Aromatic Amino Acid Pathway Research Articles

Engineering

The production of aromatic amino acids using fermentation processes with recombinant microorganisms can be an advantageous approach to reach their global demands. In addition, a large array of compounds with alimentary and pharmaceutical applications can potentially be synthesized from intermediates of this metabolic pathway. However, contrary to other amino acids and primary metabolites, the artificial channelling of building blocks from central metabolism towards the aromatic amino acid pathway is complicated to achieve in an efficient manner. The length and complex regulation of this pathway have progressively called for the employment of more integral approaches, promoting the merge of complementary tools and techniques in order to surpass metabolic and regulatory bottlenecks. As a result, relevant insights on the subject have been obtained during the last years, especially with genetically modified strains of Escherichia coli. By combining metabolic engineering strategies with developments in synthetic biology, systems biology and bioprocess engineering, notable advances were achieved regarding the generation, characterization and optimization of E. coli strains for the overproduction of aromatic amino acids, some of their precursors and related compounds. In this paper we review and compare recent successful reports dealing with the modification of metabolic traits to attain these objectives.

Constitutive expression of selected genes from the pentose phosphate and aromatic pathways increases the shikimic acid yield in high-glucose batch cultures of an Escherichia coli strain lacking PTS and pykF

BackgroundDuring the last two decades many efforts have been directed towards obtaining efficient microbial processes for the production of shikimic acid (SA); however, feeding high amounts of substrate to increase the titer of this compound has invariably rendered low conversion yields, leaving room for improvement of the producing strains. In this work we report an alternative platform to overproduce SA in a laboratory-evolved Escherichia coli strain, based on plasmid-driven constitutive expression of six genes selected from the pentose phosphate and aromatic amino acid pathways, artificially arranged as an operon. Production strains also carried inactivated genes coding for phosphotransferase system components (ptsHIcrr), shikimate kinases I and II (aroK and aroL), pyruvate kinase I (pykF) and the lactose operon repressor (lacI).ResultsThe strong and constitutive expression of the constructed operon permitted SA production from the beginning of the cultures, as evidenced in 1 L batch-mode fermentors starting with high concentrations of glucose and yeast extract. Inactivation of the pykF gene improved SA production under the evaluated conditions by increasing the titer, yield and productivity of this metabolite compared to the isogenic pykF+ strain. The best producing strain accumulated up to 43 g/L of SA in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected, with SA accounting for 80% of the produced aromatic compounds. These results were consistent with high expression levels of the glycolytic pathway and synthetic operon genes from the beginning of fermentations, as revealed by transcriptomic analysis. Despite the consumption of 100 g/L of glucose, the yields on glucose of SA and of total aromatic compounds were about 50% and 60% of the theoretical maximum, respectively. The obtained yields and specific production and consumption rates proved to be constant with three different substrate concentrations.ConclusionsThe developed production system allowed continuous SA accumulation until glucose exhaustion and eliminated the requirement for culture inducers. The obtained SA titers and yields represent the highest reported values for a high-substrate batch process, postulating the strategy described in this report as an interesting alternative to the traditionally employed fed-batch processes for SA production.

Simultaneous determination of multiple amino acids in plasma in critical illness by high performance liquid chromatography with ultraviolet and fluorescence detection

There is increasing recognition that the host response to critical illness includes derangement of multiple amino acid pathways, including amino acids (AAs) central to metabolism and immune, endothelial and neurological function. To characterise concentration changes of these plasma amino acid we report the development and validation of a method for the quantification of AAs in small volumes of plasma (50μL) using HPLC with simultaneous UV and fluorescence (FL) detection. Protein precipitation and pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is followed by reversed phase HPLC separation. Calibration curves were built with norleucine as an internal standard. Thirty-three (including the 20 proteinogenic) AAs, were selected as standards and their corresponding concentrations in the plasma of healthy human controls and patients with severe falciparum malaria were quantified. This method enables the detection of perturbations in arginine metabolism, aromatic amino acid pathways, methionine transsulfuration and transmethylation pathways and other metabolic pathways.

