The LuxR family regulator SAV111 enhances avermectin production by binding to aveA1 gene promoter in Streptomyces avermitilis

Abstract

Avermectins, macrocyclic lactones produced by Streptomyces avermitilis, serve as essential therapeutic and agrochemical agents. LuxR-type transcriptional regulators are multifunctional proteins known to orchestrate antibiotic biosynthesis alongside virulence modulation, biofilm dynamics, and host immune interactions. However, no prior studies have characterized LuxR-family proteins as direct regulators of avermectin biosynthesis. This study delineates the mechanism through which SAV111, a LuxR-family transcriptional activator, enhances avermectin biosynthesis. Batch fermentation of the SAV111-overexpressing strain demonstrated dual functionality: (1) significant upregulation of avermectin titers and (2) accelerated hyphal growth kinetics. Developmental profiling revealed precocious morphological differentiation in the overexpression strain compared to wild-type controls. Electrophoretic mobility shift assays confirmed direct binding of SAV111 to the aveA1 promoter, encoding the synthase catalytic subunit within the pathway of avermectin biosynthetic. Transcriptional activation of aveA1 represents the primary mechanism underlying SAV111-mediated avermectin overproduction. These findings advance the understanding of LuxR-family regulatory networks in secondary metabolism and establish a molecular framework for engineering hyperproductive S. avermitilis strains.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00203-025-04576-w.

Introduction

Renowned for their metabolic versatility, Streptomyces synthesize diverse bioactive secondary metabolites with substantial pharmaceutical applications, including antimicrobial agents, immunosuppressants, and anticancer therapeutics (Liu et al. 2018a, b). Streptomyces avermitilis synthesizes the 16-membered macrolide antibiotic avermectin, a fermentation product renowned for its potent anthelmintic and insecticidal properties, which underpin its extensive applications in veterinary medicine, agricultural pest control, and human helminthiasis treatment (Dong and Zhang 2020, Lu et al. 2020). Antibiotic biosynthesis in Streptomyces is hierarchically governed by transcriptional regulators, spanning pathway-specific cluster-situated regulators (CSRs) and global pleiotropic controllers. Notably, certain transcription factors orchestrate both morphological development and antibiotic biosynthesis, enabling strain improvement through targeted genetic engineering (Jin et al. 2023).

The LuxR N-terminal signal-binding domain (SBD), containing an α/β fold, mediates interactions with quorum-sensing signaling molecules such as acyl-homoserine lactones (AHLs). LuxR-type regulators typically harbor a C-terminal HTH DNA-binding domain (Zhang et al. 2022). These proteins form a multi-level regulatory network in Streptomyces through sequence-specific DNA binding, signal sensing, and interactions with other regulatory factors. LuxR-type regulators could affect various metabolic processes, including morphology. LuxR protein or LuxR-type transcriptional regulators typically control biofilm formation in pathogens, such as Klebsiella pneumonia (Wilksch et al. 2011) and the species Vibrio (Ball et al. 2017). An orphan LuxR receptor PpoR participates in the biofilms formation and architecture in consortium with the bacterium Pseudomonas putida in the fugus Ophiostoma piceae (Subramoni et al. 2009; Fernández-Piñar et al. 2011; Alberto et al. 2021). Some of the pleiotropic LuxR-family controllers simultaneously regulate morphological differentiation and antibiotic production. In Sterptomyces coelicolor, LAL-regulators (Large ATP-binding regulators of the LuxR family) SCO0877 and SCO7173 could affect actinorhodin biosynthesis and morphological differentiation through PhoRP system (Guerra et al. 2012; Allenby et al. 2012). In Streptomyces albulus, the LuxR homolog WysPII shortens the spore formation time by activating the expression of genes related to aerial mycelium formation and fermentation time to reach the maximum Wuyiencin production (Liu et al. 2018a, b). In Streptomyces ahygroscopicus, overexpression of PAS-LuxR family gene can enhance metabolic flux. This regulatory effect may be achieved through the formation of a complex network with other regulatory factors (e.g., SARP family proteins) (Yan et al. 2024). Recent studies have identified LuxR homologs and acyl-homoserine lactone (AHL) synthase-like genes in six Streptomyces species suggesting they may regulate secondary metabolism through AHL-like signaling molecules. The SARP family protein ActII-orf4 and LuxR homologs in S. coelicolor synergistically activate the expression of actinorhodin biosynthetic genes by sharing direct repeats in the promoter region (Salehi-Najafabadi et al. 2024). RapH (LAL-type) overexpression boosts rapamycin yield by 46.5% in Streptomyces rapamycinicus (He et al. 2022).

