Papaver somniferum plants accumulate the secondary products codeine and morphine. P. bracteatum accumulates their precursor, thebaine. The aims of the project were to use tissue cultures for the production of these alkaloids and for the biotransformation of thebaine to codeine and morphine. Methods were evaluated for the extraction, separation and quantification of mg or mug amounts of morphinan alkaloids from plant material. TLC, IIPLC and RIA were useful. Poppy cells fron a range of seed sources and explants were grown in static and suspension culture. Manipulations were made in atterpts to induce morphinan biogenesis. These included inmobilisation of cells and changes in the growth medium. Morphinans were absent from unspecialised cells in all but one instance. The biotransformation of thebaine was tested in cell suspensions of P. somniferum and Nicotiana alata. Using thebaine (biosynthesised from CO2) these experiments were extended to organs of the P. somniferum plant. A thebaine-biotransfomation product arose in N. alata (but not P. somniferum) suspensions that also arose in excised P. somniferum capsules. A non-specific enzymic activity is proposed. No codeine or morphine were produced. Plant regeneration was demonstrated, in good yield, by embryogenesis fron meristenoid tissue of P. bracteatum. In P. somniferum the process was initiated but was not routinely successful. Regeneration may be useful for plant improvenent via cloning or as a source of variation. On reorganisation into plantlets, capacity for morphinan alkaloid accumulation was realised. Capacity for alkaloid accumulation is discussed in tenns of a requisite minimum level of cytodifferentiation, perhaps of laticifer-like cells. The uptake or binding of radiolabelled morphine by suspension cultures was investigated, since binding may be a reason for failure to detect morphinans in cultures extracted by the usual methods. Evidence was found that exogenous morphine binds to an insoluble fraction in P. somniferum and I. tabacum but cells did not contain any endogenous bound morphincins.

No description available.

No description available.

No description available.

Streptomycetes are Gram positive, soil dwelling filamentous bacteria, known for their production of secondary metabolites. Genome sequencing of Streptomyces coelicolor identified 26 known or predicted secondary metabolite gene clusters ranging from antibiotics to siderophores, lipids, pigments and lantibiotics. Most studies investigating secondary metabolite production in Streptomyces, as well as other bacteria, are undertaken in liquid or on solid media. There is little gene expression data available from in situ studies. This study determined expression in a totally different growth medium, soil, to gain insight into growth and adaptation under more 'normal' habitat conditions and the effect changing environmental factors has on the expression of secondary metabolite gene clusters. In order to do this, S. coelicolor was grown in soil microcosms from which RNA was extracted and amplified using an optimised T7 polymerase-based RNA amplification protocol. The amplified RNA was used to determine gene expression profiles via endpoint RT-PCR and RT-qPCR. In a complementary approach, S. coelicolor sand microcosms were subjected to a novel protein extraction procedure to determine protein expression profiles in soil. This study elucidated how carbon, nitrogen and metal availability, the environmentally bioactive entomopathogenic fungus Metarhizium anisopliae and nematode Steinernema kraussei affect secondary metabolite gene expression in soil. In contrast to the consensus of secondary metabolism commencing after reduction or cessation of growth, this study revealed expression of secondary metabolite biosynthetic genes and proteins related to secondary metabolism before the onset of exponential growth. Some secondary metabolite genes/proteins were even expressed constitutively. Soil microcosms have been shown to be an important tool for gene expression analysis. The results of these novel transcriptomic and proteomic approaches therefore have given new insight into secondary metabolism and its role under natural habitat conditions.

Most microbial organic molecules are secondary metabolites which consist of diverse chemical structures and a range of biological activities. Actinobacteria form a large group of Eubacteria that are prolific producers of these metabolites. The recurrence of pathogens resistant to antibiotics and a wider use of these metabolites apart from their use as anti-infectives, has been the impetus for pharmaceutical companies to search for compounds produced by rare and existing actinobacterial cultures. Accessing microbial biosynthetic pathway diversity has been possible through the use of sensitive and innovative molecular detection methodologies. The present study evaluated the use of molecular based screening as a rational approach to detect secondary metabolite biosynthetic genes (SMBG) in uncharacterised natural Actinobacterial populations. A polymerase chain reaction (PCR) approach was selected for ease of application and high sample processivity. Rational designed screening approaches using PCR in the discovery of SMBG, involved identifying common functions in secondary metabolite biosynthetic pathways, such as condensation reactions in polyketide synthesis, genes encoding these functions, and using conserved regions of these genes as templates for the design of primers to detect similar sequences in uncharacterised actinobacteria. Design of primers involved rigorous in silico analysis followed by experimentation and validation. PCR screening was applied to 22 uncharacterised environmental isolates, eight of these displayed the presence of the ketosynthase (KS) gene belonging to the type I polyketide synthases and eight contained the ketosynthase (KSĄ) gene belonging to the type II polyketide synthases, six of the isolates contained the presence of a presumptive dTDP-glucose synthase (strD) gene which is involved in the formation of deoxysugar components of aminoglycoside antibiotics and one isolate contained the presence of a presumptive isopenicillin N synthase (pcbC) gene involved in beta-lactam synthesis. Alignments of partially sequenced PCR products from isolates A1488 and A3023 obtained using type II PKS primers showed close similarities with KSĄ genes from antibiotic producing actinobacteria. Similarly, alignments of sequences from isolates A1113 and A0350 showed regions of similarities to KS genes from antibiotic producing actinobacteria. Fermentation techniques were used for inducing expression of secondary metabolites from the uncharacterised actinobacteria isolates. By using antimicrobial guided screening it was determined that most of the isolates possessed the capacity to produce antimicrobial metabolites. Dominant antagonistic activity was detected against Gram positive bacteria and to a minor extent against fungi. Optimal fermentation liquid media were identified for certain isolates for the production of antimicrobial metabolites. Two alternative fermentation methods; solid-state and liquid-oil fermentations were evaluated to improve secondary metabolite production in the uncharacterised isolates. Solid-substrate fermentation showed that it could induce a complex metabolite pattern by TLC analysis, however this pattern varied according to the substrate being used. Liquid media supplemented with refined oils, showed a positive response indicated by higher antibacterial activities detected. Evaluation of semi-purified organic extracts identified two isolates A1113 and A0350 producing similar antimicrobial metabolites as detected by HPLC/UV/MS, a literature database search of similar compounds containing the same molecular weight identified the compound as belonging to the actinomycin group of compounds. A complex metabolic pattern was identified for isolate A2381, database searching identified some of the compounds as having similar molecular weights to actinopyrones, trichostatins, antibiotics PI 220, WP 3688-5 and YL 01869P. Drug discovery screening can serve to benefit from PCR detection of biochemical genotypes in initial screens, providing a rapid approach in identifying secondary metabolite producing capabilities of microorganisms prior to the commencement of costly and time consuming fermentation studies. Additionally the identification of biochemical genotypes allows a directed approach in using fermentation media designed to induce biosynthetic pathways of specific classes of compounds.