Transcriptomic analysis of UV-treated rice leaves reveals UV-induced phytoalexin biosynthetic pathways and their regulatory networks in rice

Rice produces diterpenoid and flavonoid phytoalexins for defense against pathogen attack. The production of phytoalexins in rice is also induced by UV-irradiation. To understand the metabolic networks involved in UV-induced phytoalexin biosynthesis and their regulation, phytochemical and transcriptomic analyses of UV-treated rice leaves were performed. In response to UV treatment, the accumulation of flavonoids was observed in rice leaves, which may serve as antioxidants against UV-induced oxidative stress. The phytochemical analysis confirmed sakuranetin accumulation and also demonstrated the induction of phenylamide synthesis in rice leaves by UV-irradiation. Transcriptomic analysis established that aromatic amino acid biosynthetic genes were immediately up-regulated after UV treatment. The genes involved in the phenylpropanoid pathway and flavonoid biosynthesis were also up-regulated. These findings suggest that the aromatic amino acid and flavonoid biosynthetic pathways are coordinately activated for the production of flavonoids and phenolic phytoalexins such as sakuranetin and phenylamides. An in silico analysis of UV-induced O-methyltransferase and acyltransferase genes suggested that these genes may be implicated in sakuranetin and phenylamide synthesis, respectively. The transcriptomic analysis also showed up-regulation of both methylerythritol phosphate pathway and the diterpenoid phytoalexin biosynthetic genes in response to UV treatment. A functional gene network analysis of phytoalexin biosynthetic and UV-induced genes for signaling components and transcription factors using RiceNet suggested that regulatory networks comprising signal perceiving receptor kinases, G-proteins, signal transducing mitogen-activated protein kinases and calcium signaling components, and transcription factors control flavonoid and phytoalexin biosynthesis in rice leaves under UV-C stress conditions.

Model-driven design of a Saccharomyces cerevisiae platform strain with improved tyrosine production capabilities

Saccharomyces cerevisiae, a eukaryotic model organism, is considered the ideal host for microbial production of plant secondary metabolites such as polyketides and alkaloids. However, industrial scale production of these valuable products using S. cerevisiae is limited by the availability of their precursor, aromatic amino acid tyrosine. Here, we describe a framework which uses a combination of computational modeling techniques to design an in silico metabolic engineering strategy that improves the flux through the aromatic amino acid pathway (shikimate pathway) in S. cerevisiae. The predicted yeast strain can be used as a platform strain for production of any heterologous products which require tyrosine, or any other aromatic amino acid pathway metabolites as precursors. The initial genome-scale strain design was performed using steady-state constraint-based modeling methods, Optknock and GDLS. The resulting design required deletion of multiple genes and was difficult to validate experimentally. In order to obtain an experimentally feasible design, a small-scale kinetic model was developed using Ensemble Modeling, and was used to prioritize the knockouts predicted by steady-state models for experimental validation.

Genome-wide transcriptional responses of Escherichia coli to glyphosate, a potent inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase

The shikimate pathway enzymes offer attractive targets for the development of antimetabolites. Glyphosate is an effective antimetabolite that inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the shikimate pathway, thereby resulting in a shortage of the chorismate-derived essential aromatic amino acids. However, little is known about the genome-wide transcriptional responses of bacteria to glyphosate shock. In the current study, a transcriptome analysis of Escherichia coli (E. coli) exposed to glyphosate identified the differential expression of 1040 genes, which represent 23.2% of the genome. The differentially expressed genes are primarily involved in amino acid metabolism, cell motility, and central carbon metabolism, indicating that the impact of glyphosate on the shikimate pathway also extends to other metabolic pathways. Expectedly, almost all genes encoding the proteins for the shikimate and specific aromatic amino acid pathways were downregulated after the addition of glyphosate. Furthermore, the expression of many energy- and metabolism-related genes was repressed. In contrast, glyphosate treatment induced the coordinated upregulation of at least 50 genes related to cell motility and chemotaxis. The reverse transcription-quantitative real-time PCR (RT-qPCR) data showed that the expression profiles of selected genes from the referred pathways were found to be consistent with the microarray data. The results suggest that the presence of glyphosate during growth induces metabolic starvation, an energy drain and other non-target effects.

Representation of probabilistic scientific knowledge

The theory of probability is widely used in biomedical research for data analysis and modelling. In previous work the probabilities of the research hypotheses have been recorded as experimental metadata. The ontology HELO is designed to support probabilistic reasoning, and provides semantic descriptors for reporting on research that involves operations with probabilities. HELO explicitly links research statements such as hypotheses, models, laws, conclusions, etc. to the associated probabilities of these statements being true. HELO enables the explicit semantic representation and accurate recording of probabilities in hypotheses, as well as the inference methods used to generate and update those hypotheses. We demonstrate the utility of HELO on three worked examples: changes in the probability of the hypothesis that sirtuins regulate human life span; changes in the probability of hypotheses about gene functions in the S. cerevisiae aromatic amino acid pathway; and the use of active learning in drug design (quantitative structure activity relation learning), where a strategy for the selection of compounds with the highest probability of improving on the best known compound was used. HELO is open source and available at https://github.com/larisa-soldatova/HELO

