Studies on the aroB, aroC and aroL genes of Escherichia coli

The arom pentafunctional enzyme found in fungi catalyses five consecutive reactions of the shikimate pathway. The same aromatic biosynthetic functions in bacteria are achieved by five monofunctional enzymes, products of individual unlinked genes. The evolutionary implications and questions raised by these two extremes of structural organisation can be addressed by comparison of the primary structures of the five monofunctional activities with that of the multifunctional enzyme. This thesis is concerned with contributing some of the required bacterial data. The complete amino acid sequence of the Escherichia coli 3-dehydroquinate synthase (aroB gene product) has been determined by a combined nucleotide and direct amino acid sequencing strategy. The aroB gene was sub-cloned from the plasmid pJB14, its nucleotide sequence was determined and an overexpressing (inducible) strain constructed. The gene product DHQ synthase was purified from this strain and its N-terminal amino acid sequence determined. E. coli DHQ synthase is 362 amino acids long with a calculated of 38,880. Analysis of the aroB nucleotide sequence and its 5' and 3' flanking regions has identified the aroB promoter and possible 3' terminator sequences. The aroL gene encoding the tyrR regulated shikimate kinase II was cloned from E. coli K12. The aroL gene has been identified by nucleotide sequencing and direct N-terminal amino acid sequencing. Construction of overexpressing strains has allowed purification (for the first time) of a monofunctional shikimate kinase. E. coli shikimate kinase II is monomeric with a calculated of 18,937. The amino acid sequence contains a region with some homology to sequences found in other kinases and ATP-requiring enzymes. Transcript mapping has identified a possible operator sequence overlapping the aroL promoter which could constitute the tyrR repressor binding site. The aroC gene encoding chorismate synthase has been cloned from E. coli. The construction of overexpressing strains has allowed purification for the first time of E. coli chorismate synthase. The aroC gene has been identified by nucleotide sequencing and confirmed by N-terminal amino acid sequencing of the purified protein. E. coli chorismate synthase is 357 amino acids long with a calculated of 38,183. Analysis of the 3' flanking sequences has identified possible terminator elements. The bacterial sequences for the aroB and aroL gene products have been compared with the S. cerevisiae and A. nidulans arom amino acid sequences. Discrete non-overlapping regions of the fungal polypeptide are homologous with both E. coli DHQ synthase (aroB), shikimate kinase (aroL) and the remaining aroA, aroD and aroE gene products. This evidence supports the hypothesis which forms the basis of this study: that the fungal arom multifunctional enzyme is a mosaic of five independently folded 'domains'

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol

<p>Abstract</p> <p>Background</p> <p>Glycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of <it>Escherichia coli</it> physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes.</p> <p>Results</p> <p>Transcriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: <it>lamB</it>, <it>malE</it>, <it>mglB</it>, <it>mglC</it>, <it>galP</it> and <it>glk</it> and some members of the RpoS regulon (<it>pfkA</it>, <it>pfkB</it>, <it>fbaA</it>, <it>fbaB</it>, <it>pgi, poxB</it>, <it>acs</it>, <it>actP</it> and <it>acnA</it>). Inactivation of <it>rpoS</it> had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of <it>poxB</it>, <it>pta</it>, <it>acs</it> genes, glyoxylate shunt genes (<it>aceA, aceB</it>, <it>glcB</it> and <it>glcC</it>) and <it>actP</it>, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated <it>poxB</it> or <it>pckA</it> genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated <it>tnaA</it> gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLB<it>aroG</it>^<it>fbr</it><it>tktA</it>, when growing on glycerol, as compared to glucose.</p> <p>Conclusions</p> <p>The overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.</p

Fermentative production of chlorinated and brominated L-tryptophan by metabolically engineered Corynebacterium glutamicum

Veldmann K. Fermentative production of chlorinated and brominated L-tryptophan by metabolically engineered Corynebacterium glutamicum. Bielefeld: Universität Bielefeld; 2020

