Bacterial type I modular polyketide synthases (PKS) are large multifunctional enzyme systems responsible for the biosynthesis of complex polyketide natural products such as erythromycin, pikromycin, and borrelidin. Type I systems are comprised of a loading module which generally selects an appropriate acyl group starter unit, and multiple discrete extension modules, responsible for each single round of acyl group incorporation into the final polyketide core structure. These modules can exist naturally as either single discrete polypeptides, such as modules 5 and 6 from the pikromycin PKS (PikA3 and PikA4 respectively), or as multimodular polypeptides fused together by short intrapolypeptide linkers such as the loading module and the first and second extension modules of the erythromycin and pikromycin PKSs (DEBS1 and PikAI respectively). While short peptide linkers between modules on the same polypeptide facilitate the transfer of polyketide intermediates from one module to the next via their close proximity to one another, docking domains found at the C-terminus of one module and the N-terminus of the next subsequent module facilitate the needed protein-protein interactions for the passage of biosynthetic intermediates between modules on separate polypeptides. The ability to utilize docking domains in place of intrapolypeptide linkers was explored in the pikromycin and erythromycin PKSs by dissecting the tri-modular PikAI and DEBS1 polypeptides with matched docking domains. It has been shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin PKS are used in place of the intrapolypeptide linker. In both cases the yields of pikromycin produced by the S. venezuelae host mutant, which is a PikAI deletion strain were 50% of that of an S. venezuelae strain expressing the native trimodular PikAI. Additionally, expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a 10-fold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. The utilization of docking domains to separate linked modules was also demonstrated in the erythromycin PKS. Expression of the first protein involved in erythromycin biosynthesis (DEBS1) with the DEBS thioesterase fused to the C-terminal (DEBS1-TE) in S. venezuelae results in the production of triketide lactone products. Separation of DEBS1-TE resulted in 50% triketide lactone production, consistent with the observations in the pikromycin system. Published work has shown that the DEBS loading module has relaxed substrate specificity, and is capable of incorporating acetate, butyrate and isobutyrate in addition to the normally observed propionate starter unit, which typically predominates. However, in the current study when the DEBS loading module is separated from module 1 with matched docking domains, a dramatic shift in the starter unit, favoring the isobutyrate derived tri-ketide lactone is observed. This apparent shift in starter unit preference for a dissected PKS system has resulted in insights into the kinetics of acyl group loading, off loading, as well as the hydrolysis and transfer from the AT to ACP domains. In addition to the separation of multimodular PKS polypeptides with docking domains, it has also been shown that the individual catalytic domains of single discrete module, BorA5 from the borrelidin PKS can be expressed as stand alone proteins while retaining catalytic functionality in vitro. This work has provided a basis for future studies of this module, which has been proposed to function iteratively, catalyzing three rounds of chain elongation.

Thesis (Ph. D.)--University of Idaho, 2005. / Also available online in PDF format. Abstract. "December 2005." Includes bibliographical references.

Systematic fractionations of crude extracts from various strains of Aspergillus versicolor were carried out by a combination of techniques, including liquid-liquid partition, column chromatography and thin-layer chromatography. As a result of these fractionations a total of eleven metabolites were obtained in pure form. Ten of these were identified as the known compounds, sterigmatocystin, de-0-methylsterigmatocystin, 5-methoxysterigmatocystin, versicolorin A, versicolorin C, aversin, averufin, griseofulvin, dechlorogriseofulvin and 3,8- dihydroxy-6 -methoxy-1-methylxanthone. A new metabolite was isolated and identified as 6,8-di-O-methylnidurufin. Two high-performance liquid chromatography systems were developed for the analysis of these metabolites, using microparticle silica gel and microparticle polar bonded phase columns. Studies were also carried out on the reduction of the hemi-acetal derivatives of sterigmatocystin and versicolorin A. Reduction with a limited amount of sodium borohydride yielded in each case a mixture of two major products. The fully reduced products were identified as diols, while the partially reduced products were found to be new hemiacetals. The structure elucidation of these compounds was carried out with the aid of carbon magnetic resonance spectroscopy and of chemical conversions. A modified pathway for the biosynthesis of versicolorin A from averufin is proposed that incorporates nidurufin as an intermediate. / Ph. D.

Thesis (M.Sc.) - Carleton University, 2005. / Includes bibliographical references (p. 101-112). Also available in electronic format on the Internet.

Thesis (M.S.)--University of North Carolina at Wilmington, 2006. / Includes bibliographical references (leaves: 71-75)

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2003. / Appendix consists of reprints and manuscripts of five papers co-authored with others. Includes bibliographical references. Also partially available online in PDF format; online version lacks appendix.

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biological Sciences, Technikon Natal, 2000. / This study focussed on the anti-bacterial, anti-fungal and anti-protozoal activity of four plant extracts, maackiain and formononetin from Virgilia oroboides and chlorophorin and lroko from Chlorophora excelsa / M