Three Sites and You Are Out: Ternary Synergistic Allostery Controls Aromatic Amino Acid Biosynthesis in Mycobacteriumtuberculosis

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, the pathway responsible for the biosynthesis of the aromatic amino acids Trp, Phe, and Tyr. Unlike many other organisms that produce up to three isozymes, each feedback-regulated by one of the aromatic amino acid pathway end products, Mycobacterium tuberculosis expresses a single DAH7PS enzyme that can be controlled by combinations of aromatic amino acids. This study shows that the synergistic inhibition of this enzyme by a combination of Trp and Phe can be significantly augmented by the addition of Tyr. We used X-ray crystallography, mutagenesis, and isothermal titration calorimetry studies to show that DAH7PS from M. tuberculosis possesses a Tyr-selective site in addition to the Trp and Phe sites, revealing an unusual and highly sophisticated network of three synergistic allosteric sites on one enzyme. This ternary inhibitory response, by a combination of all three aromatic amino acids, allows a tunable response of the protein to changing metabolic demands.

Comparison of initial photocatalytic degradation pathway of aromatic and linear amino acids

Amino acids are at the origin of the formation of odourous compounds after chlorination treatment. Our objectives were to test the efficiency of a photocatalytic process to remove these types of compounds. Five amino acids (AA) – phenylalanine, tyrosine, tryptophan, glycine and alanine – have been chosen as model molecules. The photocatalytic degradation has been investigated in aqueous solutions containing TiO2 suspensions as photocatalysts, in order to assess the adsorption, the disappearance rate and the mechanism of degradation. Results showed that only a small amount of amino acid is adsorbed corresponding to less than 1% of OH covered for aromatic amino acids and about 10% for linear amino acids. A comparison of disappearance rate, total organic carbon (TOC) removal and intermediate for these five amino acids are presented and discussed, taking into account their nature.

Metabolomics data exploration guided by prior knowledge

In metabolomics research, it is often important to focus the data analysis to specific areas of interest within the metabolome. In this paper, we describe the application of consensus principal component analysis (CPCA) and canonical correlation analysis (CCA) as a means to explore the relation between metabolome data and (i) biochemically related metabolites and (ii) an amino acid biosynthesis pathway. CPCA searches for major trends in the behavior of metabolite concentrations that are in common for the metabolites of interest and the remainder of the metabolome. CCA identifies the strongest correlations between the metabolites of interest and the remainder of the metabolome. CPCA and CCA were applied to two different microbial metabolomics data sets. The first data set, derived from Pseudomonas putida S12, was relatively simple as it contained metabolomes obtained under four environmental conditions only. The second data set, obtained from Escherichia coli, was much more complex as it consisted of metabolomes obtained under 28 different environmental conditions. In case of the simple and coherent P. putida S12 data set, CCA and CPCA gave similar results as the variation in the subset of the selected metabolites and the remainder of the metabolome was similar. In contrast, CCA and CPCA yielded different results in case of the E. coli data set. With CPCA the trends in the selected subset – the phenylalanine biosynthesis pathway – dominated the results. The main trends were related to high and low phenylalanine productivity, and the metabolites showing a similar behavior in concentration were metabolites regulating the phenylalanine biosynthesis route in the subset and metabolites related to general amino acid metabolism in the remainder of the metabolome. With CCA, neither subset truly dominated the data analysis. CCA described the differences between the wild type and the overproducing strain and the differences between the succinate and glucose grown cells. For the difference between the wild type and the overproducing strain, metabolites from the beginning and the end of aromatic amino acid pathways like erythrose-4-phosphate, tryptophan, and phenylalanine were important for the selected metabolites. CCA and CPCA proved to be complementary data analysis tools that enable the focusing of the data analysis on groups of metabolites that are of specific interest in relation to the remainder of the metabolome. Compared to an ordinary PCA, focusing the data analysis on biologically relevant metabolites lead especially for the complex E. coli data to a better biological interpretation of the data.

