The genetics of Sorangium cellulosum So ce26 and the biosynthesis of the antifungal polyketide soraphen A

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Title: The genetics of Sorangium cellulosum So ce26 and the biosynthesis of the antifungal polyketide soraphen A
Author: Zirkle, Ross Eric
Advisors: James M. Ligon, Committee Co-Chair
Abstract: Sorangium cellulosum is a gram-negative soil bacterium belonging to the order Myxococcales. The myxobacteria are distinguished by their complex morphological differentiation and ability to move on agar by gliding. Under conditions of starvation, myxobacteria form aggregates known as fruiting bodies in which vegetative cells are transformed into myxospores. The myxobacteria also produce a large number of diverse natural products. Many of these natural products have antimicrobial, antifungal and insectiscidal activities. The genus Sorangium is a particularly rich source of secondary metabolites. While strains of Sorangium are a good source of secondary metabolites, relatively little has been accomplished in the development of genetic systems for these organisms. Due to poor growth characteristics and the relative lack of genetic tools, experiments with S. cellulosum have been especially difficult. The results presented here describe the cloning and disruption of mglA, a gene from S. cellulosum So ce 26. An mglA mutant strain of S. cellulosum So ce26 displays a non-swarming phenotype and allows the isolation of single colonies. DNA gene transfer into S. cellulosum can only be achieved through conjugation, followed by homologous recombination of cloned DNA fragments on a transmissible plasmid and the corresponding chromosomal locus. The results presented here demonstrate an improvement of the efficiency of this system in S. cellulosum So ce26. Soraphen A is a secondary metabolite produced by S. cellulosum So ce26. Soraphen A is an antifugal macrolide that is synthesized by a type I polyketide synthase. The genes responsible for the synthesis of the soraphen polyketide synthase have been previously cloned and sequenced. The role in soraphen A biosynthesis of genes upstream of the core soraphen polyketide synthase genes has been proposed, but not experimentally determined. These genes include sorC, which is proposed to be involved in the production of the unusual extender unit, methoxymalonate, utilized by the soraphen polyketide synthase. The gene orf2, which is upstream of sorC, is proposed to have no role in the production of soraphen A. In the results presented here, both sorC and orf2 were disrupted and data obtained from the resulting strains demonstrates that sorC is required for the biosynthesis of soraphen A while orf2 has no apparent role in soraphen A biosynthesis. Due to the relative lack of satisfactory genetic systems in S. cellulosum, the heterologous production of soraphen A in a more amenable host was sought. Heterologous production would allow for detailed mechanistic studies of soraphen A biosynthesis as well as the production of engineered soraphen derivatives. The soraphen polyketide synthase genes sorA and sorB along with the methyltansferase sorM were cloned into expression vectors and integrated into the chromosome of Streptomyces lividas ZX7. SorC, sorD, sorE and sorR, all of which are genes upstream of sorA, were cloned into an expression plasmid with badA, a benzoate-coenzyme A ligase from Rhodopseudomonas palustris. This expression plasmid was then transformed into the St. lividans strain containing the integrated sorA, sorB and sorM. The resulting strains produced soraphen A when putative precursors for the production of the benzoyl-coenzyme A starter unit, sodium benzoate or trans-cinnamic acid, were supplied to the fermentations.
Date: 2004-04-15
Degree: PhD
Discipline: Microbiology
URI: http://www.lib.ncsu.edu/resolver/1840.16/5500


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