title: Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement.
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.
authors.affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI, 53705, USA.|Xiangya International Academy of Translational Medicine, Central South University, Changsha, Hunan, 410013, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL, 33458, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL, 33458, USA.
authors.forename: Guohui
authors.initials: G
authors.lastname: Pan
authors.affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI, 53705, USA.|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.forename: Gong-Li
authors.initials: GL
authors.lastname: Tang
authors.affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI, 53705, USA. shenb@scripps.edu.|Department of Chemistry, The Scripps Research Institute, Jupiter, FL, 33458, USA. shenb@scripps.edu.|Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, 33458, USA. shenb@scripps.edu.|Natural Products Library Initiative at The Scripps Research institute, The Scripps Research Institute, Jupiter, FL, 33458, USA. shenb@scripps.edu.
authors.forename: Ben
authors.initials: B
authors.lastname: Shen
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics*; D019295:Computational Biology; D015870:Gene Expression; D020868:Gene Silencing; D005809:Genes, Regulator; D007769:Lactams / Q000378:metabolism*; D018942:Macrolides / Q000378:metabolism*; D060847:Metabolic Engineering; D005810:Multigene Family; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism*; D013871:Thiones / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D007769:Lactams; D018942:Macrolides; D013844:Thiazoles; D013871:Thiones; D014157:Transcription Factors; C059000:leinamycin
keywords: Biosynthesis; Crp/Fnr regulator; Leinamycin; Regulation; Titer improvement