Chorismate synthase from Staphylococcus aureus

The aroC gene encoding chorismate synthase and the ndk gene encoding nucleoside diphosphate kinase were cloned from Staphylococcus aureus by complementation of the Aro- phenotype of the aroC E. coli strain, GLW40. Two partial open reading frames (ORFs) encoding 3-dehydroquinate synthase and a protein which had similarity with the putative gerCC gene product from B. subtilis were also cloned. In S. aureus the aroC gene is likely to form the first gene in an operon which includes the aroB and aroA genes. It has been demonstrated in this study that the ndk genes and the gerCAgerCBgerCC genes in S. aureus are situated upstream from the aroCaroB genes; this gene organisation has also been observed in B. subtilis. The S. aureus aroC gene was expressed from the T7 promoter on plasmid pTB361. This expression system resulted in the accumulation of very high levels of soluble S. aureus chorismate synthase and facilitated the purification of the enzyme to near homogeneity, producing 100mg of enzyme from 13g of cells. No detectable immunological crossreactivity was observed between S. aureus chorismate synthase and antibodies raised against E. coli chorismate synthase. This contrasts with other chorismate synthases and indicates that there are structural differences between the chorismate synthases from S. aureus and E. coli. S. aureus chorismate synthase was determined to be a homotetramer using gel filtration and chemical crosslinking. The pH optimum determined for S. aureus chorismate synthase was found to be non-symmetrical in MOPS buffer with an optimum of activity around pH 7.0. The apparent Km for EPSP of the S. aureus enzyme was calculated to be 12.7muM and the apparent Km for FMN was calculated to be 4.8muM. The apparent Km value for FMN for the S. aureus enzyme is two orders of magnitude greater compared to other chorismate synthases, excluding the B. subtilis enzyme. S. aureus chorismate synthase was investigated using pre-steady state kinetics and a flavin intermediate was observed during turnover with a difference spectrum resembling that obtained with the E. coli enzyme. The spectral characteristics of the S. aureus flavin intermediate were different, however, with respect to its maxima, minima and its overall shape. The rate of decay (6.5s-1) of the intermediate was eight times slower than that observed for the E. coli enzyme (52s-1) and this compares well with a 7 fold lower Vmax. Tyrosine 121 of S. aureus chorismate synthase was changed to phenylalanine or alanine using site-directed mutagenesis. The conversion to alanine resulted in a loss of activity while the phenylalanine mutant retained 10% of wild-type activity

Structure and stability studies of shikimate kinase

This thesis describes a structural and functional study of shikimate kinase, the enzyme which catalyses the fifth step in the biosynthetic shikimate pathway in which shikimate is converted to shikimate-3 -phosphate. Mutagenesis studies were undertaken to discover more about the roles of the assumed catalytic residues in particular the single lysine (K15) within the active site and the neighbouring cysteine (C13). The cysteine remote from the active site (C162) was also mutated to prevent intermolecular disulphide formation which had previously been observed during crystallographic studies. Biophysical techniques including circular dichroism (CD), fluorescence and microcalorimetry were used to monitor the conformational changes of shikimate kinase accompanying ligand binding and to measure ligand binding. The single tryptophan (W54) was found to be a very useful fluorophore for monitoring conformational changes. Calculated Kd values from this compared well with the Km values measured by steady state kinetics. The dissociation constant (Kd) for shikimate was 300 muM and for ATP 700 muM. When monitored by fluorescence the binding of the ATP to the enzyme showed a degree of co-operativity. The data did not fit a standard Michaelis Menten binding curve but gave a sigmoidal plot with a Hill co-efficient of approximately 2. The binding of ADP gave a Hill co-efficient of approximately 1.5. Fluorescence quenching studies using the quenching agents of succinimide and sodium iodide allowed the calculation of Stern-Volmer constants. The results provided information about the environment of the Trp-54. ATP binding to the enzyme was also measured by Isothermal titration calorimetry (ITC) This gave Kd values of only 25-40 muM whereas apparent steady state kinetics gave a Km value of 700 muM. This discrepancy has not been explained but it may reflect the existence of a second ATP binding site. Thermal stability was measured using differential scanning calorimetry (DSC) which was analysed to give the apparent melting temperature (Tm). Ligand binding (ATP) was found to stabilize the structure of shikimate kinase illustrated by an increase in the Tm from 39 to 47°C. However the shikimic acid provides a lower degree of stabilisation. The mutant K15M had a higher Tm than the wild type enzyme; this may be due to the formation of an extra ion pair in the structure, formed between the side chains of Asp 32 and Arg 11. Thermal stability was also measured by using CD to monitor the loss of secondary structure at 222nm. Denaturation and refolding studies were carried out using guanidine hydrochloride (GdnHCl) as the denaturant. The concentration of GdnHCl needed to bring about 50% of the total change of the native structure in both secondary, measuring the change in CD, and tertiary structure measuring the changes in fluorescence was 1.25M. This was observed as a single transition. Total loss of structure was observed at concentrations of 4M and above. Refolding of the enzyme was monitored by both fluorescence and CD using both manual mixing and stopped flow techniques. The data indicated that refolding took place in a number of distinctive stages. One of the intermediates appeared to have a high affinity for the fluorescent probe 1-anilino-8-napthalenesulphonate (ANS). An outline model for the refolding of the enzyme could be proposed

