Bottleneck Enzymes Research Articles

Overexpression of BAPT and DBTNBT genes in Taxus baccatain vitro cultures to enhance the biotechnological production of paclitaxel.

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated-β-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.

De novo biosynthesis of berberine and halogenated benzylisoquinoline alkaloids in Saccharomyces cerevisiae

Berberine is an extensively used pharmaceutical benzylisoquinoline alkaloid (BIA) derived from plants. Microbial manufacturing has emerged as a promising approach to source valuable BIAs. Here, we demonstrated the complete biosynthesis of berberine in Saccharomyces cerevisiae by engineering 19 genes including 12 heterologous genes from plants and bacteria. Overexpressing bottleneck enzymes, fermentation scale-up, and heating treatment after fermentation increased berberine titer by 643-fold to 1.08 mg L-1. This pathway also showed high efficiency to incorporate halogenated tyrosine for the synthesis of unnatural BIA derivatives that have higher therapeutical potentials. We firstly demonstrate the in vivo biosynthesis of 11-fluoro-tetrahydrocolumbamine via nine enzymatic reactions. The efficiency and promiscuity of our pathway also allow for the simultaneous incorporation of two fluorine-substituted tyrosine derivatives to 8, 3’-di-fluoro-coclaurine. This work highlights the potential of yeast as a versatile microbial biosynthetic platform to strengthen current pharmaceutical supply chain and to advance drug development.

Bioproduction of Linalool From Paper Mill Waste.

A key challenge in chemicals biomanufacturing is the maintenance of stable, highly productive microbial strains to enable cost-effective fermentation at scale. A “cookie-cutter” approach to microbial engineering is often used to optimize host stability and productivity. This can involve identifying potential limitations in strain characteristics followed by attempts to systematically optimize production strains by targeted engineering. Such targeted approaches however do not always lead to the desired traits. Here, we demonstrate both ‘hit and miss’ outcomes of targeted approaches in attempts to generate a stable Escherichia coli strain for the bioproduction of the monoterpenoid linalool, a fragrance molecule of industrial interest. First, we stabilized linalool production strains by eliminating repetitive sequences responsible for excision of pathway components in plasmid constructs that encode the pathway for linalool production. These optimized pathway constructs were then integrated within the genome of E. coli in three parts to eliminate a need for antibiotics to maintain linalool production. Additional strategies were also employed including: reduction in cytotoxicity of linalool by adaptive laboratory evolution and modification or homologous gene replacement of key bottleneck enzymes GPPS/LinS. Our study highlights that a major factor influencing linalool titres in E. coli is the stability of the genetic construct against excision or similar recombination events. Other factors, such as decreasing linalool cytotoxicity and changing pathway genes, did not lead to improvements in the stability or titres obtained. With the objective of reducing fermentation costs at scale, the use of minimal base medium containing paper mill wastewater secondary paper fiber as sole carbon source was also investigated. This involved simultaneous saccharification and fermentation using either supplemental cellulase blends or by co-expressing secretable cellulases in E. coli containing the stabilized linalool production pathway. Combined, this study has demonstrated a stable method for linalool production using an abundant and low-cost feedstock and improved production strains, providing an important proof-of-concept for chemicals production from paper mill waste streams. For scaled production, optimization will be required, using more holistic approaches that involve further rounds of microbial engineering and fermentation process development.

Co\u2011cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling.

