Activation of Secondary Metabolite Gene Clusters in Streptomyces clavuligerus by the PimM Regulator of Streptomyces natalensis.

Yolanda Martínez-Burgo,Andrew W. Truman,Fernando Reyes,José Rubén Tormo,Jesús F. Aparicio,Eva G. Barreales,Antonio Rodríguez-García,Javier Santos-Aberturas,Paloma Liras

doi:10.3389/fmicb.2019.00580

Abstract

Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex.

Highlights

PimM is a PAS-LuxR type regulator that is critical for the regulation of the biosynthetic gene cluster of pimaricin, a polyene macrolide antifungal compound produced by Streptomyces natalensis (Antón et al, 2007; Barreales et al, 2018)
Given the functional conservation of PASLuxR regulators across different strains, and their capacity to cross-regulate the production of polyene antibiotics and other structurally different compounds (Olano et al, 2014; Vicente et al, 2015), we introduced the pimM gene of S. natalensis into S. clavuligerus and studied its effect on both the transcription of biosynthetic pathways and the production of secondary metabolites
The PAS-LuxR type regulator PimM of S. natalensis was initially described as a pathway-specific transcriptional regulator of pimaricin biosynthesis (Antón et al, 2007; Santos-Aberturas et al, 2011a)

Summary

Introduction

PimM is a PAS-LuxR type regulator that is critical for the regulation of the biosynthetic gene cluster of pimaricin, a polyene macrolide antifungal compound produced by Streptomyces natalensis (Antón et al, 2007; Barreales et al, 2018). Homologous regulators are encoded by genes located in the clusters for other polyene macrolides, e.g., for amphotericin B in Streptomyces nodosus (AmphRIV; Carmody et al, 2004), for filipin in Streptomyces avermitilis (PteF; Omura et al, 2001; Ikeda et al, 2003) and Streptomyces filipinensis (FilF; Payero et al, 2015), and for candicidin in Streptomyces griseus (FscRI; Chen et al, 2003). These positive regulators are required for the production of the respective antibiotics in the producer strains Production of the polyketide/non-ribosomal peptide antimycin was activated by PimM in S. albus (Olano et al, 2014)

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Conclusion