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

BMC Biotechnology

Elisa Binda¹  Giorgia Letizia Marcone¹  Francesca Berini¹  Loredano Pollegioni¹  Flavia Marinelli¹ 

[1] “The Protein Factory” Research Center, Politecnico of Milano, ICRM CNR Milano and University of Insubria, Varese, Italy

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

Others  :  1134516
DOI  :  10.1186/1472-6750-13-24

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

【 摘 要 】

Background

VanY_n, encoded by the dbv7 gene (also known as vanY_n) 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 VanY_n role in glycopeptide resistance in the producer actinomycete an appropriate host-vector expression system is required.

Results

The cloning strategy of vanY_n 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-vanY_n) or at the N-terminus (N-His₆-vanY_n). 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. VanY_n is a transmembrane protein which was easily detached and recovered from the cell wall fraction. Purified C-His₆-VanY_n showed D,D-carboxypeptidase and D,D-dipeptidase activities on synthetic analogs of bacterial peptidoglycan (PG) precursors. C-His₆-VanY_n 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.

Conclusions

Heterologous expression of vanY_n 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 VanY_n 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】

   
2013 Binda et al.; licensee BioMed Central Ltd.

【 图 表 】

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