Eustáquio Alessandra S, Li Shu-Ming, Heide Lutz
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Microbiology (Reading). 2005 Jun;151(Pt 6):1949-1961. doi: 10.1099/mic.0.27669-0.
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG. The predicted gene product of novG shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism Streptomyces spheroides was expressed in the heterologous host Streptomyces coelicolor M512, and additional strains were produced which lacked the novG gene within the heterologously expressed cluster. These Delta novG strains produced only 2% of the novobiocin formed by the S. coelicolor M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of novG into the mutant. The presence of novG on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His(6)-tagged NovG showed specific DNA-binding activity for the novG-novH and the cloG-cloY intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N)(11)CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins.
氨基香豆素抗生素新生霉素的生物合成基因簇包含两个假定的调控基因，即novE和novG。novG的预测基因产物显示出一个假定的螺旋-转角-螺旋DNA结合基序，并且与StrR具有序列相似性，StrR是一种对链霉素生物合成进行了充分研究的途径特异性转录激活因子。在此，通过遗传和生化方法提供了功能证据，证明NovG作为新生霉素生物合成的正调控因子的作用。产生菌球形链霉菌的整个新生霉素基因簇在异源宿主天蓝色链霉菌M512中表达，并产生了在异源表达的基因簇中缺少novG基因的其他菌株。这些缺失novG的菌株产生的新生霉素仅为携带完整新生霉素基因簇的天蓝色链霉菌M512菌株所形成新生霉素的2%。通过将完整的novG拷贝引入突变体中，可以恢复产量。在含有完整基因簇的菌株中，novG存在于多拷贝质粒上导致抗生素产量几乎增加了三倍，这表明新生霉素的生物合成受到NovG蛋白可用性的限制。此外，纯化的N端带有His(6)标签的NovG分别对新生霉素和氯新生霉素生物合成基因簇的novG-novH和cloG-cloY基因间区域显示出特异性DNA结合活性。通过比较与NovG结合的片段的DNA序列，在两个片段中都鉴定出了保守的反向重复序列，类似于被鉴定为StrR结合位点的序列。StrR和假定的NovG结合位点的共有序列为GTTCRACTG(N)(11)CRGTYGAAC。因此，NovG和StrR显然属于同一类DNA结合调控蛋白家族。
