Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters.
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.
Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR47UH, UK.Show MeSH
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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).
Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR47UH, UK.