Affiliation:
1. grid.10392.39 0000000121901447 Pharmaceutical Institute University of Tübingen Auf der Morgenstelle 8 72076 Tübingen Germany
2. grid.10267.32 0000000121940956 Central European Institute of Technology Masaryk University and National Centre for Biomolecular Research Kamenice 753/5 625 00 Brno Czech Republic
3. grid.10392.39 0000000121901447 Institute of Microbiology and Infection Medicine University of Tübingen Auf der Morgenstelle 28 72076 Tübingen Germany
4. grid.418095.1 0000000110153316 Institute of Microbiology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague 4 Czech Republic
Abstract
Abstract
Coumermycin A1 is an aminocoumarin antibiotic produced by Streptomyces rishiriensis. It exhibits potent antibacterial and anticancer activity. The coumermycin A1 molecule contains two terminal 5-methyl-pyrrole-2-carboxylic acid moieties and one central 3-methylpyrrole-2,4-dicarboxylic acid moiety (CPM). While the biosynthesis of the terminal moieties has been elucidated in detail, the pathway leading to the CPM remains poorly understood. In this work, the minimal set of genes required for the generation of the CPM scaffold was identified. It comprises the five genes couR1, couR2a, couR2b, couR3, and couR4 which are grouped together in a contiguous 4.7 kb region within the coumermycin A1 biosynthetic gene cluster. The DNA fragment containing these genes was cloned into an expression plasmid and heterologously expressed in Streptomyces coelicolor M1146. Thereupon, the formation of CPM could be shown by HPLC and by HPLC-MS/MS, in comparison to an authentic CPM standard. This proves that the genes couR1–couR4 are sufficient to direct the biosynthesis of CPM, and that the adjacent genes couR5 and couR6 are not required for this pathway. The enzyme CouR3 was expressed in Escherichia coli and purified to near homogeneity. The protein exhibited an ATPase activity similar to that reported for its close ortholog, the threonine kinase PduX. However, we could not show a threonine kinase activity of CouR3, and; therefore, the substrate of CouR3 in CPM biosynthesis is still unknown and may be different from threonine.
Publisher
Oxford University Press (OUP)
Subject
Applied Microbiology and Biotechnology,Biotechnology,Bioengineering
Cited by
1 articles.
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