Non-ribosomally synthesised natural products derived mainly from bacteria and fungi act as important therapeutic agents. Due to their complex structures it is difficult to chemically synthesise such compounds, therefore the engineering of biosynthetic and biocatalytic pathways that are vital for their production are the aims of this thesis. The first project involved the study of the biosynthesis of 3-O-methyl aspartic acid (OmAsp) in the antibiotic A54145. We demonstrated that LptL functions as an asparagine hydroxylase. We also predicted that LptJ and LptK are involved in the biosynthesis of OmAsp, although we did not find evidence of this non-standard amino acid in the antibiotic CDA when we over-expressed both proteins in Streptomyces coelicolor. The second project involved the study of the chlorination and mannosylation of hydroxyphenyl glycine (Hpg) residues in ramoplanin. We over-expressed the putative chlorinase and mannosyl transferase separately in the enduracidin-producer, in which the production of enduracidin has been characterised. We then found that trichlorinated and mannosylated enduracidin analogues were produced. The final project involved the re-engineering of a preQ1 riboswitch. We successfully created ten preQ1 riboswitch mutants fused to the reporter gene lacZ in Bacillus subtilis, all of which showed different levels of β-galactosidase activity. Subsequently, we found that the preQ1 C17U mutant riboswitch can respond specifically to the synthetic ligands 5-(aminomethyl)furo[2,3-d]pyrimidine-2,4-diamine (D6) and 2,4-diamino-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (D9) to control gene expression in a dose dependent manner. The results described here show the successful production of variant antibiotics by bio-engineering and the use of an engineered preQ1 riboswitch as a tool for regulating gene expression.

Thesis advisor: Steven D. Bruner / Peptidic natural products are produced by diverse organisms ranging from bacteria to humans. These secondary metabolites can be assembled by the ribosome or by nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. The architectural complexity and biological activity of such compounds make them interesting targets for study. Frequently, nonribosomal peptides contain nonproteinogenic amino acid building blocks, and the biosynthetic routes to both ribosomal and nonribosomal peptides often utilize tailoring enzymes. These specialized enzymes catalyze mechanistically challenging reactions and provide peptidic natural products with structural motifs not normally found in proteins. Structural studies of these tailoring enzymes will further our understanding of biosynthetic pathways, and engineered tailoring enzymes could find use as promiscuous catalysts for the chemoenzymatic synthesis of natural product analogs. The L-tyrosine 2,3-aminomutase <italic>Sg</italic>TAM catalyzes the formation of &beta;-tyrosine from L-tyrosine, and is used in the biosynthetic pathway to the enediyne antitumor antibiotic C-1027. This enzyme contains the rare electrophilic prosthetic group 4-methylideneimidazole-5-one (MIO) and is homologous to the histidine ammonia lyase family of enzymes. While lyases form &alpha;,&beta;-unsaturated carboxylates as products, <italic>Sg</italic>TAM catalyzes additional chemical steps that result in an overall 2,3-amino shift. The precise mechanistic role of MIO in the ammonia lyase and aminomutase families of enzymes was actively debated for over 50 years. Here, we report the first x-ray crystal structure of an MIO-dependent aminomutase and detail the synthesis and characterization of mechanistic probes for this enzyme. Furthermore, we report several structures of <italic>Sg</italic>TAM bound to substrate analogs. These co-crystal structures reveal how <italic>Sg</italic>TAM achieves substrate recognition and suggest a specific role for MIO in catalysis. The results of our studies allow for the rational engineering of MIO-based aminomutases and ammonia lyases with altered physical properties and substrate specificities. Additionally, we are currently studying several enzymes involved in the biosynthesis of the tricyclic depsipeptide microviridin J. This ribosomal peptide natural product contains two lactones and one lactam, which are introduced by two enzymes belonging to the ATP-grasp ligase superfamily of proteins. Here, we detail the overexpression of these enzymes, MdnJ-B and MdnJ-C, in <italic>E. coli</italic> and report the optimization of conditions which lead to the crystallization of both enzymes. The structural characterization of MdnJ-B and MdnJ-C will lead to a greater understanding of macrocycle formation in ribosomal peptide biosynthesis, and engineered variants of these enzymes may find use as macrocylcization catalysts. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.

No description available.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.

The non-ribosomal peptide synthase (NRPS) blue pigment synthase (BpsA) has been shown in several heterologous hosts to mediate the production of the blue pigment indigoidine from two molecules of L-glutamine. Activation of BpsA is mediated by transfer of a coenzyme A (CoA) by a 4′-phosphopantetheinyl transferase (4′-PPTase). In this thesis, I explored heterologous co-expression of BpsA and the Pseudomonas aeruginosa 4′-PPTase (PaPcpS) and their co- localization to either cytoplasm or chloroplast stroma of the green model microalga Chlamydomonas reinhardtii. The alga represents a potentially sustainable production host for indigoidine, as it is able to grow using CO2 as a sole carbon source and (sun)light for its energy. Both heterologous proteins (BpsA and PaPcpS) could be expressed as full-length fusion proteins with either the mVenus yellow fluorescent reporter or spectinomycin resistance (aadA) selection marker in both subcellular localisations. Dual transformants were identified and subjected to multiple growth conditions to determine whether indigoidine was produced. Under no condition tested was indigoidine detected, indicating that either activation of BpsA or the catalysis of L-glutamine to indigoidine was not occurring in alga. Future work will be required to determine whether it is possible to activate the BpsA in C. reinhardtii. However, this represents the first documented example of expression of a heterologous NRPS in a eukaryotic alga and may serve as foundational work for other target NRPS expression projects.

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage < 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin.

Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N-formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily.

The soil bacterium Rhodococcus is a member of the phylum Actinobacteria and is related to Streptomyces, which is known for its production of many secondary metabolites. Recent genomic investigation of Rhodococcus has uncovered many silent gene clusters that appear to code for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKS) of unknown function. Previous work, showed that Rhodococcus species strain KCHXC3 produces an inhibitory compound in agar culture extracts that displays prominent activity against several Gram positive and Gram negative species including the pathogens Rhodococcus equi, Shigella dysenteriae and Pseudomonas aeruginosa. Using the engineered Rhodococcus transposon vector, pTNR, the goal of this investigation is to screen random mutants of KCHXC3 for strains that no longer produce the inhibitory molecule. A library of 1825 random insertion mutants was produced via electroporation then screened for production of the inhibitory molecule by a disk diffusion assay against Shigella dysenteriae. From this screening, 7 mutants which no longer produce the compound of interest were identified.