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首页> 外文期刊>BMC Biotechnology >Streptomyces spp. as efficient expression system for a d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance
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Streptomyces spp. as efficient expression system for a d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance

机译：链霉菌作为参与糖肽抗生素耐药性的d，d-肽酶/ d，d-羧肽酶的有效表达系统

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Background VanYn, 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 VanYn 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. 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. 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 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.
机译：由致力于A40926产生的生物合成簇的dbv7基因（也称为vanY n ）编码的背景VanY n 是一种参与自我机制的新型蛋白质。野野菌的抗性ATCC39727。这种丝状放线菌是一种不常见的微生物，难以操作，但具有生物技术价值，因为它可产生糖肽抗生素A40926，它是第二代达巴万星在临床开发的第三阶段的前体。为了研究VanY n 在生产性放线菌中对糖肽抗性的作用，需要适当的宿主-载体表达系统。结果表达载体pIJ86中vanY n 基因的克隆策略（GC比例为73.3％）产生了一个重组蛋白，在C端添加了一个编码组氨酸六聚体的标签（C-His 6 -vanY n ）或在N端（N-His 6 -vanY n ）。这些质粒用于转化3个链霉菌。 ，它们是通过基因处理的高GC含量革兰氏阳性细菌，在分类学上与同源生产商Nonomuraea sp。有关。获得了最高的蛋白质表达和纯化产量（在3？L生物反应器规模下，每升培养液含12？mg蛋白质）委内瑞拉链霉菌ATCC 10595中的那一种是快速生长的链霉菌，对糖肽敏感。 VanY n 是一种跨膜蛋白，很容易从细胞壁部分分离和回收。纯化的C-His 6 -VanY n 对细菌肽聚糖（PG）前体的合成类似物具有d，d-羧肽酶和d，d-二肽酶活性。 C-His 6 -VanY n 的过表达赋予委内瑞拉葡萄球菌糖肽抗性。相反，在蛋白质的N末端添加His 6 -标签在体外或体内测定中均取消了其生物学活性。结论Nonomuraea sp。的vanY n 的异源表达。在委内瑞拉葡萄球菌中成功获得ATCC 39727并赋予宿主更高水平的糖肽抗性。重组VanY n 的细胞定位及其作为ad，d-肽酶/ d和d-羧肽酶的酶活性与其在从五肽PG前体中去除最后一个d-Ala并重新编程细胞壁的作用相符如先前在糖肽抗性病原体中报道的那样进行生物合成。

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