Metabolic engineering of Synechocystis sp. PCC 6803 for improved bisabolene production

Terpenoids are a wide class of organic compounds with industrial relevance. The natural ability of cyanobacteria to produce terpenoids via the methylerythritol 4-phosphate (MEP) pathway makes these organisms appealing candidates for the generation of light-driven cell factories for green chemistry. Here we address the improvement of the production of (E)-α-bisabolene, a valuable biofuel feedstock, in Synechocystis sp. PCC 6803 via sequential heterologous expression of bottleneck enzymes of the native pathway. Expression of the bisabolene synthase is sufficient to complete the biosynthetic pathway of bisabolene. Expression of a farnesyl-pyrophosphate synthase from Escherichia coli did not influence production of bisabolene, while enhancement of the MEP pathway via additional overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and IPP/DMAPP isomerase (IDI) significantly increased production per cell. However, in the absence of a carbon sink, the overexpression of DXS and IDI leads to significant growth impairment. The final engineered strain reached a volumetric titre of 9 ​mg ​L−1 culture of bisabolene after growing for 12 days. When the cultures were grown in a high cell density (HCD) system, we observed an increase in the volumetric titres by one order of magnitude for all producing-strains. The strain with improved MEP pathway presented an increase twice as much as the remaining engineered strains, yielding more than 180 ​mg ​L−1 culture after 10 days of cultivation. Furthermore, the overexpression of these two MEP enzymes prevented the previously reported decrease in the bisabolene specific titres when grown in HCD conditions, where primary metabolism is usually favoured. We conclude that fine-tuning of the cyanobacterial terpenoid pathway is crucial for the generation of microbial platforms for terpenoid production on industrial-scale.

Improvement in D-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering.

As the effects of climate change become apparent, metabolic engineers and synthetic biologists are exploring sustainable sources for transportation fuels. The design and engineering of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce our dependence on fossil fuels as well as lower the emissions of greenhouse gases. Over the past 2 decades, a considerable amount of work has led to the development of microbial strains for the production of advanced fuel compounds from both C5 and C6 sugars. In this work, we combined two strategies-adaptive laboratory evolution and rational metabolic engineering-to improve the yeast Saccharomyces cerevisiae's ability to utilize D-xylose, a major C5 sugar in biomass, and produce the advanced biofuel isobutanol. Whole genome resequencing of several evolved strains followed by reverse engineering identified two single nucleotide mutations, one in CCR4 and another in TIF1, that improved the yeast's specific growth rate by 23% and 14%, respectively. Neither one of these genes has previously been implicated to play a role in utilization of D-xylose. Fine-tuning the expression levels of the bottleneck enzymes in the isobutanol pathway further improved the evolved strain's isobutanol titer to 92.9 ± 4.4mg/L (specific isobutanol production of 50.2 ± 2.6mg/g DCW), a 90% improvement in titer and a 110% improvement in specific production over the non-evolved strain. We hope that our work will set the stage for an economic route to the advanced biofuel isobutanol and enable efficient utilization of xylose-containing biomass.

CaMeRe: A Novel Tool for Inference of Cancer Metabolic Reprogramming.

Metabolic reprogramming is prevalent in cancer, largely due to its altered chemical environments such as the distinct intracellular concentrations of O2, H2O2 and H+, compared to those in normal tissue cells. The reprogrammed metabolisms are believed to play essential roles in cancer formation and progression. However, it is highly challenging to elucidate how individual normal metabolisms are altered in a cancer-promoting environment; hence for many metabolisms, our knowledge about how they are changed is limited. We present a novel method, CaMeRe (CAncer MEtabolic REprogramming), for identifying metabolic pathways in cancer tissues. Based on the specified starting and ending compounds, along with gene expression data of given cancer tissue samples, CaMeRe identifies metabolic pathways connecting the two compounds via collection of compatible enzymes, which are most consistent with the provided gene-expression data. In addition, cancer-specific knowledge, such as the expression level of bottleneck enzymes in the pathways, is incorporated into the search process, to enable accurate inference of cancer-specific metabolic pathways. We have applied this tool to predict the altered sugar-energy metabolism in cancer, referred to as the Warburg effect, and found the prediction result is highly accurate by checking the appearance and ranking of those key pathways in the results of CaMeRe. Computational evaluation indicates that the tool is fast and capable of handling large metabolic network inference in cancer tissues. Hence, we believe that CaMeRe offers a powerful tool to cancer researchers for their discovery of reprogrammed metabolisms in cancer. The URL of CaMeRe is http://csbl.bmb.uga.edu/CaMeRe/.

