Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein
- 1 November 1992
- journal article
- Published by Wiley in Molecular Microbiology
- Vol. 6 (21) , 3237-3249
- https://doi.org/10.1111/j.1365-2958.1992.tb01778.x
Abstract
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.Keywords
This publication has 42 references indexed in Scilit:
- Genes for Polyketide Secondary Metabolic Pathways in Microorganisms and PlantsPublished by Wiley ,2007
- Mutactin, a novel polyketide from Streptomyces coelicolor. Structure and biosynthetic relationship to actinorhodinThe Journal of Organic Chemistry, 1990
- Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolorGene, 1988
- The use of a chromosome integration vector to map erythromycin resistance and production genes in Saccharopolyspora erythraea (Streptomyces erythraeus)Gene, 1988
- Three‐dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometryEuropean Journal of Biochemistry, 1988
- Visualizing Gene Expression in Time and Space in the Filamentous Bacterium Streptomyces coelicolorScience, 1988
- Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous hostNature, 1984
- TECHNOLOGICAL EXAMINATION OF LOW‐FIRED TERRACOTTA STATUES FROM AYIA IRINI, KEAArchaeometry, 1982
- Genetics of Actinorhodin Biosynthesis by Streptomyces coelicolor A3(2)Journal of General Microbiology, 1979
- Biosynthesis of branched long-chain fatty acids from the related short-chain α-keto acid substrates by a cell-free system of Bacillus subtilisCanadian Journal of Microbiology, 1973