Strains of both <I>Streptomyces hygroscopicus </I>and <I>S. griseochromogenes</I> produce the antiparasitic compound milbemycin. Milbemycin is a macrocyclic polyketide synthesised by a Polyketide Synthase (PKS) in a manner analogous to the synthesis of long chain fatty acids by Fatty Acid Synthases (FAS). DNA libraries were constructed of <I>S. hygroscopicus</I> and <I>S. griseochromogenes</I> genomic DNA and probed with oligonucleotide probes for key PKS activities. A number of overlapping cosmids were identified with inserts potentially encoding the milbemycin PKS. The cosmids derived from <I>S. hygroscopicus</I> seemed to form three distinct groups based on homology with each other, while all of the <I>S. griseochromogenes </I>cosmids appeared to be derived from a single cluster. The DNA sequence was determined for a single fragment from each group of cosmids and the derived amino acid sequences were use to search the SwissProt protein sequence database. This analysis revealed that all of the fragments appeared to encode modular PKSs. Recombinant bacteriophage and plasmids were engineered to disrupt the chromosomal homologues of all of the sequenced fragments. Potential recombinants were isolated and assayed for milbemycin production by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Attempts to disrupt the <I>S. griseochromogenes</I> PKS genes were unsuccessful and the function of the cloned DNA remains unproven. In <I>S. hygroscopicus</I> disruption of two of the putative PKS clusters resulted in mutant phenotypes both exhibiting partial loss of milbemycin synthesis. Hence the milbemycin PKS gene cluster has been conclusively identified and the way paved for determination of its genetic organisation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:638560 |
| Date | January 1999 |
| Creators | Powell, N. G. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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