Systems metabolic engineering of Corynebacterium glutamicum for the bioproduction of biliverdin via protoporphyrin independent pathway

BackgroundBiliverdin, a prospective recyclable antioxidant and one of the most important precursors for optogenetics, has received growing attention. Biliverdin is currently produced by oxidation of bilirubin from mammalian bile using chemicals. However, unsustainable procedures of extraction, chemical oxidation, and isomer separation have prompted bio-based production using a microbial cell factory.ResultsIn vitro thermodynamic analysis was performed to show potential candidates of bottleneck enzymes in the pathway to produce biliverdin. Among the candidates, hemA and hemL were overexpressed in Corynebacterium glutamicum to produce heme, precursor of biliverdin. To increase precursor supply, we suggested a novel hemQ-mediated coproporphyrin dependent pathway rather than noted hemN-mediated protoporphyrin dependent pathway in C. glutamicum. After securing precursors, hmuO was overexpressed to pull the carbon flow to produce biliverdin. Through modular optimization using gene rearrangements of hemA, hemL, hemQ, and hmuO, engineered C. glutamicum BV004 produced 11.38 ± 0.47 mg/L of biliverdin at flask scale. Fed-batch fermentations performed in 5 L bioreactor with minimal medium using glucose as a sole carbon source resulted in the accumulation of 68.74 ± 4.97 mg/L of biliverdin, the highest titer to date to the best of our knowledge.ConclusionsWe developed an eco-friendly microbial cell factory to produce biliverdin using C. glutamicum as a biosystem. Moreover, we suggested that C. glutamicum has the thermodynamically favorable coproporphyrin dependent pathway. This study indicated that C. glutamicum can work as a powerful platform to produce biliverdin as well as heme-related products based on the rational design with in vitro thermodynamic analysis.

Metabolic engineering of Pseudomonas taiwanensis VLB120 with minimal genomic modifications for high-yield phenol production

Aromatic chemicals are important building blocks for the production of a multitude of everyday commodities. Currently, aromatics production relies almost exclusively on petrochemical processes. To achieve sustainability, alternative synthesis methods need to be developed. Here, we strived for an efficient production of phenol, a model aromatic compound of industrial relevance, from renewable carbon sources using the solvent-tolerant biocatalyst Pseudomonas taiwanensis VLB120. First, multiple catabolic routes for the degradation of aromatics and related compounds were inactivated, thereby obtaining the chassis strain P. taiwanensis VLB120Δ5 incapable of growing on 4-hydroxybenzoate (ΔpobA), tyrosine (Δhpd), and quinate (ΔquiC, ΔquiC1, ΔquiC2). In this context, a novel gene contributing to the quinate catabolism was identified (quiC2). Second, we employed a combination of reverse- and forward engineering to increase metabolic flux towards the product, using leads obtained from the analysis of aromatics producing Pseudomonas putida strains previously generated by mutagenesis. Phenol production was enabled by the heterologous expression of a codon-optimized and chromosomally integrated tyrosine phenol-lyase encoding gene from Pantoea agglomerans AJ2985 (PaTPL2). The genomic modification of endogenous genes encoding TrpEP290S, AroF-1P148L, and PheAT310I, and the deletion of pykA improved phenol production 17-fold, while also minimizing the burden caused by plasmids and auxotrophies. The additional overexpression of known bottleneck enzymes (AroGfbr, TyrAfbr) derived from Escherichia coli further enhanced phenol titers. The best producing strain P. taiwanensis VLB120Δ5-TPL36 reached yields of 15.8% and 18.5% (Cmol/Cmol) phenol from glucose and glycerol, respectively, in a mineral medium without addition of complex nutrients. This is the highest yield ever reported for microbially produced phenol.

