摘要
通过使用fosmid文库对enkB周围的区域（Ent53B结构基因）测序来分析肠道菌素NKR-5-3B（Ent53B）（一种新型环状细菌素）的推定的生物合成基因簇。获得长度约9kb的区域，并鉴定编码参与Ent53B的产生，成熟和分泌的推定的生物合成蛋白的enkB1，enkB2，enkB3和enkB4基因。我们还确定了介导对Ent53B作用的自身免疫的蛋白质的身份。成功建立了各种异源宿主中的异源表达系统，如粪肠球菌和乳酸乳球菌菌株。成熟的Ent53B的产生和分泌需要五种基因的协同功能。 Ent53B仅由那些表达enkB，enkB1，enkB2，enkB3和enkB4基因的蛋白质产物的异源宿主产生。此外，对Ent53B的抗微生物作用的自身免疫性由至少两个独立的机制赋予。具有完整的enkB4基因和/或完整的enkB1和enkB3基因的组合的异源宿主对Ent53B的抑制作用是免疫的。

重要性除了它们作为食品防腐剂的潜在应用之外，圆形细菌素现在被认为是治疗性抗生素的可能替代物，因为它们的圆形结构赋予了特殊的稳定性。环状细菌素的成功实际应用只有在其生物合成的分子细节被完全理解的情况下才能成为可能。本研究的结果为循环细菌素生物合成的可能机制提供了新的视角。另外，由于一些肠球菌菌株与致病性，毒力和耐药性相关，建立第一个多重宿主异源生产的Ent53B具有非常高的实际意义，因为它拓宽了可能的Ent53B应用的范围。

A putative biosynthetic gene cluster of the enterocin NKR-5-3B (Ent53B), a novel circular bacteriocin, was analyzed by sequencing the flanking regions aroundenkB, the Ent53B structural gene, using a fosmid library. A region approximately 9 kb in length was obtained, and the enkB1, enkB2, enkB3, and enkB4 genes, encoding putative biosynthetic proteins involved in the production, maturation, and secretion of Ent53B, were identified. We also determined the identity of proteins mediating self-immunity against the effects of Ent53B. Heterologous expression systems in various heterologous hosts, such as Enterococcus faecalisand Lactococcus lactis strains, were successfully established. The production and secretion of the mature Ent53B required the cooperative functions of five genes. Ent53B was produced only by those heterologous hosts that expressed protein products of the enkB, enkB1, enkB2, enkB3, and enkB4 genes. Moreover, self-immunity against the antimicrobial action of Ent53B was conferred by at least two independent mechanisms. Heterologous hosts harboring the intact enkB4 gene and/or a combination of intact enkB1 and enkB3 genes were immune to the inhibitory action of Ent53B.
IMPORTANCE In addition to their potential application as food preservatives, circular bacteriocins are now considered possible alternatives to therapeutic antibiotics due to the exceptional stability conferred by their circular structure. The successful practical application of circular bacteriocins will become possible only if the molecular details of their biosynthesis are fully understood. The results of the present study offer a new perspective on the possible mechanism of circular bacteriocin biosynthesis. In addition, since some enterococcal strains are associated with pathogenicity, virulence, and drug resistance, the establishment of the first multigenus host heterologous production of Ent53B has very high practical significance, as it widens the scope of possible Ent53B applications.
http://jb.asm.org/content/198/2/291.short