Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product

Abstract

Avermectins produced by Streptomyces avermitilis are commercially important anthelmintic agents. The detailed regulatory mechanisms of avermectin biosynthesis remain unclear. Here, we identified SAV3619, a TetR-family transcriptional regulator designated AveT, to be an activator for both avermectin production and morphological differentiation in S. avermitilis. AveT was shown to indirectly stimulate avermectin production by affecting transcription of the cluster-situated activator gene aveR. AveT directly repressed transcription of its own gene (aveT), adjacent gene pepD2 (sav_3620), sav_7490 (designated aveM), and sav_7491 by binding to an 18-bp perfect palindromic sequence (CGAAACGKTKYCGTTTCG, where K is T or G and Y is T or C and where the underlining indicates inverted repeats) within their promoter regions. aveM (which encodes a putative transmembrane efflux protein belonging to the major facilitator superfamily [MFS]), the important target gene of AveT, had a striking negative effect on avermectin production and morphological differentiation. Overexpression of aveT and deletion of aveM in wild-type and industrial strains of S. avermitilis led to clear increases in the levels of avermectin production. In vitro gel-shift assays suggested that C-5-O-B1, the late pathway precursor of avermectin B1, acts as an AveT ligand. Taken together, our findings indicate positive-feedback regulation of aveT expression and avermectin production by a late pathway intermediate and provide the basis for an efficient strategy to increase avermectin production in S. avermitilis by manipulation of AveT and its target gene product, AveM. Soil-dwelling species of Streptomyces produce about half of currently known antibiotics (including antibacterial, anticancer, anthelmintic, and immunosuppressive agents) during their complex morphological differentiation cycle (1) and have many important medical and commercial applications. Antibiotic biosynthesis is controlled by large gene clusters, usually including cluster-situated regulators (CSRs). These CSRs are at the lowest level of the complex regulatory network for antibiotic biosynthesis and are controlled by various higher-level pleiotropic regulators in response to developmental state, population density, environmental signals, and physiological signals (2-4).The species Streptomyces avermitilis produces avermectins, a series of 16-membered macrocyclic lactones (termed A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b) that are excellent anthelmintic agents with high potency, broad-spectrum activity against various arthropod and nematode parasites, and a low level of side effects on the host (5, 6). Of the eight avermectin components, B1a has the highest insecticidal activity (7). Avermectins are a commercially important group of antibiotics with annual worldwide sales of ϳ$850 million (8) and are widely applied in the agricultural, veterinary, and medical fields. The 82-kb ave gene cluster that controls avermectin biosynthesis includes 18 open reading frames (ORFs) (9). The gene aveR, loc...

The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

“…The biosynthetic genes of antibiotics are generally clustered together on chromosomes or plasmids (31, 32) and usually contain one or more CSRs (33) that provide a direct contributions to antibiotic production through transcriptional regulation of other biosynthetic genes in the clusters (18,34). In the present study, we identified and characterized LmbU, a novel transcriptional regulator that is involved in the regulation of lincomycin biosynthesis.…”

AvaR2, a pseudo γ‐butyrolactone receptor homologue from Streptomyces avermitilis, is a pleiotropic repressor of avermectin and avenolide biosynthesis and cell growth

“…We reported that PhoP, the response regulator of the two‐component PhoR‐PhoP system that responds to phosphate limitation, directly inhibits avermectin production by binding to the PHO box in the aveR promoter (Yang et al ., ). Some characterized regulators shown to control avermectin biosynthesis through indirect effects on aveR transcription are AveI (Chen et al ., ), SAV7471 (Liu et al ., ), SAV576 (Guo et al ., ), SAV151 (He et al ., ), SAV577 (Guo et al ., ), AveT (Liu et al ., ) and extracytoplasmic function (ECF) σ 25 (Luo et al ., ). To date, no other regulators have been reported to directly regulate avermectin production via aveR .…”

Sodium Lactate Negatively Regulates Shewanella putrefaciens CN32 Biofilm Formation via a Three-Component Regulatory System (LrbS-LrbA-LrbR)

“…Sputcn32_0304, the second response regulator in the three-component regulatory system, has a predicted effector domain with a helix-turn-helix (HTH) motif that is essentially similar to the HTH domains in the LuxR/GerE family transcriptional regulators. In previous studies, a transcription factor was shown to regulate the expression of its own gene (39,42). To determine whether Sputcn32_0304 directly regulates the expression of its own gene and Sputcn32_0305, an electrophoretic mobility shift assay (EMSA) was performed.…”

“…Two single mutants of Sputcn32_0304 and Sputcn32_0305, Δ0304 and Δ0305, were constructed. The deletion mutant of Sputcn32_0304 was constructed according to previous studies (39)(40)(41), which can avoid the polar effect on expression of the downstream gene Sputcn32_0303 and facilitate the investigation on its own expression changes. The biofilm biomasses of both single mutants were significantly increased compared to that of the WT strain, and biofilms of both complementation strains C-0304 and C-0305 were restored to the WT level ( Fig.…”

“…All other deletion mutants were constructed according to previous studies (39)(40)(41). To construct the Δ0304 mutant, PCR was used to generate a 1,587-bp DNA fragment upstream (positions Ϫ1443 to 144 relative to the Sputcn32_0304 start codon) and a 1,529-bp DNA fragment downstream (positions 565 to 2094 relative to the Sputcn32_0304 start codon) of the Sputcn32_0304 gene (reserving 144 bp after the start codon for real-time RT-PCR analysis) using primers 0304-5F/0304-5R and 0304-3F/0304-3R, respectively.…”