Metabolic engineering of Pichia pastoris for production of isobutanol and isobutyl acetate

BackgroundInterests in renewable fuels have exploded in recent years as the serious effects of global climate change become apparent. Microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered Pichia pastoris, an industrial workhorse in heterologous enzyme production, to produce the biofuel isobutanol from two renewable carbon sources, glucose and glycerol. Our strategy exploited the yeast’s amino acid biosynthetic pathway and diverted the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production. To further demonstrate the versatility of our yeast platform, we incorporated a broad-substrate-range alcohol-O-acyltransferase to generate a variety of volatile esters, including isobutyl acetate ester and isopentyl acetate ester.ResultsThe engineered strain overexpressing the keto-acid degradation pathway was able to produce 284 mg/L of isobutanol when supplemented with 2-ketoisovalerate. To improve the production of isobutanol and eliminate the need to supplement the production media with the expensive 2-ketoisovalerate intermediate, we overexpressed a portion of the amino acid l-valine biosynthetic pathway in the engineered strain. While heterologous expression of the pathway genes from the yeast Saccharomyces cerevisiae did not lead to improvement in isobutanol production in the engineered P. pastoris, overexpression of the endogenous l-valine biosynthetic pathway genes led to a strain that is able to produce 0.89 g/L of isobutanol. Fine-tuning the expression of bottleneck enzymes by employing an episomal plasmid-based expression system further improved the production titer of isobutanol to 2.22 g/L, a 43-fold improvement from the levels observed in the original strain. Finally, heterologous expression of a broad-substrate-range alcohol-O-acyltransferase led to the production of isobutyl acetate ester and isopentyl acetate ester at 51 and 24 mg/L, respectively.ConclusionsIn this study, we engineered high-level production of the biofuel isobutanol and the corresponding acetate ester by P. pastoris from readily available carbon sources. We envision that our work will provide an economic route to this important class of compounds and establish P. pastoris as a versatile production platform for fuels and chemicals.

Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10 production in Rhodobacter sphaeroides

In this report, UbiE and UbiH in the quinone modification pathway (QMP) were identified in addition to UbiG as bottleneck enzymes in the CoQ10 biosynthesis by Rhodobacter sphaeroides. The CoQ10 content was enhanced after co-overexpression of UbiE and UbiG, however, accompanied by the accumulation of the intermediate 10P-MMBQ. UbiH was then co-overexpressed to pull the metabolic flux towards downstream, resulting in an elevated CoQ10 productivity and decreased biomass. On the other hand, the expression levels of UbiE and UbiG were tuned to eliminate the intermediate accumulation, however at the sacrifice of productivity. To alleviate the detrimental effect on either productivity or cell growth, we tried to fuse UbiG with UbiE and localize them onto the membrane to elevate intermediate conversion. By fusing UbiE and UbiG to pufX, CoQ10 was accumulated to 108.51±2.76mg/L with a biomass of 12.2±0.9g/L. At last, we combined the optimized QMP and the previously engineered 2-methyl-d-erythritol-4-phosphate pathway (MEP) to further boost CoQ10 biosynthesis, resulting in a strain with 138±2.64mg/L CoQ10 production.

Characterization of the human AGE1.HN cell line: a systems biology approach

Background With the emergence of functional genomics approaches in the past decade, powerful tools for the discovery and understanding of cellular mechanisms are accessible. The main purpose of those analyses is to investigate the cell on the level of the transcriptome, proteome and metabolome to retrieve information on possible limitations for growth or productivity. In subsequent steps, these limitations could be addressed by genetic modifications (e.g. overexpression of genes coding for bottle neck enzymes) or supplementation of the media. In this work, we present a systems biology approach for the analysis of the human protein expression cell line AGE1.hn. The focal point of the analyses was on the central energy metabolism, namely glycolysis, TCA and oxidative phosphorylation. In addition to the metabolome analysis transcriptome and proteome data were obtained. Combining the above mentioned techniques we could gather valuable insights in the cellular processes of a human protein production cell line.

