Keller Ullrich, Schauwecker Florian
Technical University of Berlin, Institute of Chemistry, Research Group Biochemistry, Franklinstrasse 29, D-10587 Berlin, Germany.
Comb Chem High Throughput Screen. 2003 Sep;6(6):527-40. doi: 10.2174/138620703106298707.
Non-ribosomal peptide synthetases (NRPS) are modular assembly lines catalysing the synthesis of many small peptides in microbes. Genetic replacements of domains or modules in NRPS encoded by gene clusters in Bacillus sp. with corresponding domains or modules from foreign NRPS have led in several cases to the in vivo synthesis of peptides with predicted amino acid substitutions. Fusion points were in variable regions between C- and A- or between T- and C-domains. Successful insertions of whole modules using fusion points in conserved regions internal to functional domains have also been reported. For studying the role of C-, A-, T- and TE (thioesterase)-domains in NRPS, several bi- and trimodular model-NRPS derived from natural NRPS systems were constructed and obtained after expression in E. coli with coexpression of a 4'- phosphopantetheine transferase or in suitable hosts such as the Streptomyces. Such enzymes were shown to catalyse in vitro synthesis of di- and tripeptides, respectively, with and without turnover depending on the presence of Te-domains. The enzymatic analysis revealed the mechanisms of the domains and proved their functional autonomy suggesting the possibility to use any NRPS interdomain region for fusions. Nevertheless, recombinant synthesis of longer and more complex peptides will still be restricted to alteration of existing structures by manipulations of NRPS gene clusters located on chromosomes or artificial chromosomes. Besides targeted replacements of domains and modules, reprogramming of NRPS by altering the substrate specificities of A-domains is a promising tool for the future to get novel peptides.
非核糖体肽合成酶（NRPS）是模块化装配线，催化微生物中许多小肽的合成。用来自外源NRPS的相应结构域或模块对芽孢杆菌属基因簇编码的NRPS中的结构域或模块进行基因替换，在一些情况下导致了体内合成具有预测氨基酸取代的肽。融合点位于C结构域和A结构域之间或T结构域和C结构域之间的可变区域。也有报道称，利用功能域内部保守区域的融合点成功插入了整个模块。为了研究C、A、T和TE（硫酯酶）结构域在NRPS中的作用，构建了几种源自天然NRPS系统的双模块和三模块模型NRPS，并在大肠杆菌中表达，同时共表达4'-磷酸泛酰巯基乙胺转移酶，或在链霉菌等合适宿主中表达后获得。这些酶被证明分别催化二肽和三肽的体外合成，根据Te结构域的存在情况，有或没有周转。酶促分析揭示了这些结构域的机制，并证明了它们的功能自主性，这表明可以使用任何NRPS结构域间区域进行融合。然而，更长、更复杂肽的重组合成仍将限于通过操纵位于染色体或人工染色体上的NRPS基因簇来改变现有结构。除了结构域和模块的靶向替换外，通过改变A结构域的底物特异性对NRPS进行重新编程是未来获得新型肽的一种有前途的工具。
