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Research Article

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Chaoxian Bai, Yang Zhang, Xuejin Zhao, Yiling Hu, Sihai Xiang, Jin Miao, Chunbo Lou, and Lixin Zhang
PNAS first published September 15, 2015; https://doi.org/10.1073/pnas.1511027112
Chaoxian Bai
aChinese 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;
bUniversity of Chinese Academy of Science, Beijing 100149, China;
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Yang Zhang
aChinese 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;
bUniversity of Chinese Academy of Science, Beijing 100149, China;
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Xuejin Zhao
aChinese 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;
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Yiling Hu
aChinese 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;
cSchool of Life Sciences, Anhui University, Hefei 230601, China
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Sihai Xiang
aChinese 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;
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Jin Miao
aChinese 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;
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Chunbo Lou
aChinese 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;
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  • For correspondence: zhanglixin@im.ac.cn louchunbo@gmail.com
Lixin Zhang
aChinese 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;
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  • For correspondence: zhanglixin@im.ac.cn louchunbo@gmail.com
  1. Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved August 26, 2015 (received for review June 5, 2015)

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Significance

To meet the increasing demands of drug discovery and biosynthetic studies, we established a precise quantitative method based on flow cytometry at single-cell (protoplast) resolution in Streptomyces for the identification of regulatory elements. A series of native or synthetic promoters and ribosomal binding sites has been characterized. Moreover, an insulator was demonstrated to eliminate element–element interference. As a proof of concept, a native silent gene cluster was activated by the synthetic modular regulatory elements in a predictable manner. The universality of these elements is of high value to the synthetic biology of 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.

  • synthetic biology
  • natural product
  • flow cytometry
  • single-cell resolution
  • modular regulatory elements

Footnotes

  • ↵1C.B., Y.Z., and X.Z. contributed equally to this work.

  • ↵2To whom correspondence may be addressed. Email: zhanglixin{at}im.ac.cn or louchunbo{at}gmail.com.
  • Author contributions: C.L. and L.Z. designed research; C.B., Y.Z., X.Z., and Y.H. performed research; C.B., Y.Z., X.Z., Y.H., S.X., and J.M. analyzed data; and C.B., C.L., and L.Z. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1511027112/-/DCSupplemental.

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Measuring regulatory parts of Streptomyces by FACS
Chaoxian Bai, Yang Zhang, Xuejin Zhao, Yiling Hu, Sihai Xiang, Jin Miao, Chunbo Lou, Lixin Zhang
Proceedings of the National Academy of Sciences Sep 2015, 201511027; DOI: 10.1073/pnas.1511027112

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Measuring regulatory parts of Streptomyces by FACS
Chaoxian Bai, Yang Zhang, Xuejin Zhao, Yiling Hu, Sihai Xiang, Jin Miao, Chunbo Lou, Lixin Zhang
Proceedings of the National Academy of Sciences Sep 2015, 201511027; DOI: 10.1073/pnas.1511027112
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