Selection of Best Indicators for Ranking and Determination of Bottleneck Enzymes in Metabolic Reaction Systems

Dynamic logarithmic gain and its modifications can be theoretically used as a bottleneck ranking (BR) indicator in a metabolic reaction system. However, it is not sufficiently explicit whether they can be successfully used in practical applications. The present work therefore focuses on the selection of best BR indicators in both instantaneous and overall cases. A modified ethanol fermentation model is used as a case study. The results indicate that the mathematical product of dynamic logarithmic gain and desired product concentration at a given time is the best instantaneous BR indicator, whereas its value at the end time of the fermentation process is the best overall BR indicator. The former is useful for observing the time course of the desired product concentration and determining a process time to be terminated. The latter is for ranking bottleneck enzymes to determine which enzyme activity should be changed to attain a higher desired product concentration. Moreover, discussion is made on the utilization of the overall BR indicators to predict how much the final desired product concentration is increased.

Identification of bottleneck enzymes with negative dynamic sensitivities: Ethanol fermentation systems as case studies

Dynamic logarithmic gains (normalised time-varying sensitivities) or their products with metabolite concentrations, called an instantaneous bottleneck ranking indicator (instantaneous BR indicator), can express the time courses of bottleneck enzyme ranking. The integrated value of the instantaneous BR indicator, called an overall BR indicator, provides a ranking of bottleneck enzymes over the entire reaction period of time. Owing to the property of the logarithmic gains, these BR indicators are allowed to include both positive and negative values and in turn raise several questions on the identification of a bottleneck enzyme. To eliminate these questions, the present work studies the identification of bottleneck enzymes in four ethanol fermentation systems using suspended and immobilised cells at pH 4.5 and 5.5 as case studies. These systems consist of both intracellular reactions without cell growth (intracellular reaction systems) and those with cell growth (cell growth systems). The analyses of the intracellular reaction systems reveal that both the suspended and immobilised cell systems at pH 5.5 are not robust. In the cell growth systems, therefore, the analyses are conducted only for the suspended and immobilised cell models at pH 4.5. The enzyme glyceraldehyde 3-phosphate dehydrogenase (gapd) initially has the largest logarithmic gains under all conditions, whereas the system becomes insensitive to it over time. Interestingly, the instantaneous BR indicator for gapd is negatively large. Also, other indicators change in positive and/or negative regions. To deal with these values of different signs, all the indicators are integrated to obtain and rank the overall BR indicators in the order of the magnitudes of their absolute values. The result shows that the ranking of enzymes are completely identical to that of the final ethanol concentrations obtained by finitely increasing or decreasing the relevant enzyme activities in accordance with the signs of overall BR indicators. This result indicates that the enzymes must be ranked in the order of the magnitudes of their absolute values to determine a bottleneck enzyme. As a result, the analyses of the intracellular reaction systems reveal that the bottleneck enzyme is switched from a membrane-bound glucose transporter enzyme to phosphofructokinase when the cell-retaining condition is changed from suspension to immobilisation. In the cell growth systems, gapd is identified as the most likely bottleneck enzyme, suggesting that the gapd activity must be decreased to increase the ethanol productivity.

Genes encoding hub and bottleneck enzymes of the Arabidopsis metabolic network preferentially retain homeologs through whole genome duplication

BackgroundWhole genome duplication (WGD) occurs widely in angiosperm evolution. It raises the intriguing question of how interacting networks of genes cope with this dramatic evolutionary event.ResultsIn study of the Arabidopsis metabolic network, we assigned each enzyme (node) with topological centralities (in-degree, out-degree and between-ness) to measure quantitatively their centralities in the network. The Arabidopsis metabolic network is highly modular and separated into 11 interconnected modules, which correspond well to the functional metabolic pathways. The enzymes with higher in-out degree and between-ness (defined as hub and bottleneck enzymes, respectively) tend to be more conserved and preferentially retain homeologs after WGD. Moreover, the simultaneous retention of homeologs encoding enzymes which catalyze consecutive steps in a pathway is highly favored and easily achieved, and enzyme-enzyme interactions contribute to the retention of one-third of WGD enzymes.ConclusionsOur analyses indicate that the hub and bottleneck enzymes of metabolic network obtain great benefits from WGD, and this event grants clear evolutionary advantages in adaptation to different environments.

