title: Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces.
abstract: There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Science, Beijing 100149, China;
authors.forename: Chaoxian
authors.initials: C
authors.lastname: Bai
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Science, Beijing 100149, China;
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
authors.forename: Xuejin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; School of Life Sciences, Anhui University, Hefei 230601, China.
authors.forename: Yiling
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
authors.forename: Sihai
authors.initials: S
authors.lastname: Xiang
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
authors.forename: Jin
authors.initials: J
authors.lastname: Miao
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; zhanglixin@im.ac.cn louchunbo@gmail.com.
authors.forename: Chunbo
authors.initials: C
authors.lastname: Lou
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; zhanglixin@im.ac.cn louchunbo@gmail.com.
authors.forename: Lixin
authors.initials: L
authors.lastname: Zhang
mesh_terms: D001665:Binding Sites / Q000235:genetics; D001688:Biological Products / Q000032:analysis / Q000302:isolation & purification*; D055808:Drug Discovery / Q000379:methods* / Q000639:trends; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D005810:Multigene Family / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011419:Propidium; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D013302:Streptomyces / Q000737:chemistry*; D058615:Synthetic Biology / Q000379:methods* / Q000639:trends
chemical_list: D001688:Biological Products; D049452:Green Fluorescent Proteins; D011419:Propidium
keywords: flow cytometry; modular regulatory elements; natural product; single-cell resolution; synthetic biology