The development of high throughput metabolomics to aid the synthetic biology 'design-build-test-learn' cycle

Metabolomics offers a comprehensive insight to metabolite profiles within biological systems. It often uses mass spectrometry as an analytical technique, enabling detection with high sensitivity and selectivity. Metabolomics has been successfully utilized to study a range of fields, including synthetic biology. The basis of synthetic biology is the design and construction of new biological systems, and the re-design of existing natural systems for a specific purpose. Due to its complexity, synthetic biology often requires several iterations of the ‘design-build-test-learn’ cycle to optimise a bioprocess. Despite major advances in strain engineering throughput, there are few publications demonstrating the same improvement in strain analysis. Therefore, due to lengthy analytical techniques, the ‘test’ phase remains a bottleneck. To explore this under-developed area, a combination of high throughput metabolomics and low-cost automation were used for strain optimisation. High throughput metabolomics was implemented using a targeted method on a triple quadrupole instrument using flow injection. This method was optimised using multiple reaction monitoring, allowing 36 metabolites in the pathways of interest to be identified. This method has a short run time of 2 minutes per sample, enabling a full 96 well plate to be run in less than 4 hours. A liquid handling robot, built from LEGO® and programmed using MINDSTORMS® software, was implemented and illustrates that low-cost automation is a viable option to increase throughput. As an example of strain optimisation, the metabolism of Escherichia coli NST74 was examined. This strain is capable of overproducing L-phenylalanine due to feedback de-regulation. Therefore, eight inhibitors were used to probe the metabolism of this strain, with the aim to increase the yield of L-phenylalanine and provide potential targets for further genetic modification. One inhibitor, L-tryptophan - a feedback inhibitor of anthranilate synthase - increased the yield of extracellular Lphenylalanine by five-fold compared to the control, whilst another, L-tyrosine, increased the yield by seven-fold, also through feedback inhibition. This illustrates successful yield optimisation. In summary, this research aimed to reduce the bottleneck of the synthetic biology ‘design-build-test-learn’ cycle. Automation and high throughput mass spectrometry methods were implemented and decreased the time needed for sample preparation and data acquisition. Optimisation of the strain resulted in a seven-fold increase in the desired product. The combination of high throughput metabolomics and low-cost automation were used for strain optimisation successfully, representing a starting point for a high throughput platform for strain optimisation, and rapid testing using metabolomics

