A study of the fungal ecology of <I>Picea sitchensis </I>stumps on mineral soils on first rotation sites in Scotland was carried out to determine fungal colonization, succession and the mechanisms of fungal interaction. Fungal and bacterial colonization of stump and buttress roots of stumps 0, 7, 28 days, 12, 16 and 48/53 months old was assessed. <I>Melanotus proteus </I>was found in all 12 month old stumps; <I>Sistotrema brinkmanni</I> was recorded most frequently in 16 and 48/53 month old stumps. A series of <I>in vitro </I>experiments was carried out to identify interactions occurring between pairs of <I>P. sitchensis </I>fungi on Norkrans agar, <I>P. sitchensis </I>sawdust, root blocks and billets, to determine possible modes of interaction occurring <I>in vivo. </I>Fungi exhibiting antagonism toward <I>Heterobasidion annosum in vitro </I>were noted to determine possible <I>in vivo </I>applications as curative/preventative biological controls against <I>H. annosum. </I>Sitka spruce stumps were highly receptive to <I>H. annosum </I>basidiospore infection up to 24 hours after felling and showed a significant level of receptivity 7 days after felling. <I>M. proteus </I>infection was lower in live stumps, compared to dead or moribund stumps, and was reduced or inhibited in stumps inoculated with <I>Resinicium bicolor </I>sawdust inoculum. <I>In vitro </I>experiments indicated that 5% urea prevented <I>M. proteus </I>basidiospore germination and hyphal growth. Treating stumps or billets with a 20% urea solution, however, had no significant effect on <I>M. proteus </I>colonization. Antifungal metabolites were detectable in 85% of the 25 fungal species tested representing members of the Basidiomycotina, Deuteromycotina and Ascomycotina, when bioassayed with <I>Cladosporium cucumerinum. </I>The production of antifungal metabolites in Sitka spruce stumps by <I>H. annosum, R. bicolor, Stereum sanguinolentum, M. proteus </I>and <I>Hypholoma fasciculare </I>was demonstrated.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / During the last few decades, Streptomycetes have shown to be an important and adaptable group of bacteria for the production of various beneficial secondary metabolites. One such secondary metabolite, epsilon polylysine (ε-PL), produced by Streptomyces albulus is of particular interest due to its antimicrobial activity. This work aimed to study different facets surrounding ε-PL and its production. Firstly, to grow S. albulus CCRC 11814, using economically viable crude glycerol as a carbon source and subsequently measure ε-PL production using an anionic dye, trypan blue. Secondly, to evaluate the antimicrobial activity of ε-PL against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Penicillium simplissium. Thirdly, to determine whether there is economic feasibility of ε-PL as a food preservative in South Africa. Lastly, to develop and optimise tools for metabolic engineering such as recombineering and group II introns to improve ε-PL production. The results obtained in this study fall into three different areas: In terms of growth studies, S. albulus grew in the presence of crude glycerol, although the growth was suboptimal, 0.48 g/L as compared to 1.04 g/L produced using pure glycerol or glucose. This is due to the pressures on the bacteria from the impurities of crude glycerol such as methanol and salts. ε-PL antimicrobial activity was effective at a concentration of 100 μg/ml against S. aureus, E. coli and A. niger. It was however, ineffective against P. aeruginosa owing to the low outer membrane permeability of the bacteria. Due to the ability of S. albulus to grow in crude glycerol, it could be used as a financially viable option to produce ε-PL as a natural food preservative in South Africa. The economic feasibility of ε-PL as a food preservative in South Africa showed potential in terms of market research as well as the financial evaluations. However; the production volumes are low due to the use of the crude glycerol and may not cater for the large food industry in the country. For these reasons, metabolic engineering could be employed to improve these production volumes. The first step to metabolic engineering was to develop novel tools which can be used for genetic modifications in S. albulus. The group II intron tools for gene knockouts were developed by the construction of a vector which subsequently requires sequencing and testing to perform gene knockouts. Based on current knowledge, this is the first experiment of its kind. In terms of introduction of genetic material post gene knockouts, iii transformation was shown to be a more effective gene transfer technique as opposed to electroporation, producing 7.75 transformants/μg and 0.038 transformants/μg of DNA, respectively. Future work would involve the use of biocatalysis for metabolic engineering of S. albulus by either removing genes inhibiting ε-PL or overexpressing the enzyme responsible for its production. This research has developed the groundwork for future ε-PL production improvement using biocatalysis and economically viable crude glycerol as a carbon source for applications of the secondary metabolite as a food preservative.

