Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein.
A methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing-over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high-frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.
Khosla
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Department of Genetics, John Innes Institute, John Innes Centre, Norwich, UK.
Ebert-Khosla
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Hopwood
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D000213:Acyl Carrier Protein / Q000235:genetics* / Q000502:physiology; D000483:Alleles; D000595:Amino Acid Sequence; D000880:Anthraquinones; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D064429:Fatty Acid Synthases / Q000235:genetics / Q000502:physiology; D015321:Gene Rearrangement / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000502:physiology; D005810:Multigene Family / Q000235:genetics* / Q000502:physiology; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D010641:Phenotype; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D000095622:Benzoisochromanequinones
D000213:Acyl Carrier Protein; D000880:Anthraquinones; D001426:Bacterial Proteins; D064429:Fatty Acid Synthases; D009097:Multienzyme Complexes; C013390:actinorhodin; D000095622:Benzoisochromanequinones