Time-resolved metabolomics reveals metabolic modulation in rice foliage

BackgroundTo elucidate the interaction of dynamics among modules that constitute biological systems, comprehensive datasets obtained from "omics" technologies have been used. In recent plant metabolomics approaches, the reconstruction of metabolic correlation networks has been attempted using statistical techniques. However, the results were unsatisfactory and effective data-mining techniques that apply appropriate comprehensive datasets are needed.ResultsUsing capillary electrophoresis mass spectrometry (CE-MS) and capillary electrophoresis diode-array detection (CE-DAD), we analyzed the dynamic changes in the level of 56 basic metabolites in plant foliage (Oryza sativa L. ssp. japonica) at hourly intervals over a 24-hr period. Unsupervised clustering of comprehensive metabolic profiles using Kohonen's self-organizing map (SOM) allowed classification of the biochemical pathways activated by the light and dark cycle. The carbon and nitrogen (C/N) metabolism in both periods was also visualized as a phenotypic linkage map that connects network modules on the basis of traditional metabolic pathways rather than pairwise correlations among metabolites. The regulatory networks of C/N assimilation/dissimilation at each time point were consistent with previous works on plant metabolism. In response to environmental stress, glutathione and spermidine fluctuated synchronously with their regulatory targets. Adenine nucleosides and nicotinamide coenzymes were regulated by phosphorylation and dephosphorylation. We also demonstrated that SOM analysis was applicable to the estimation of unidentifiable metabolites in metabolome analysis. Hierarchical clustering of a correlation coefficient matrix could help identify the bottleneck enzymes that regulate metabolic networks.ConclusionOur results showed that our SOM analysis with appropriate metabolic time-courses effectively revealed the synchronous dynamics among metabolic modules and elucidated the underlying biochemical functions. The application of discrimination of unidentified metabolites and the identification of bottleneck enzymatic steps even to non-targeted comprehensive analysis promise to facilitate an understanding of large-scale interactions among components in biological systems.

Regulation of ammonia assimilation in ammonia-limited chemostat cultures of Escherichia coli ML 30: evidence of bistability.

In a preceding paper evidence of two stationary stable states (bistability) in the specific activity of glutamine synthetase (GS) in ammonia-limited steady-state cultures of Escherichia coli ML 30 at dilution rates (D) about 0.15 h-1 was described (Müller et al. 1977). For better understanding of the regulation mechanisms leading to GS bistability chemostat experiments were performed over a wide range of dilution rates up to D = 0.8 h-1. For each steady state the specific activities of GS and glutamate dehydrogenase (GDH)--the other key enzyme of the two NH3 assimilation routes in E. coli--and in addition the remaining NH3 concentration in the culture liquid were determined. Parallel to GS bistability two states of GDH activity and NH3 concentration are found. The higher state of GS is connected with a lower GDH activity and NH3 concentration. With rising D the GS activities decrease whereas GDH activities and NH3 concentrations increase. Since no adenylation of the GS is detectable GS bistability seems to be regulated on the level of enzyme synthesis like GDH bistability. From the experimental findings a mathematical model is derived based on the bottle neck enzyme theory of growth. It describes the dependence between the specific growth rates on the one hand and the specific enzyme activities and NH3 concentration on the other. It is shown that the specific uptake rate of the limiting NH3 and the specific growth rates, respectively, depend on the simultaneous action of two bottle neck enzymes which are connected by a regulative link.