In a program aimed at the identification of compounds responsible for the immunosuppressive and antifertility activities of the perennial twining vine, Tripterygium wilfordii. 5 new and 13 known compounds were isolated from the TRP-4a tissue culture cell line developed from Tripterygium wilfordii. The structures of the new compounds were determined by a combination of spectral analysis, chemical correlation and single crystal X-ray diffraction analysis. 22β-Hydroxy-3-oxoolean-12-en-29-oic acid (137), 22α-hydroxy-3-oxoolean- 12- en-29-oic acid (138) and 3β, 22β-dihydroxyolean-12-en-29-oic acid (139) are new triterpenes possessing an oleanene-type skeleton and were chemically correlated with 3β, 22α-dihydroxyolean-12-en-29-oic acid (51), the structure of which was confirmed by single crystal X-ray diffraction analysis. Oleanolic acid (127), β-sitosterol (128) and polpunonic acid (55), were isolated previously from the TRP-4a cell line in earlier studies in this laboratory. α-Amyrin (145), β-amyrin (146), 3β, 29-dihydroxyolean-12-ene (151) and 3β, llα-dihydroxyolean-12-ene (152) are known triterpenes possessing an oleanene-type skeleton and are isolated for the first time from the TRP-4a cell line. Tingenone (148) and 22β-hydroxytingenone (150) are quinone methide triterpenes, also isolated for the first time from the TRP-4a cell line. Similarly, the novel diterpene, 12-methoxyabieta-8, 11, 13- trien-3α-ol (147) and the novel triterpene, methyl-22β-hydroxy-3, 21-dioxo-D:A-friedo-29-noroleanan-24-oate (149), a member of the friedelane family, are isolated for the first time. A biosynthetic pathway, based on the isolation of 149 and its structural similarity to polpunonic acid (55) and 22β-hydroxytingen6ne (150), is postulated for the quinone methides. The cytotoxic diterpenes, tripdiolide (1) and triptolide (2) and the hydroxy acid, 160, isolated as the methyl ester, 124, have been previously reported from this laboratory. Tripdiolide (1) and triptolide (2) have been shown to possess strong antifertility and immunosuppressive activities. In another aspect of our program, biotransformation studies of the synthetic precursors, 19 (4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-ol (171) and 19-hydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (91), and the radioactive congeners, 182 and 209, were carried out using the TRP-4a cell line. It was hoped that the data obtained might shed some light on the "late stage" biosynthetic pathway of the diterpene triepoxides, tripdiolide (1) and triptolide (2). Synthesis of 171 was achieved in 5 steps from dehydroabietic acid (80). The radioactive congener, 182, was synthesised using ¹⁴C-paraformaldehyde with 0.4% incorporation of the radiolabel. Biotransformation of 171 using the TRP-4a cell line yielded 19(4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-al (185) and 19(4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-oic acid (186) for spectral identification. Biotransformation of 182 yielded the aldehyde, 183 (33.2%) and the acid, 184 (51.9%), the radioactive congeners of 185 and 186 respectively. Synthesis of 91 was achieved in 4 steps from dehydroabietic acid (80). The radioactive congener, 209, was synthesised using ¹⁴C-methyl iodide via ¹⁴C-dimethylsulphonium methylide, with 0.6% incorporation of the radiolabel. Biotransformation of 91 using TRP-4a tissue cultures yielded 19-hydroxy-7-oxo-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (214), 2β, 19-dihydroxy-7-oxo-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (215), 7β, 19-dihydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (216) and 2β 19-dihydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (96), for spectral identification. Biotransformation of 209 yielded the ketone, 210 (56.7%), the hydroxy ketone, 211 (5.9%), the benzylic alcohol, 212 (9.6%) and the C2 alcohol, 213 (6.8%), the radioactive congeners of 214,215,216 and 96 respectively. A biosynthetic pathway to the diterpene triepoxides is postulated based on the oxygenated biotransformation products. [formulas omitted] / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

The search for new antibiotics continues in a rather overlooked hunting ground. In the course of screening for new antibiotic-producing microorganisms, seventy-nine isolates showing antimicrobial activity were isolated from soil samples from various habitats in the KwaZulu-Natal midlands, South Africa. Existing methods of screening for antibiotic producers together with some novel procedures were reviewed. Both modified agar-streak and agar-plug methods were used in the primary screens. The use of selective isolation media, with or without antibiotic incorporation and/or heat pretreatment, enhanced the development of certain actinomycete colonies on the isolation plates. Winogradsky's nitrite medium (Winogradsky, 1949), M3 agar (Rowbotham and Cross, 1977), and Kosmachev's medium (Kosmachev, 1960), were found to be selective for actinomycetes. Statistical analysis showed highly significant interactions between isolates, assay media and the test organisms. The diameters of inhibition zones were found to be larger on Iso-sensitest agar (ISTA)[Oxoid, England] than in nutrient agar plates. Of the 79 isolates that showed antimicrobial activity, 44 isolates were selected for confirmatory screening. Of these, 13 were selected for secondary screening. Criteria for selection were based on significant inhibition of at least two test organisms and/or the inhibition of the specifically targeted organisms, Pseudomonas and Xanthomonas species. Following secondary screening eight isolates were considered for further investigation. The isolates were tentatively identified . on the basis of morphological features, using both light microscopy and scanning electron microscopy(SEM); their ability to utilize various carbon sources; and selected physiological and staining tests. Suspected actinomycetes were further characterized on the basis of selected chemical properties using thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) techniques. High pressure liquid chromatography analysis (Beckman 6300 analyzer) detected the presence of diaminopimelic acid (DAP) in whole-cell hydrolysates of six of the isolates while TLC analysis confirmed the type ofDAP present. The isolates N2, N12, N16, N19 and N35 were tentatively identified as Thermomonospora, Saccharopolyspora, Nocardiodes, Corynebacterium and Promicromonospora, respectively. Isolate N30 was identified as belonging to the coryneform group ofbacteria, possibly an Arthrobacter species. Isolate, N8, tentatively identified as Actinosynnema, was unique among the isolates tested as it showed good antimicrobial activity against all the Gram- positive and Gram-negative bacteria, and yeasts used as test organisms in the present investigation. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.