No description available.

No description available.

Cyanobacteria are rich in biologically active secondary metabolites, many of which have potential application as anticancer or antimicrobial drugs or as useful probes in cell biology studies. A Jamaican isolate of the marine cyanobacterium, Lyngbya majuscula was the source of a novel antifungal and cytotoxic secondary metabolite, hectochlorin. The structure of hectochlorin suggested that it was derived from a hybid PKS/NRPS system. Unique features of hectochlorin such as the presence of a gem dichloro functionality and two 2,3-dihydroxy isovaleric acid prompted efforts to clone and characterize the gene cluster involved in hectochlorin biosynthesis. Initial attempts to isolate the hectochlorin biosynthetic gene cluster led to the identification of a mixed PKS/NRPS gene cluster, LMcryl, whose genetic architecture did not substantiate its involvement in the biosynthesis of hectochlorin. This gene cluster was designated as a cryptic gene cluster because a corresponding metabolite remains as yet unidentified. The expression of this gene cluster was successfully demonstrated using RT-PCR and these results form the basis for characterizing the metabolite using a novel interdisciplinary approach. A 38 kb region putatively involved in the biosynthesis of hectochlorin has also been isolated and characterized. The hct gene cluster consists of eight open reading frames (ORFs) and appears to be colinear with regard to hectochlorin biosynthesis. An unusual feature of this gene cluster includes the presence of a ketoreductase domain in an NRPS module and appears to be the first report of such an occurrence in a cyanobacterial secondary metabolite gene cluster. Other tailoring enzymes present in the gene cluster are two cytochrome P450 monooxygenases and a putative halogenase. The juxtaposition of two ORF's with identical modular organization suggests that this gene cluster may have resulted from a gene duplication event. Furthermore, biochemical characterization of two adenylation domains from this cluster strengthens its involvement in the biosynthesis of hectochlorin. / Graduation date: 2006

This thesis details my investigations of marine cyanobacterial natural products that resulted in the discovery of thirteen new secondary metabolites, the isolation of over fifteen previously reported metabolites and the biosynthetic investigation of two additional cyanobacterial compounds. Two novel lipopeptides were identified from a Lyngbya majuscula and Schizothrix sp. assemblage collected in the Fiji Islands. Somamide A is a depsipeptide consisting of a hexanoate moiety extended by seven amino acids, including two nonstandard units characteristic of cyanobacterial peptides. In contrast, somocystinamide A is a unique linear disulfide dimer displaying potent cytotoxicity against a mammalian neuroblastoma cell line. The organic extract from a Puerto Rican L. majuscula proved remarkably rich in chemistry, producing twelve known compounds as well as four new secondary metabolites. Among these new isolates were the novel sodium channel blocker antillatoxin B, a new chlorinated quinoline derivative and the new ��-pyrone malyngamide T. A collection of L. majuscula from Antany Mora, Madagascar, led to the isolation of the previously reported antineoplastic agent dolastatin 16 and the discovery of a new series of lipopeptides, the antanapeptins. These new molecules are characterized by the presence of the unique ��-hydroxy acid 3-hydroxy-2-methyloctynoate, or its reduced double- or single-bond equivalent. Wewakazole is a novel cyclic dodecapeptide isolated from a Papua New Guinea collection of L. majuscula. This large molecule contains both a methyloxazole and two oxazoles, residues rarely observed in marine cyanobacterial metabolites. Extensive utilization of 1D and 2D NMR techniques were required to elucidate the structure of this distinctive peptide. Biosynthetic investigations of two halogenated cytotoxins were also conducted on a cultured L. majuscula strain originally isolated from Hector Bay, Jamaica. Stable isotope feeding experiments demonstrated that both jamaicamide A and hectochlorin derive from mixed PKS and NRPS biosynthetic origins but are comprised of primary precursors unique to each molecule. / Graduation date: 2003