Prophylaxis of disease caused by bacterial pathogens of man

This thesis reports research undertaken which will lead to improved pretreatments and therapies for disease caused by Clostridium perfringens, Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. C. perfringens is thought to be the most widely distributed bacterial pathogen and is the most important Clostridial species associated with enteric disease in domesticated animals. During warfare C. perfringens has been a significant causes of mortality. Between 1 and 10% of wounded personnel developed gas gangrene during the 1st and 2nd world wars. The ability of the bacterium to cause a range of diseases is due largely to the differential production of toxins. The first reported cloning and nucleotide sequencing of three of the four major toxins (α, β and ε-toxins) is documented in this thesis. The regulation of expression of α-toxin in C. perfringens has been investigated and methods for the expression of recombinant proteins in E. coli have been devised This information has been used to develop improved PCR-based diagnostic tests, and to investigate structure-function relationships. A high resolution crystal structure of a-toxin (phospholipase C) is reported. Using molecular and biophysical techniques, the functions of the two domains of the protein have been determined. Residues that play roles in the interaction of the toxin with host cell membranes have been identified using site-directed mutagenesis. This work has also provided a major insight into the structures and functions of related phospholipases C (the zincmetallophospholipases C) from other bacterial pathogens. This pioneering work with α-toxin is recognised by invitations to write reviews and book chapters on this subject and on bacterial phospholipases C. C. perfringens β-toxin has been shown to be related to pore forming toxins such as Staphylococcus aureus α-toxin. This finding suggests, for the first time, the mode of action of β-toxin. The interaction of C. perfringens ε-toxin with host cells has been investigated and progress made in identifying the cell-surface receptor for the toxin. Genetically engineered toxoids have been devised which induce high-level protection against α and ε-toxins. These vaccines are currently being developed by industry for veterinary use. Similar approaches have been used to devise a recombinant vaccine against Clostridium botulinum toxin F. The wider applications of toxins as therapeutics have also been investigated, and a novel cancer drug delivery system based on targeted lysis of drug-containing liposomes by α-toxin has been devised and patented. F. tularensis is the etiological agent of tularemia, a disease of man that is found in most countries in the Northern hemisphere and most frequently in Scandinavia, N. America, Japan and N. Russia. In this thesis the efficacy of antibiotics for the prevention and treatment of experimental tularemia is documented. Two surface antigens (lipopolysaccharide and FopA) have been evaluated as sub-unit vaccines. Of these, lipopolysaccharide shows potential as a protective antigen. However, because of the paucity of information available on this bacterium, a wider approach to vaccine development, involving the determination of the genome sequence of a fully virulent strain of F. tularensis has been undertaken. A preliminary analysis of the genome sequence is reported here, which has allowed the identification of targets for the development of a rationally attenuated mutant for use as a live vaccine. Y. pestis is generally recognised to have caused three major pandemics of disease, and credible estimates indicate that together these resulted in 200 million deaths. WHO figures indicate that there is a continuing public health problem from plague, especially in Africa, Asia and South America. In this thesis existing vaccines and antibiotics have been evaluated for the prevention and treatment of plague and found to have limitations. A number of approaches to the development of an improved vaccine have been investigated including rationally attenuated strains of the bacterium and isolated surface antigens. A sub-unit vaccine against plague has been devised based on recombinant forms of the F1- and V-antigens. This vaccine provides high level protection against both bubonic and pneumonic plague. This recombinant sub-unit vaccine has been patented and is currently in phase I clinical trials in man. This vaccine has been formulated for single oral or intranasal delivery, using microencapsulated or Salmonella-based delivery systems. Methods for enhancing the stability and efficacy of these vaccines have been investigated. Reviews on plague and plague vaccines have been written, confirming the status of the author as a world leader in this field. The work to devise an improved vaccine has also provided insight into the molecular basis of pathogencity of Y. pestis. A phoP / phoQ regulatory system has been discovered in the bacterium, which plays a key role in survival of the bacterium within macrophages. The V-antigen has been shown to be surface located to play a key role in the translocation of effector proteins into host cells. The biogenesis of the F1-capsular antigen has been investigated at a genetic and biophysical level. In order to underpin future work with this pathogen, the genome sequence is currently being determined. This work has already provided major new insights into the evolution of this pathogen. B. pseudomallei (formerly Pseudomonas pseudomallei) is found primarily in S. E. Asia, N. Australia and other tropical areas of the world. Melioidosis has recently appeared in temperate zones, including mainland France and the UK possible as a consequence of increased international travel. Acute disease can be treated with antibiotics but the bacterium can persist in the host and subsequent disease episodes can occur. In this thesis ciprofloxacin and doxycyline have been are evaluated and shown to have significant limitations for the treatment of melioidodis. In the longer term there is a requirement for an effective vaccine against melioidosis, and work is reported here to devise the genetic tools which will be necessary for the genetic manipulation of the bacterium, with a view towards the identification of virulence determinants