Intracellular Expression and Purification of a Recombinant Enzyme Involved in Bioconversion of Arteannuin B to Artemisinin in Escherichia coli Expression System

Streptomyces pactum produces an enzyme that converts Arteannuin B to Artemisinin that is an important antimalarial. The gene encoding this novel enzyme from Streptomyces pactum MTCC 3664 has been cloned and expressed in Escherichia coli BL21 (DE3). The enzyme is expressed in the form of inclusion bodies at 370C or in a soluble form at 280C. The recombinant enzyme was purified by Immobilized Metal Ion Affinity Chromatography (IMAC). The purified enzyme produced by constitutive expression was found to be functionally active as it showed the conversion of arteannuin B to artemisinin as detected by TLC and HPLC

Investigating the biosynthesis of the streptomycete antibiotic pacidamycin

Abstract There is an ever increasing need for the development of new antibiotics to fight the emergence of antibacterial resistant strains of pathogens. Developing antimicrobials with ‘novel scaffolds’ and modes of action is an effective way to combat pathogens that are resistant to compounds currently in clinical use. The pacidamycins are a member of the uridyl peptide class of antibiotics that are produced by the soil dwelling bacterium Streptomyces coeruleorubidus. They show specific activity against the pathogen Pseudomonas aeruginosa, using a currently unexploited mode of action against a cell wall biosynthetic enzyme target. This thesis reports the investigation into the biosynthesis of pacidamycin, more specifically, into the function of the hypothetical protein genes present in the pacidamycin gene cluster and the biosynthesis of the non-proteinogenic amino acid, (2S, 3S)-diaminobutyric acid (DABA), which is at the core of the pacidamycin structure and other related antimicrobials. A multidisciplinary approach has been taken in this investigation, utilising biophysical, biochemical and genetic approaches. Protein crystallographic studies have deduced the structure of Pac17, postulated to be a lyase involved in DABA biosynthesis along with structural determination of the protein bound to the proposed substrate aspartate. Site directed mutagenesis of a number of the Pac17 active site amino acids also showed their essentiality for aspartate binding. In vitro biochemical approaches to study the enzymatic activity of the DABA biosynthetic proteins were inconclusive, with no activity observed. Genetic disruptions of the genes under investigation revealed the function of pac13 as a dehydratase, responsible for dehydrating the furan ring of the uridyl nucleoside present in the pacidamycin structure. Further to this, these studies established the essentiality of the DABA biosynthetic genes pac19 and pac20 for pacidamycin production in the native producer

Antibacterial Activity of and Resistance to Small Molecule Inhibitors of the ClpP Peptidase

There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. β-Lactones with certain substituents on the α- and β-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analogue of a bioactive β-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the growth inhibitory β-lactones also inactivate the M. tuberculosis ClpP1P2 peptidase in vitro via formation of a covalent adduct at the ClpP2 catalytic serine. Given the potent antibacterial activity of these compounds and their medicinal potential, we sought to identify innate mechanisms of resistance. Using a genome mining strategy, we identified a genetic determinant of β-lactone resistance in Streptomyces coelicolor, a non-pathogenic relative of M. tuberculosis. Collectively, these findings validate the potential of ClpP inhibition as a strategy in antibacterial drug development and define a mechanism by which bacteria could resist the toxic effects of ClpP inhibitors.National Institutes of Health (U.S.) (Grant GM-101988

