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Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement

  • Applied genetics and molecular biotechnology
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Abstract

The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.

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Acknowledgments

We thank Kyowa Hakko Kogyo Co. Ltd. (Tokyo, Japan) for the wild-type S. atroolivaceus S-140 strain. This work was supported in part by NIH Grant CA106150.

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Author notes

  1. Dong Yang

    Present address: Xiangya International Academy of Translational Medicine, Central South University, Changsha, Hunan, 410013, China

  2. Gong-Li Tang

    Present address: State Key Laboratory of Bioorganic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Rd, Shanghai, 200032, China

Authors and Affiliations

  1. Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI, 53705, USA

    Yong Huang, Gong-Li Tang & Ben Shen

  2. Xiangya International Academy of Translational Medicine, Central South University, Changsha, Hunan, 410013, China

    Yong Huang

  3. Department of Chemistry, The Scripps Research Institute, Jupiter, FL, 33458, USA

    Dong Yang, Guohui Pan & Ben Shen

  4. Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, 33458, USA

    Ben Shen

  5. Natural Products Library Initiative at The Scripps Research institute, The Scripps Research Institute, Jupiter, FL, 33458, USA

    Ben Shen

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  1. Yong Huang
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Correspondence to Ben Shen.

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Yong Huang and Dong Yang contributed equally to this work.

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Huang, Y., Yang, D., Pan, G. et al. Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement. Appl Microbiol Biotechnol 100, 10555–10562 (2016). https://doi.org/10.1007/s00253-016-7864-2

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