No description available.

Zuzana Jindřišková The Study of Secondary Metabolites in Explant Culture of Trifolium pratense L. The basic prerequisite for a successful elicitation that is used to increase the production of secondary metabolites is, among others, finding a suitable elicitor, its concentration and optimal duration of effect of the elicitor on the plant in vitro culture, which was the main subject of this diploma thesis. The focus of our observations was the influence of 6-, 24- , 48- and 168-hour effect of nickel chloride solution (in the concentrations of 0.1 mmol, 1 mmol, 10 mmol and 100 mmol) and zinc sulphate (in the concentrations of 0.1 µmol, 1 µmol, 10 µmol a 100 µmol) on the production of flavonoids and isoflavonoids in the suspension culture of Trifolium pratense L. variety Tempus. The culture was cultivated on the Gamborg nutrien medium with the addition of 2 mg.l-1 2,4- dichlorophenoxyacetic acid and 2 mg.l-1 6- benzylaminopurine at 25řC and the light period of 16 hours light/ 8 hours dark. The maximum content of flavonoids, which was found out by the photometric determination of the Czech Pharmacopoeia 2009, was proved in the suspension culture of Trifolium pratense L. variety Tempus (0.406%) after 48-hour elicitation of nickel chloride solution in the concentration of 0.1 mmol, when there was a...

Unbranched heterocytous cyanobacteria exhibit complex filament and colony architectures and variable life strategies from symbionts to free living planktic and non-planktic species. They are counted among microbial groups showing an extensive production of secondary metabolites, resulting in both pharmaceutically important and toxic compounds. The main focus of this thesis is to broaden our knowledge on bioactive secondary metabolite potential in this widespread group of cyanobacteria. An effective combination of methods including whole genome sequencing, bioinformatic analysis, and analytical chemistry techniques are applied to accomplish this task. The discrepancies in distribution of various classes of compounds among ecological groups defined by different life strategies are discussed. Additionally, the thesis endeavours to test multidisciplinary approaches to tackle taxonomic assignments of unresolved unbranched heterocytous cyanobacteria using morphological, phylogenetic and ecophysiological methods, including a meta-analysis of morphological traits.

Many bacterial diseases were controlled by antibiotics since mid 1900s. However, over use of the drugs leads to the prevalence of resistant strains, some of them are resistant to essentially all of the commercial antibiotics except for one or two. Therefore, new drugs are needed to combat the die-hard resistant pathogens. This study is aimed at isolating bacteria with antimicrobial activities from the intertidal soils of Taiwan. A total of 25 samples were collected from 5 locations, including Peng-Hu, Da-Peng Bay, Ken-ding, Gueishan Island and Little Liu-chiu. Marine Agar 2216 and Actinomycete Isolation Agar were used for cultivation. Of 313 bacterial isolated, 47 of them showed antimicrobial activities against at least one of the 7 indicators (Bacillus cereus, Staphylococcus aureus, Klebsiella pneumonia, Salmonella typhimurium, Pseudomonas aeruginosa, Vibrio harveyi, Escherichia coli). The 47 strains were classified based on 16S rRNA phylogeny. The majority of them are Pseudoalteromonas spp. (31) and Bacillus spp. (9) as well as Virgibacillus sp. (1), Vibrio sp. (1), Streptomyces sp. (1) and Microbulbifer spp. (4). Over all, 10/47 are Gram positive and 37/47 require added salts for growth. The titers of antimicrobial substances as judged by ethyl acetate extraction were influenced by cultivation conditions, such as: growth temperature, types of media and time.