Zhao Xuejin, Wei Weijia, Zong Yeqing, Bai Chaoxian, Guo Xian, Zhu Hua, Lou Chunbo
State Key Laboratory of Microbial Resources, CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Synth Syst Biotechnol. 2022 Jun 6;7(3):972-981. doi: 10.1016/j.synbio.2022.05.010. eCollection 2022 Sep.
The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter P, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal P promoter was determined, and a P promoter library with a wide range of strengths was constructed. Furthermore, a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in ISP5230. Theoretically, the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes. The yield of thaxtomin A was significantly improved to 202.1 15.3 g/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in ISP5230. Besides, the applicability of this transcription system was also tested in Streptomyces alb J1074, and the titer of thaxtomin A was raised to as high as 239.3 ± 30.6 μg/mL. Therefore, the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeeping σ factors for the heterologous expression of valuable products in diverse hosts
珍贵的天然产物帚曲霉素A是一种来自马铃薯疮痂病致病菌株的强效生物除草剂，但其天然产生菌的低产量严重阻碍了它的应用。在此，我们开发了一种正交转录系统，利用胞外功能（ECF）σ因子17（ECF17）及其同源启动子P来表达帚曲霉素基因簇并提高帚曲霉素A的产量。确定了最小P启动子，并构建了具有广泛强度范围的P启动子文库。此外，还开发了一种香豆酸盐诱导系统，用于在ISP5230中对ECF17转录系统进行精确的时间控制。理论上，可切换的ECF17转录系统可以减少来自宿主的不必要影响，并减轻异源基因过表达带来的负担。使用可切换的ECF17转录系统在ISP5230中对帚曲霉素基因簇进行异源表达，帚曲霉素A的产量显著提高到202.1±15.3μg/mL。此外，还在白色链霉菌J1074中测试了该转录系统的适用性，帚曲霉素A的效价提高到了239.3±30.6μg/mL。因此，诱导型ECF17转录系统可以作为基于强天然组成型启动子和管家σ因子的常用转录系统的补充，用于在不同宿主中异源表达有价值的产物。