Pb2+ tolerance by Frankia sp. strain EAN1pec involves surface-binding

Several Frankia strains have been shown to be lead-resistant. The mechanism of lead resistance was investigated for Frankia sp. strain EAN1pec. Analysis of the cultures by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDAX) and Fourier transforming infrared spectroscopy (FTIR) demonstrated that Frankia sp. strain EAN1pec undergoes surface modifications and binds high quantities of Pb +2 . Both labelled and unlabelled shotgun proteomics approaches were used to determine changes in Frankia sp. strain EAN1pec protein expression in response to lead and zinc. Pb 2+ specifically induced changes in exopolysaccharides, the stringent response, and the phosphate (pho) regulon. Two metal transporters (a Cu2+-ATPase and cation diffusion facilitator), as well as several hypothetical transporters, were also upregulated and may be involved in metal export. The exported Pb2+ may be precipitated at the cell surface by an upregulated polyphosphate kinase, undecaprenyl diphosphate synthase and inorganic diphosphatase. A variety of metal chaperones for ensuring correct cofactor placement were also upregulated with both Pb+2 and Zn+2 stress. Thus, this Pb+2 resistance mechanism is similar to other characterized systems. The cumulative interplay of these many mechanisms may explain the extraordinary resilience of Frankia sp. strain EAN1pec to Pb+2. A potential transcription factor (DUF156) binding site was identified in association with several proteins identified as upregulated with heavy metals. This site was also discovered, for the first time, in thousands of other organisms across two kingdoms

Establishing modular ligation through transglutaminase engineering

The ability to precisely direct proteins into ordered complexes is highly valuable for the synthetic biologist. Tools that can direct formation of covalent bonds facilitate particularly robust assemblies, which may enable new properties in areas such as optical control or programming of cell behaviour. Decades of research have produced an extensive range of tools for covalent conjugation of proteins both in vitro and in vivo, each with specific requirements and characteristics that determine the scope of their application. However, there is still a need for systems that can conjugate endogenous proteins in the extracellular space or precisely assemble endogenous proteins in vitro. Transglutaminases are a family of enzymes characterized by their ability to catalyse formation of a covalent ε-(γ-glutamyl) lysine isopeptide bond between protein substrates. Transglutaminase 2 (TG2) is a well-studied human transglutaminase with potential to provide unique functionality to the protein-protein coupling toolbox. The moderate specificity of TG2 for glutamine donors and low specificity for lysine donors presents the opportunity for covalent decoration of endogenous protein surfaces. This thesis aimed to explore the engineering of TG2 to enhance protein-protein conjugation. In Chapter 3 I demonstrated that TG2 is a substrate for itself and forms high molecular weight covalent multimers when activated. A truncated variant of TG2 (TG2465) retained transamidase activity while self-aggregation was abolished. I also demonstrated that TG2 interacts robustly with commonly used scaffold proteins and the cell surface. I determined kinetics of transamidase catalysis in different activation states and generated a variant with prolonged activity in oxidising conditions. I introduced glutamine- and lysine bearing peptide tags to model substrates and demonstrated that these tags impart TG2-reactivity. In Chapter 4 I investigated SnoopLigase, a protein-protein conjugation system derived from RrgA of Streptococcus pneumoniae. SnoopLigase requires very stringent reaction conditions, which limits application. I demonstrated considerable improvement in reaction efficiency for the SnoopLigase2 system in several common buffers and determined the contribution of each component to the overall increase in performance. I also found that SnoopLigase2 has a strong tolerance to temperature and pH, achieving conjugation in conditions that have not been demonstrated with any competing technologies. These findings open up new possibilities for protein-protein conjugation. In Chapter 5 I established protocols for efficient assembly of TG2:cargo conjugates using SnoopLigase2. I demonstrated TG2-mediated retention of TG2:cargo constructs to protein surfaces via non-covalent interactions. I generated a TG2:TGFα construct and achieved enhanced duration of TGFα activity due to TG2-mediated localisation at the cell surface. In this thesis, I have revealed properties of TG2 and SnoopLigase2 that offer interesting functionalities for the synthetic biologist and may be useful for molecular assembly in tissue engineering and bio-photonic applications

Decrypting the programming of β-methylation in virginiamycin M biosynthesis

During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic ß-ketones into ß-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for ß-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second ß-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin ß-branching programming. Furthermore, variations in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives

蛋白質精製のための新規pH応答性ペプチドタグの開発

筑波大学 (University of Tsukuba)201

Study of a Bacillus circulans chitin-binding domain by a green fluorescent protein binding assay and detection of lysozymes by improved zymograms

