Wang Xiuling, Lin Ping, Shen Qiyao, Feng Xueyan, Xu Shouying, Zhang Qijun, Liu Yang, Ren Cailing, Yong Daojing, Duan Qiong, Huo Liujie, Zhang Youming, Li Gang, Fu Jun, Li Ruijuan
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, 266237, China.
Department of Natural Medicinal Chemistry and Pharmacognosy, School of Pharmacy, Qingdao University, Qingdao, 266071, China.
Microb Cell Fact. 2025 May 14;24(1):105. doi: 10.1186/s12934-025-02722-z.
Heterologous expression in Streptomyces provides a platform for mining natural products (NPs) encoded by cryptic biosynthetic gene clusters (BGCs) of bacteria. The BGCs are first engineered in hosts with robust recombineering systems, such as Escherichia coli, followed by expression in optimized heterologous hosts, such as Streptomyces, with defined metabolic backgrounds.
We developed a highly efficient heterologous expression platform, named Micro-HEP (microbial heterologous expression platform), that uses versatile E. coli strains capable of both modification and conjugation transfer of foreign BGCs and optimized chassis Streptomyces strain for expression. The stability of repeat sequences in these E. coli strains was superior to that of the commonly used conjugative transfer system E. coli ET12567 (pUZ8002). For optimizing expression of foreign BGCs, the chassis strain S. coelicolor A3(2)-2023 was generated by deleting four endogenous BGCs followed by introducing multiple recombinase-mediated cassette exchange (RMCE) sites in the S. coelicolor A3(2) chromosome. Additionally, modular RMCE cassettes (Cre-lox, Vika-vox, Dre-rox, and phiBT1-attP) were constructed for integrating BGCs into the chassis strain. Micro-HEP was tested using BGCs for the anti-fibrotic compound xiamenmycin and griseorhodins. Two to four copies of the xim BGC were integrated by RMCE, with increasing copy number associated with increasing yield of xiamenmycin. The grh BGC was also efficiently expressed, and the new compound griseorhodin H was identified.
We demonstrated that our Micro-HEP system enables the efficient expression of foreign BGCs, facilitating the discovery of new NPs and increasing yields.
在链霉菌中进行异源表达为挖掘细菌隐秘生物合成基因簇（BGCs）编码的天然产物（NPs）提供了一个平台。首先在具有强大重组工程系统的宿主（如大肠杆菌）中对BGCs进行工程改造，然后在具有明确代谢背景的优化异源宿主（如链霉菌）中进行表达。
我们开发了一个高效的异源表达平台，名为Micro-HEP（微生物异源表达平台），它使用能够对外源BGCs进行修饰和接合转移的通用大肠杆菌菌株以及用于表达的优化底盘链霉菌菌株。这些大肠杆菌菌株中重复序列的稳定性优于常用的接合转移系统大肠杆菌ET12567（pUZ8002）。为了优化外源BGCs的表达，通过删除四个内源性BGCs，然后在天蓝色链霉菌A3(2)染色体中引入多个重组酶介导的盒式交换（RMCE）位点，构建了底盘菌株天蓝色链霉菌A3(2)-2023。此外，还构建了模块化RMCE盒（Cre-lox、Vika-vox、Dre-rox和phiBT1-attP），用于将BGCs整合到底盘菌株中。使用用于抗纤维化化合物厦门霉素和灰紫红菌素的BGCs对Micro-HEP进行了测试。通过RMCE整合了两到四个拷贝的xim BGC，拷贝数增加与厦门霉素产量增加相关。grh BGC也得到了有效表达，并鉴定出了新化合物灰紫红菌素H。
我们证明了我们的Micro-HEP系统能够高效表达外源BGCs，有助于发现新的NPs并提高产量。