Exploring the Effects of Carbon Sources on the Metabolic Capacity for Exploring the Effects of Carbon Sources on the Metabolic Capacity for Predictions

Effects of various industrially important carbon sources (glucose, sucrose, xylose, gluconate, and glycerol) on shikimic acid (SA) biosynthesis in Escherichia coli were investigated to gain new insight into the metabolic capability for overproducing SA. At the outset, constraints-based flux analysis using the genome-scale in silico model of E. coli was conducted to quantify the theoretical maximum SA yield. The corresponding flux distributions fueled by different carbon sources under investigation were compared with respect to theoretical yield and energy utilization, thereby identifying the indispensable pathways for achieving optimal SA production on each carbon source. Subsequently, a shikimate-kinase-deficient E. coli mutant was developed by blocking the aromatic amino acid pathway, and the production of SA on various carbon sources was experimentally examined during 5 l batch culture. As a result, the highest production rate, 1.92 mmol SA/h, was obtained when glucose was utilized as a carbon source, whereas the efficient SA production from glycerol was obtained with the highest yield, 0.21 mol SA formed per mol carbon atom of carbon source consumed. The current strain can be further improved to satisfy the theoretically achievable SA production that was predicted by in silico analysis.

Listeria monocytogenes as novel carrier system for the development of live vaccines

Listeria monocytogenes is a facultative intracellular bacterium that enters a variety of non-professional mammalian cells by triggered phagocytosis (“zipper mechanism”) and replicates in the cytosol of the infected host cells. Therefore, it is a promising vaccine vector for the presentation of passenger antigens to the MHC class II and especially class I pathways. Here, we review recent progress made in our laboratory on the development of novel attenuated L. monocytogenes carrier strains for the delivery of heterologous antigens or antigen-encoding DNA and RNA to eukaryotic host cells. Based on the deletion of the chromosomal copy of the tryptophanyl-tRNA synthetase gene ( trpS) and plasmid-based in trans complementation of the same, we were able to establish a balanced-lethal plasmid system in L. monocytogenes. Safety concerns in the antigen delivery in vivo were addressed by chromosomal deletion of genes in the basic branch of the aromatic amino acid pathway, resulting in safe, attenuated L. monocytogenes carrier strains. Furthermore, plasmid-based expression of a cytosolically expressed phage lysin resulted in a self-destructing carrier strain that has been successfully used for the delivery of antigens as well as antigen-encoding plasmid DNA and particularly mRNA, therefore overcoming bottlenecks that have been shown to exist for bacteria-mediated DNA delivery.

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

BackgroundThe rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.ResultsMTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.ConclusionThe identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

Determination and metabolic engineering of rate limiting reactions in aromatic amino acid pathway in Bacillus subtilis for l-phenylalanine production

Determination and metabolic engineering of rate limiting reactions in aromatic amino acid pathway in Bacillus subtilis for l-phenylalanine production

Transcriptome analysis of a shikimic acid producing strain of Escherichia coli W3110 grown under carbon- and phosphate-limited conditions

Shikimic acid, which is produced in the aromatic amino acid pathway in plants and microorganisms, is an industrially interesting chiral starting material for the synthesis of many chemical substances, e.g. the influenza medicine Tamiflu. When produced by genetically modified Escherichia coli it has previously been found that carbon-rich conditions (e.g. phosphate-limitation) favors production of shikimic acid over shikimate pathway by-products, whereas the situation is the opposite at carbon-(glucose-) limited conditions. In the present study, gene expression patterns of the shikimate producing strain W3110.shik1 (W3110 with aroL deletion and plasmid-overexpressed aroF) and the wild type strain W3110 grown under carbon- and phosphate-limited (carbon-rich) chemostat conditions (D=0.23h(-1)) were analyzed. The study suggests that the by-product formation under carbon-limitation is explained by a set of upregulated genes coupled to the shikimate pathway. The genes, ydiB, aroD and ydiN, were strongly induced only in carbon-limited W3110.shik1. Compared to W3110 the lg(2)-fold changes were: 6.25 (ydiB); 3.93 (aroD) and 8.18 (ydiN). In addition, the transcriptome analysis revealed a large change in the gene expression when comparing phosphate- to carbon-limitation, which to a large part could be explained by anabolic-catabolic uncoupling, which is present under phosphate-limitation but not under carbon-limitation. Interestingly, there was also a larger difference between the two strains under carbon-limitation than under phosphate-limitation. The reason for this difference is interpreted in terms of starvation for aromatic amino acids under carbon-limitation which is relieved under phosphate-limitation due to an upregulation of aroK and aroA.

Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv.

Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 angstroms, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 angstroms resolution using a synchrotron-radiation source.

