotides in a time frame of 0-70 seconds. Dynamic modelling of fermentor off-gas O2/CO2-measurements allows to calculate O2 uptake and CO2 production rates in such a perturbation experiment (0-70 seconds time windows). A new LC-MSMS based method has been developed to measure large sets of intracellular metabolites in said in-vivo kinetic experiments. It has been shown for the first time that in long chemostat cultivation (up to 800 hrs.) intracellular metabolites levels drop, showing absence of a real steady state. A new kinetic format, lin log kinetics, has been developed for describing the intracellular kinetic behavior of metabolic networks. This format allows general analytical solutions of networks flux, metabolic levels. From simulation studies it appears that this approach is remarkable accurate in describing intracellular metabolite dynamics and in metabolic design questions of where to change enzyme levels and how much to achieve a desired change in fluxes and metabolite levels. At this moment these tools are being applied in metabolic engineering studies of Saccharomyces cerevisiae and Penicillin chrysogenum.

Adenosine Triphosphate↗