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Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters.

Gomez-Escribano JP, Bibb MJ - Microb Biotechnol (2010)

Bottom Line: To remove potentially competitive sinks of carbon and nitrogen, and to provide a host devoid of antibiotic activity, we deleted four endogenous secondary metabolite gene clusters from S. coelicolor M145--those for actinorhodin, prodiginine, CPK and CDA biosynthesis.We then introduced point mutations into rpoB and rpsL to pleiotropically increase the level of secondary metabolite production.Introduction of the native actinorhodin gene cluster and of gene clusters for the heterologous production of chloramphenicol and congocidine revealed dramatic increases in antibiotic production compared with the parental strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR47UH, UK.

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Pedigree of mutant strains. Steps involved in deleting the four endogenous secondary metabolite gene clusters (A) and in the introduction of point mutations in rpsL and rpoB (B). Constructs used in each step are indicated.
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f1: Pedigree of mutant strains. Steps involved in deleting the four endogenous secondary metabolite gene clusters (A) and in the introduction of point mutations in rpsL and rpoB (B). Constructs used in each step are indicated.

Mentions: We deleted the four antibiotic gene clusters from S. coelicolor M145 [a derivative of the wild‐type strain A3(2) lacking plasmids SCP1 and SCP2 (Kieser et al., 2000)] by homologous recombination by cloning 2 kb PCR‐amplified fragments from both ends of each cluster into a suicide vector (Tables S1 and S2). The extent of three of the gene clusters (act, red and cda) was taken from Challis and Hopwood (2003) while that of the cpk gene cluster was taken from Pawlik and colleagues (2006). The strategy used to make the deletions and the constructs used (Table S2 and Experimental procedures in Supporting information) are summarized in Fig. 1A.


Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters.

Gomez-Escribano JP, Bibb MJ - Microb Biotechnol (2010)

Pedigree of mutant strains. Steps involved in deleting the four endogenous secondary metabolite gene clusters (A) and in the introduction of point mutations in rpsL and rpoB (B). Constructs used in each step are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818861&req=5

f1: Pedigree of mutant strains. Steps involved in deleting the four endogenous secondary metabolite gene clusters (A) and in the introduction of point mutations in rpsL and rpoB (B). Constructs used in each step are indicated.
Mentions: We deleted the four antibiotic gene clusters from S. coelicolor M145 [a derivative of the wild‐type strain A3(2) lacking plasmids SCP1 and SCP2 (Kieser et al., 2000)] by homologous recombination by cloning 2 kb PCR‐amplified fragments from both ends of each cluster into a suicide vector (Tables S1 and S2). The extent of three of the gene clusters (act, red and cda) was taken from Challis and Hopwood (2003) while that of the cpk gene cluster was taken from Pawlik and colleagues (2006). The strategy used to make the deletions and the constructs used (Table S2 and Experimental procedures in Supporting information) are summarized in Fig. 1A.

Bottom Line: To remove potentially competitive sinks of carbon and nitrogen, and to provide a host devoid of antibiotic activity, we deleted four endogenous secondary metabolite gene clusters from S. coelicolor M145--those for actinorhodin, prodiginine, CPK and CDA biosynthesis.We then introduced point mutations into rpoB and rpsL to pleiotropically increase the level of secondary metabolite production.Introduction of the native actinorhodin gene cluster and of gene clusters for the heterologous production of chloramphenicol and congocidine revealed dramatic increases in antibiotic production compared with the parental strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR47UH, UK.

Show MeSH
Related in: MedlinePlus