In S. avermitilis, LuxR family proteins play significant roles in multiple biological processes. The LuxR family protein PteF affects the synchrony between filipin synthesis and morphological development by regulating the transcription of mycelial differentiation genes (Du et al. 2024). AveR, another important LuxR-related protein, is a pathway-specific positive regulator for avermectin biosynthesis. Additinallly, other types of regulator were reported to control both avermectin biosynthesis and morphological development in S. avermitilis. The knock-out strain of AfsKav (an eukaryotic serine/threonine protein kinase) no longer produces spores, melanin, or avermectin. bldA encodes the only tRNA that efficiently translates the rare UUA leucine codon in S. coelicolor and the bldA homologue inactivation showed a bald phenotype and did not synthesize avermectins (Tao et al. 2007). The autoregulator receptor AvaR3 positively controls avermectin production and cell morphology both in liquid and solid culture (Miyamoto et al. 2011). TetR-family regulator AveI controls avermectin and oligomycin production, and morphological differentiation (Liu et al. 2019). The heat shock response regulator HspR and the redox-sensitive transcriptional regulator SoxR directly controls avermectin production, morphological development in S. avermitilis (Lu et al. 2021; Wang et al. 2022). OmpR-family regulator MtrA (SAV5063) negatively regulates avermectin biosynthesis while positively controls morphological differentiation (Tian et al. 2024). In S. avermitilis, the functions and regulatory roles of other LuxR family proteins remain poorly understood. Through more in-depth investigations, we may identify additional regulatory proteins involved in avermectin biosynthesis.

Through iterative mutagenesis, we derived S. avermitilis 76-02-e, a hyperproductive avermectin strain. Transcriptomic profiling (Table S1) revealed SAV111, encoding a LuxR-family transcriptional activator, exhibited excess 7.88-fold upregulation during early fermentation stage on day 2, and 1.29-fold downregulation during late fermentation stage on day 6. This phenomenon raised our interest to explore the role of SAV111. In S. avermitilis, the functional role of the SAV111 protein remains uncharacterized, and its regulatory relationship with avermectin biosynthesis has not yet been reported. These findings implicate SAV111 as a putative positive regulator of avermectin biosynthesis, SAV111 may coordinate with pathway-specific regulators (e.g., AveR) to fine-tune polyketide synthase assembly and precursor supply, as observed in analogous regulatory modules governing macrolide antibiotic production in SAV111 overexpression strain. Consequently, this study elucidates SAV111’s regulatory role in avermectin biosynthesis, advancing our understanding of secondary metabolite regulation networks and facilitating rational engineering of industrial overproducers.

Materials and methods

Bacterial strains, plasmids, and culture conditions

S. avermitilis strains and Escherichia coli strains were cultivated under standardized culture conditions as previously described (Guo et al. 2018). Phenotypic characterization of S. avermitilis was conducted on YMS (Yeast Malt Extract-Starch), MM (Minimal Medium), and RM14-agar media following published protocols (Zhu et al. 2016). For routine avermectin biosynthesis, fermentation was performed in FM-I medium optimized for secondary metabolite production (Zhu et al. 2016). Bacterial growth kinetics were quantified using FM-II-liquid fermentation medium under controlled aerobic conditions (Zhu et al. 2016).

