Both fatty acid and erythromycin biosynthesis are anticipated to involve an acyl carrier protein (ACP) or an ACP domain within a larger protein. A series of oligonucleotides directed against a key amino acid sequence in <i>Escherichia coli</i> ACP were used to try and detect the gene or genes coding for ACP in '<i>Streptomyces erythreus</i>''. Two positively-hybridising fragments were detected and these were cloned into multicopy vectors in <i>E. coli</i>. The insert fragments were characterised by DNA sequencing and it was shown that neither in fact coded for an ACP. In an alternative approach, malonyl CoA-incorporating activity from '<i>Streptomyces erythreus</i>'' and <i>Streptomyces coelicolor</i> was characterised. In particular, this activity was found to be dependent on a heat stable factor which was subsequently identified as an authentic ACP. It was shown that ACP from <i>E. coli</i> could substitute for the '<i>Streptomyces erythreus</i>'' ACP. Evidently, the malonyl CoA-incorporating activities from <i>Streptomyces</i> are freely dissociable like that of <i>E. coli</i> fatty acid synthase. ACP from '<i>Streptomyces erythreus</i>'' was purified to homogeneity and the N-terminal portion of the protein sequence was determined. Also an ACP from <i>Propionibacterium shermanii</i>, (another Gram-positive actinomycete) was purified to homogeneity and sequenced to provide a comparison. The close similarity with <i>E. coli</i> fatty acid synthase, previously unexpected, should allow the assay and purification of the other components of fatty acid and erythronolide synthase. As an example, the condensing enzyme of fatty acid synthase has been fractionated and assayed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:599841 |
| Date | January 1988 |
| Creators | Hale, R. S. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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