Fermentation characteristics of l-tryptophan production by thermoacidophilic Bacillus acidocaldarius in a defined medium

A wild-type thermoacidophilic bacteria Bacillus acidocaldarius which excretes l-tryptophan (Trp) was used for the production of Trp. A fructose-based defined fermentation medium, i.e. containing 8 kg m −3 fructose, 5.0 kg m −3 (NH 4) 2HPO 4, and a salt solution, was designed by investigating the influence of carbon- and nitrogen-sources and ions, to create the physiological conditions where the regulatory mechanisms in B. acidocaldarius weakly operate. The effects of oxygen transfer were investigated within limited-oxygen transfer (LimOT), low-oxygen transfer (LOT), and medium-oxygen transfer (MOT) conditions by using the parameters Q O/ V R = 0.2 and 0.5 vvm and N = 250 and 500 min −1 in V = 3 dm 3 bioreactors, at initial pH 5.0 and T = 55 °C. The fructose, cell, Trp and the other amino acid and organic acid concentrations, the oxygen transfer rate (OTR), oxygen uptake rate (OUR), and oxygen transfer coefficients ( K L a) were determined throughout the bioprocess; moreover, the yield and maintenance coefficients as well as the kinetic constants were reported. In general, the OTR and OUR decrease as the oxygen transfer decreases towards the limited-oxygen transfer conditions, i.e. LimOT. Damköhler number ( Da) that is the maximum possible oxygen utilization rate (OD) per maximum possible oxygen-transfer rate (OTR max) decreases with the decrease in Q O/ V R and/or N, that is, the oxygen-transfer resistance increases. The fructose consumption rate is the highest at MOT and the lowest at LimOT condition. In general, the cell growth rate is maximum when both the oxygen-transfer and biochemical reaction resistances are effective, i.e. at MOT. Trp production favours limited oxygen transfer conditions as B. acidocaldarius produces 0.34 kg m −3 Trp with the highest yield at LimOT condition, where the highest total amino acid concentration was obtained as 0.36 kg m −3, and overall Trp yield on fructose was 0.3 kg kg −1 at t = 17 h. Total amino acid concentration increased with the decrease in the oxygen transfer, despite the fact that the other aromatic group amino acids, i.e. l-tyrosine and l-phenylalanine, favour MOT condition. To open a new avenue to the production of Trp probably due to the increased reaction rates and the weakness of the regulatory mechanisms in the aromatic amino acid pathway, the fermentation characteristics of B. acidocaldarius were determined to start strain improvement research using metabolic engineering techniques.

Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate‐limited and carbon‐limited conditions

Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W3110) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain (W3110) under carbon- and phosphate-limited conditions. For the shikimic acid producing strain (referred to as W3110.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 +/- 0.012 vs. 0.024 +/- 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-dehydroshikimic acid (DHS) decreased from 0.076 +/- 0.028 to 0.022 +/- 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 +/- 0.021 c-mol/c-mol), quinic acid (0.012 +/- 0.005 c-mol/c-mol), and gallic acid (0.002 +/- 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shik1, and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult.

Bioconversion of phenyllactate to phenylalanine by bacteria of the genus Pseudomonas

The nature of phenylalanine auxotrophy in Pseudomonas sp. strain M9 was studied. A metabolic defect was found after the aromatic amino acid pathway branches from chorismate, so that the latter could not be converted to phenylpiruvate. It was shown that enzymatic activity of the strain could convert phenyllactate to phenylalanine. The reaction mechanisms were studied, and approaches to increasing the rate of the process and phenylalanine yield were proposed.

Exploring the diversity of complex metabolic networks

Metabolism, the network of chemical reactions that make life possible, is one of the most complex processes in nature. We describe here the development of a computational approach for the identification of every possible biochemical reaction from a given set of enzyme reaction rules that allows the de novo synthesis of metabolic pathways composed of these reactions, and the evaluation of these novel pathways with respect to their thermodynamic properties. We applied this framework to the analysis of the aromatic amino acid pathways and discovered almost 75,000 novel biochemical routes from chorismate to phenylalanine, more than 350,000 from chorismate to tyrosine, but only 13 from chorismate to tryptophan. Thermodynamic analysis of these pathways suggests that the native pathways are thermodynamically more favorable than the alternative possible pathways. The pathways generated involve compounds that exist in biological databases, as well as compounds that exist in chemical databases and novel compounds, suggesting novel biochemical routes for these compounds and the existence of biochemical compounds that remain to be discovered or synthesized through enzyme and pathway engineering. Framework will be available via web interface at http://systemsbiology.northwestern.edu/BNICE (site under construction). vassily@northwestern.edu or broadbelt@northwestern.edu http://systemsbiology.northwestern.edu/BNICE/publications.