Abstract
BackgroundVanYn, encoded by the dbv7 gene (also known as vanYn) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727. This filamentous actinomycete is an uncommon microorganism, difficult-to-handle but biotechnologically valuable since it produces the glycopeptide antibiotic A40926, which is the precursor of the second-generation dalbavancin in phase III of clinical development. In order to investigate VanYn role in glycopeptide resistance in the producer actinomycete an appropriate host-vector expression system is required.ResultsThe cloning strategy of vanYn gene (G-C ratio 73.3%) in the expression vector pIJ86 yielded a recombinant protein with a tag encoding for a histidine hexamer added at the C-terminus (C-His6-vanYn) or at the N-terminus (N-His6-vanYn). These plasmids were used to transform three Streptomyces spp., which are genetically-treatable high G-C content Gram-positive bacteria taxonomically related to the homologous producer Nonomuraea sp.. Highest yield of protein expression and purification (12 mg of protein per liter of culture at 3 L bioreactor-scale) was achieved in Streptomyces venezuelae ATCC 10595, that is a fast growing streptomyces susceptible to glycopeptides. VanYn is a transmembrane protein which was easily detached and recovered from the cell wall fraction. Purified C-His6-VanYn showed d,d-carboxypeptidase and d,d-dipeptidase activities on synthetic analogs of bacterial peptidoglycan (PG) precursors. C-His6-VanYn over-expression conferred glycopeptide resistance to S. venezuelae. On the contrary, the addition of His6-tag at the N-terminus of the protein abolished its biological activity either in vitro or in vivo assays.ConclusionsHeterologous expression of vanYn from Nonomuraea sp. ATCC 39727 in S. venezuelae was successfully achieved and conferred the host an increased level of glycopeptide resistance. Cellular localization of recombinant VanYn together with its enzymatic activity as a d,d-peptidase/d,d-carboxypeptidase agree with its role in removing the last d-Ala from the pentapeptide PG precursors and reprogramming cell wall biosynthesis, as previously reported in glycopeptide resistant pathogens.
Highlights
VanYn, encoded by the dbv7 gene of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp
Genome sequencing of representative microbes belonging to this group has shown that they possess a vast array of genes devoted to the production and secretion of enzymes, due to the role they play in recycling organic material in the biosphere [2]
Streptomyces spp. are used as host systems for the production of heterologous proteins and of whole biosynthetic clusters originating from less easy-to-handle actinomycetes, such as those belonging to Nonomuraea, Actinoplanes, Planomonospora, Planobispora, Microbispora genera [1]
Summary
VanYn, encoded by the dbv gene ( known as vanYn) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. Streptomyces spp. are used as host systems for the production of heterologous proteins and of whole biosynthetic clusters originating from less easy-to-handle actinomycetes, such as those belonging to Nonomuraea, Actinoplanes, Planomonospora, Planobispora, Microbispora genera [1]. These uncommon actinomycetes possess a still-untapped richness of metabolic pathways - and some of them are valuable producers of new drugs - but their exploitation is often limited by the lack of genetic manipulation tools [5,6]
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