A fluorescent binding assay was developed to investigate the effects of site-directed mutagenesis on the binding affinity and binding specificity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. The chitin-binding domain (ChBD) was genetically fused to the N-terminus of the green fluorescent protein, GFP. The polyhistidine-tagged hybrid protein was expressed in Escherichia coli under the dose-dependent regulation of the araBAD promoter and purified using metal affinity-, chitin- or ion-exchange chromatography. Residues suggested to be involved in binding from previous three-dimensional studies were mutated and their contributions to binding and substrate specificity were evaluated by depletion assays. Purified fusion proteins were incubated with chitin beads, polysaccharide-protein complexes were removed by centrifugation and the free protein concentration was measured fluorometrically. The experimental binding isotherms were analyzed by non-linear regression using a modified Langmuir equation. Binding affinity and specificity were alternatively studied by affinity electrophoresis under non-denaturing conditions. Non-conservative substitution of tryptophan residue (W687) with alanine abolished chitin-binding affinity. Double mutation E668K/P689A also impaired binding significantly. Other substitutions in the binding site had little effect on overall affinity for chitin. Interestingly, mutation T682A led to a higher specificity towards chitinous substrates than observed for the wild-type. Furthermore, the ChBD-GFP hybrid protein proved to be useful for specifically labeling cell walls of fungi and yeast and for the detection of fungal infections in tissue samples. Additionally, an improved method for detecting cell lytic activity by a colorbased zymogram was developed. Proteins were separated by electrophoresis in SDS-polyacrylamide gels copolymerized with Remazol-brilliant-blue labeled whole cells of Micrococcus lysodeikticus. After electrophoresis, the enzymes were allowed to refold and lyse the blue-labeled cells embedded in the gel, producing clearing zones in an otherwise bluish gel. This improved zymogram method allows the rapid, sensitive and simultaneous determination of cell lytic specificity, relative activity and molecular weight. This assay should be useful for many research disciplines investigating the role of lysozymes and other cell wall hydrolases capable of refolding after SDS treatment

Investigations of Streptomyces coelicolor A3(2) siderophore binding proteins

Siderophores are small, high-affinity ferric iron chelators released by many microorganisms and some plants to solubilize iron. They are of great interest due to their clinical use to treat iron overload in humans, and also in relation to the development of novel antibiotics that target the biosynthetic and uptake pathways for iron in pathogens. Pathogens such as Bacillus anthracis excrete more than one type of siderophore. This is linked to increased pathogenicity. The Gram-positive soil bacterium Streptomyces coelicolor A3(2) excretes three siderophores: desferrioxamine B, desferrioxamine E and coelichelin. These displace iron from insoluble ferric hydroxides, and the resulting ferric complexes are transported into the cell via siderophore-binding proteins (lipoprotein receptors) associated with ATP-binding cassette (ABC) transporters. Previous studies showed that some of the genes in the biosynthetic clusters of the desferrioxamines (des) and coelichelin (cch) were required for efficient uptake of ferrioxamine E and ferri-coelichelin respectively and a third ABC transporter gene cluster (cdt), not associated with siderophore biosynthesis genes, was implicated in the import of ferrioxamine B. In this study, the lipoprotein receptors encoded within the des, cch and cdt clusters - DesE, CchF and CdtB – were recombinantly overproduced in E. coli and purified by immobilized metal affinity chromatography. Also, ferri-coelichelin was purified from cultures of S. coelicolor. The binding of the ferric complexes of the three cognate siderophores, as well as the xenosiderophores ferrichrome and ferrialbomycin, to the lipoprotein receptors was monitored by intrinsic fluorescence quenching. Dissociation constants of receptor-siderophore complexes were found to be in the nanomolar range, and a revised model of cognate siderophore transport in S. coelicolor was proposed. In collaboration with researchers at St. Andrews University, an X-ray crystal structure was solved for apo-DesE and DesE bound to ferrioxamine B, which demonstrated the similarity of DesE to other bacterial siderophore-binding proteins and the negligible conformational change on substrate binding. Ferrioxamine B also exhibited an unusual configuration not observed before in X-ray crystals of this ferri-siderophore. Also, a forcefield was constructed to model the structure and distortions ferric-tris-hydroxamate complexes, which could be used in the future to investigate the molecular basis of the tight and specific binding of ferri-siderophores to siderophore-binding proteins