Double-reporter-guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527
Double-reporter-guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527
复制标题
DOI：
10.1002/bit.28347
复制
发表时间：
2023-02-18
影响因子：
3.8
通讯作者：
Yu，Xiaoping
中科院分区：
文献类型：
--
作者：
Shi，Yue;Zhang，Jinyao;Yu，Xiaoping
在龟裂链霉菌M527中，土霉素（OTC）生物合成基因簇在实验室条件下不表达。在这项研究中，使用报告指导的突变体选择（RGMS）程序来激活簇。构建了双报告基因质粒pAGT，其中编码β-葡萄糖醛酸酶的gusA和编码硫链丝菌素抗性甲基转移酶的tsrN被置于天然启动子ofoxyA基因（PoxyA）的控制下。将质粒pAGT导入S. rimosus M527，产生初始菌株M527-pAGT。随后，通过使用核糖体工程技术产生M527-pAGT的突变体。根据GUS活性和硫链丝菌素抗性的目测观察，选择了具有激活的OTC基因簇的突变体。最后，选择产生浓度为235.2 mg/L的OTC的突变体M527-pAGT-R7。在该突变体中，与野生型菌株相比，oxysr基因特别是oxyAsr基因的转录水平增加。rimosus M527.突变体M527-pAGT-R7对革兰氏阴性和革兰氏阳性菌株显示出拮抗活性。所有数据表明，OTC基因簇被成功激活使用RGMS方法。
InStreptomyces rimosusM527, the oxytetracycline (OTC) biosynthetic gene cluster is not expressed under laboratory conditions. In this study a reported‐guided mutant selection (RGMS) procedure was used to activate the cluster. The double‐reporter plasmid pAGT was constructed in whichgusAencoding a β‐glucuronidase andtsrencoding a thiostrepton resistance methyltransferase were placed under the control of the native promoter ofoxyAgene (PoxyA). Plasmid pAGT was introduced and integrated into the chromosome ofS. rimosusM527 by conjugation, yielding initial strain M527‐pAGT. Subsequently, mutants of M527‐pAGT were generated by using ribosome engineering technology. The mutants harboring activated OTC gene cluster were selected based on visual observation of GUS activity and thiostrepton resistance. Finally, mutant M527‐pAGT‐R7 was selected producing OTC in a concentration of 235.2 mg/L. In this mutant transcriptional levels ofoxysrgenes especialoxyAsrgene were increased compared to wild‐type strainS. rimosusM527. The mutant M527‐pAGT‐R7 showed antagonistic activities against Gram‐negative and Gram‐positive strains. All data indicate that the OTC gene cluster was successfully activated using the RGMS method.