Sioud Samiha, Karray-Rebai Ines, Aouissaoui Hedi, Aigle Bertrand, Bejar Samir, Mellouli Lotfi
Laboratory of Prokaryotic Enzymes and Metabolites, Centre of Biotechnology of Sfax, Road of Sidi Mansour Km 6, P.O. Box K, 3038 Sfax, Tunisia.
J Biomed Biotechnol. 2007;2007(6):91409. doi: 10.1155/2007/91409.
We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro) diketopiperazine (DKP). To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp) was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS). The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro) biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.
我们之前从突尼斯土壤中分离出一种名为链霉菌属US24的新型放线菌菌株，并表明它能产生两种生物活性分子，其中包括环（L-苯丙氨酸，L-脯氨酸）二酮哌嗪（DKP）。为了鉴定负责合成这种DKP衍生物的结构基因，以链霉菌属US24基因组DNA为模板，使用与编码肽合成酶（NRPS）的基因类似设计的两个简并寡核苷酸进行了PCR扩增（696 bp）。然后，通过使用源自接合质粒pSET152并包含PCR产物的自杀载体，通过同源重组进行基因破坏，实现了对链霉菌属US24菌株DKP衍生物生物合成途径的检测。对野生型链霉菌属US24菌株以及通过这种基因靶向破坏方法获得的三个突变体的上清液培养物进行色谱分析、生物学测试和光谱研究表明，扩增的DNA片段是链霉菌属US24菌株中环（L-苯丙氨酸，L-脯氨酸）生物合成所必需的。这种DKP衍生物似乎要么直接通过非核糖体途径产生，要么是在较长肽的非核糖体合成过程中作为副产物产生。
