Streptomyces spp. as efficient expression system for a d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance

BMC Biotechnology

Research Article

Elisa Binda¹  Giorgia Letizia Marcone¹  Francesca Berini¹  Loredano Pollegioni¹  Flavia Marinelli¹ 

[1] Department of Biotechnology and Life Sciences, University of Insubria, via J. H. Dunant 3, Varese, Italy;“The Protein Factory” Research Center, Politecnico of Milano, ICRM CNR Milano and University of Insubria, Varese, Italy;

关键词: Streptomyces;    Heterologous protein production;    ,-carboxypeptidases;    Glycopeptide production;    Glycopeptide resistance;    Dalbavancin;   

DOI  :  10.1186/1472-6750-13-24

 received in 2012-11-26, accepted in 2013-03-10,  发布年份 2013

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Binda et al.; licensee BioMed Central Ltd. 2013