Abstract
The bacterial species Streptomyces avermitilis is an important industrial producer of avermectins, which are widely utilized as effective anthelmintic and insecticidal drugs. We used gene deletion, complementation, and overexpression experiments to identify SAV4189, a MarR-family transcriptional regulator (MFR) in this species, as an activator of avermectin biosynthesis. SAV4189 indirectly stimulated avermectin production by altering expression of cluster-situated activator gene aveR, and directly repressed the transcription of its own gene (sav_4189) and adjacent cotranscribed gene sav_4190 (which encodes an unknown transmembrane efflux protein). A consensus 13-bp palindromic sequence, 5′-TTGCCYKHRSCAA-3′ (Y = T/C; K = T/G; H = A/C/T; R = A/G; S = C/G), was found within the SAV4189-binding sites of its own promoter region, and shown to be essential for binding. The SAV4189 regulon was thus predicted based on bioinformatic analysis. Night new identified SAV4189 targets are involved in transcriptional regulation, primary metabolism, secondary metabolism, and stress response, reflecting a pleiotropic role of SAV4189. sav_4190, the important target gene of SAV4189, exerted a negative effect on avermectin production. sav_4189 overexpression and sav_4190 deletion in S. avermitilis wild-type and industrial strains significantly increased avermectin production. SAV4189 homologs are widespread in other Streptomyces species. sav_4189 overexpression in the model species S. coelicolor also enhanced antibiotic production. The strategy of increasing yield of important antibiotics by engineering of SAV4189 homologs and target gene may potentially be extended to other industrial Streptomyces species. In addition, SAV4189 bound and responded to exogenous antibiotics hygromycin B and thiostrepton to modulate its DNA-binding activity and transcription of target genes. SAV4189 is the first reported exogenous antibiotic receptor among Streptomyces MFRs.
Highlights
Streptomyces are filamentous soil bacteria characterized by complex life cycle and production of numerous antibiotics
We examined the regulatory role of SAV4189, and found that it functions as an indirect activator of avermectin production
On the end of fermentation day 10, overexpression of sav_4189 increased avermectin yield by ∼2.5-fold, and deletion of sav_4189 led to a clear reduction (∼40%) in avermectin yield (Figure 1B). These findings indicate that SAV4189 plays an activator role in avermectin production
Summary
Streptomyces are filamentous soil bacteria characterized by complex life cycle and production of numerous antibiotics (van Wezel and McDowall, 2011). At least 20 families of transcriptional regulators are found in Streptomyces, including TetR, LuxR, LysR, AraC, GntR, SARP, ROK, MerR, and MarR (multiple antibiotic resistance regulator). MFRs control a variety of cellular processes, including multidrug resistance, pathogenicity, stress responses, metabolic pathways, and degradation of aromatic compounds (Perera and Grove, 2010; Romero-Rodríguez et al, 2015). They have characteristic winged helix-turn-helix (wHTH) DNA-binding domains and typically act as homodimeric transcriptional repressors (sometimes as activators, or both) by binding to palindromic sequences within target promoter regions. MFRs are the fourth most abundant family of transcriptional regulators in Streptomyces (average 50 per genome), but only a few members have been characterized in this genus, including Streptomyces coelicolor OhrR (Oh et al, 2007), TamR (Huang and Grove, 2013), PecS (Huang et al, 2013), and PcaV (Davis et al, 2013), S. exfoliatus PenR, S. treptomyces arenae PntR (Zhu et al, 2013), and S. roseosporus DptR3 (Zhang et al, 2015)
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