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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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Comparative metabolic profiling. HPLC chromatograms of supernatants of M145 and the quadruply deleted mutant M1146, and of the same strains containing the congocidine gene cluster (indicated as ‘+pCGC002’) after growth in liquid GYM medium. Note the simplified chromatogram of M1146 compared with M145, and the higher levels of congocidine production in the mutant strains, especially M1154. Fractions that eluted at around 10 min were collected from the M1154 + pCGC002 sample and analysed by MS‐MS to verify the production of congocidine (Fig. S3).
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f4: Comparative metabolic profiling. HPLC chromatograms of supernatants of M145 and the quadruply deleted mutant M1146, and of the same strains containing the congocidine gene cluster (indicated as ‘+pCGC002’) after growth in liquid GYM medium. Note the simplified chromatogram of M1146 compared with M145, and the higher levels of congocidine production in the mutant strains, especially M1154. Fractions that eluted at around 10 min were collected from the M1154 + pCGC002 sample and analysed by MS‐MS to verify the production of congocidine (Fig. S3).

Mentions: The HPLC chromatogram of a culture supernatant from S. coelicolor M145 contains a large number of peaks and a high baseline. Removal of the four endogenous gene clusters (to yield M1146) resulted in a much simpler chromatogram with fewer peaks, and a lower and more stable baseline (Fig. 4). Consequently, when combined with liquid chromatography and mass spectrometry, these engineered strains are likely to markedly facilitate the discovery of new compounds from heterologously expressed gene clusters. Indeed, their utility was demonstrated by the relatively facile identification of peaks corresponding to the congocidine gene cluster. This would have been much more difficult with M145. The high level of congocidine production obtained with M1154 also allowed facile fractionation and MS/MS analysis to confirm the identity of the heterologously produced compound (Figs 4 and S3).


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

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

Comparative metabolic profiling. HPLC chromatograms of supernatants of M145 and the quadruply deleted mutant M1146, and of the same strains containing the congocidine gene cluster (indicated as ‘+pCGC002’) after growth in liquid GYM medium. Note the simplified chromatogram of M1146 compared with M145, and the higher levels of congocidine production in the mutant strains, especially M1154. Fractions that eluted at around 10 min were collected from the M1154 + pCGC002 sample and analysed by MS‐MS to verify the production of congocidine (Fig. S3).
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Related In: Results  -  Collection

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

f4: Comparative metabolic profiling. HPLC chromatograms of supernatants of M145 and the quadruply deleted mutant M1146, and of the same strains containing the congocidine gene cluster (indicated as ‘+pCGC002’) after growth in liquid GYM medium. Note the simplified chromatogram of M1146 compared with M145, and the higher levels of congocidine production in the mutant strains, especially M1154. Fractions that eluted at around 10 min were collected from the M1154 + pCGC002 sample and analysed by MS‐MS to verify the production of congocidine (Fig. S3).
Mentions: The HPLC chromatogram of a culture supernatant from S. coelicolor M145 contains a large number of peaks and a high baseline. Removal of the four endogenous gene clusters (to yield M1146) resulted in a much simpler chromatogram with fewer peaks, and a lower and more stable baseline (Fig. 4). Consequently, when combined with liquid chromatography and mass spectrometry, these engineered strains are likely to markedly facilitate the discovery of new compounds from heterologously expressed gene clusters. Indeed, their utility was demonstrated by the relatively facile identification of peaks corresponding to the congocidine gene cluster. This would have been much more difficult with M145. The high level of congocidine production obtained with M1154 also allowed facile fractionation and MS/MS analysis to confirm the identity of the heterologously produced compound (Figs 4 and S3).

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