This thesis describes investigations of marine algal secondary metabolites, with particular interest in their biomedical and agrichemical potential. Invaluable in such pursuits have been the access and application of advanced spectroscopic techniques, such as NMR, and the ability to assess the biological activity of the algal samples in a variety of diverse protocols, through in-house evaluation and industrial collaborations. As an in-house bioassay, a survey of algal extracts for molluscicidal activity has led to the isolation of the previously reported chondrocole C (Portieria hornemanni), tanikolide (Lyngbya majuscula), and debromoaplysiatoxin (L majuscula). Debromoaplysiatoxin is 100 times more potent than niclosamide, the commercially utilized molluscicide. Such activity may make debromoaplysiatoxin an attractive agent for molluscan biological control to prevent the spread of schistosomiasis in artificial waterways such as irrigation channels and rice paddies. The isolation of chondrocole C led to further chemical investigations of P. hornemanni. A total of six related polyhalogenated monoterpenes were isolated from this collection. While four of these compounds were previously reported, taviochtodene represents the newest member of this class of secondary metabolites. Previously, this alga has yielded compounds with great potential anticancer utility, but naturally and synthetically elusive. The discovery of this class of chemistry potentially locates new geographic territory to search for such anticancer metabolites. While there has been little reported biological activity attributed to the malyngamides, they form the most prevalent class of secondary metabolites isolated from Lyngbya majuscula. To this list we have added malyngamides L, Q, and R. Of particular note, malyngamides Q and R were the first malyngamides to have been reported with altered stereochemistry at the vinyl chloride carbon. Subsequently, and in part stimulated by this finding, this alternate stereochemistry has been defined for some newly reported malyngamides. Also from L majuscula, tortugin and lyngbyabellin B were isolated as toxic cyclic depsipeptides. Both of these compounds displayed relatively potent biological activity (brine shrimp and antifungal). Each possessing particular structural motifs previously seen in invertebrate secondary metabolites, they lend further evidence for cyanobacteria as the producer of many of the polyhalogenated compounds often attributed to de novo invertebrate biosynthesis. / Graduation date: 2002

Marine cyanobacteria have been shown to produce a variety of biologically active and stucturally diverse secondary metabolites. These compounds are of interest to natural products researchers mainly because of their potential application as biomedicinals, biochemical probes, and agrichemicals. The metabolic pathways utilized by the cyanobacterium Lyngbya majuscula to generate curacin A, a potent antimitotic and its cometabolite, the molluscicidal barbamide, have been studied. Application of methods including radioisotope and stable isotope labeling have revealed the role of acetate and the amino acids methionine, valine, cysteine, and leucine as potential precursors in the biosynthesis of curacin and barbamide. An analytical technique based upon GC-EIMS methodology has also been developed to monitor the production levels of curacin A from cultures of Lyngbya majuscula with respect to growth. This method which makes use of the thiazole analog of curacin A, curazole as an internal standard, has also been preliminarily applied to the curacin A production in response to environmental factors associated with changes in geographical locations at or near sites where the original collections of the cyanophyte were made in Curacao, Netherlands Antillies. This was performed by a series of transplantation experiments envolving high and trace curacin A producing strains of L. majuscula. Interest in the bioactive profile of curacin A prompted a pilot scale up of the cultured tissue for isolation of the metabolite. These efforts provided a framework of methods that can be used industrially to obtain large quantites of the compound to meet possible future pharmaceutical or dignostics demands. / Graduation date: 1997

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 4

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 54-61).