Overexpress SAV111 in S. avermitilis

To generate the SAV111 overexpression strain, we used genomic DNA from the wild-type S. avermitilis ATCC31267^T as the template and amplified the SAV111 gene by polymerase chain reaction (PCR). The primers used for amplification were 5’-CGGGATCCCCGCTTCCGTGCTGCGCC-3’ and 5’-CCCAAGCTTTCACAGGCCCAGGGAGAT-3’. The amplified SAV111 gene was subsequently digested with restriction enzymes HindIII and BamHI (TaKaRa, Japan) and ligated downstream of promoter of the erythromycin resistance gene (ermE*p) in pJL117 vector. The resulting fusion fragment, containing SAV111 and ermE*p, was then inserted into the pSET152 vector (digested with BglII) to construct the recombinant plasmid pSET152-111. This plasmid was introduced into S. avermitilis ATCC31267^T by transformation. The overexpression strain was successfully obtained by screening and verification.

Fermentation and analysis of avermectin production

Fermentation of both the wild-type and the overexpression strains was conducted as previously described (Luo et al. 2014). Briefly, 1.0 mL of fermentation broth was extracted with 4.0 mL methanol for 30 min, and subjected to centrifugation at 4,000 × g for 15 min. The supernatant was analyzed using high-performance liquid chromatography (HPLC) (Model 600; Waters, Milford, CT, USA) equipped with a C18 column (10 μm, 4.6 × 150 mm), with the mobile phase consisted of methanol: water (90:10, v/v) at a flow rate of 1.0 mL/min and a column temperature of 40 °C. The injection volume was 20 µL, and avermectins in the supernatant were detected by UV absorption spectrum at 246 nm. Commercial authentic avermectin B1 was used as an internal standard for quantification. Inoculate wild-type S. avermitilis and overexpression strains into FM-II medium for shake-flask fermentation (28 °C, 220 rpm). Transfer 1 mL of fermentation broth into 4 mL of methanol, and treated as previously described. Centrifuge the remaining broth at 4,000 × g for 20 min, discard the supernatant, dry the pellet in an oven at 80 °C for 24 h followed by 50 °C until constant weight, then weigh. Calculate the specific avermectin productivity using the formula: (Avermectin titer (mg/mL) × 50 mL)/(Cell dry weight (g).

Heterologous expression of SAV111 in E. coli and protein purification

The coding sequence of the SAV111 gene (330 bp, encoding 110 amino acids) was amplified by PCR. The PCR product was cloned into the pGEX-4T vector to generate the recombinant plasmid pGEX-111, which encodes an N-terminal glutathione S-transferase (GST)-tagged SAV111 protein and was verified by DNA sequencing. Then the plasmid pGEX-111 was transformed into E. coli BL21 (DE3). Protein overexpression was induced by isopropyl-β-D-thiogalactoside (IPTG) and incubating at 16 °C for 8 h. Cells were harvested and lysed by sonication in STE buffer (150.0 mM NaCl, 1.0 mM EDTA, 10.0 mM Tris-HCl, pH 8.0). After centrifugation, the supernatant was purified using a glutathione-Sepharose column (Tiangen) and extensively washed with PBS buffer (150.0 mM NaCl, 38.7 mM Na₂HPO₄, 11.3 mM NaH₂PO₄, pH 7.4), and the GST-SAV111 protein was eluted with elution buffer (150.0 mM NaCl, 20.0 mM glutathione, 10.0 mM Tris-HCl, 1.0 mM EDTA, pH 8.0). The concentration of GST-SAV111 was determined using the Bradford assay, and the purified protein was stored at -80 °C.

RNA isolation and real-time quantitative PCR (RT-qPCR) analysis

The mycelia cultures grown in medium FM-II for the exponential phase (two days) and the stationary phase (six days) were harvested. Total RNA was extracted with TRIzol reagent (Tiangen, China). RNA samples were assessed using a NanoVue™ Plus spectrophotometer. For cDNA synthesis, RNA was reverse transcribed using M-MLV reverse transcriptase (RNase-free, Promega), random hexamers, and a dNTP mixture. RT-qPCR was performed to quantify the expression levels of aveR and aveA1. The 16 S rRNA gene was served as an internal control. Reactions were carried out using reagent of FastStart Universal SYBR Green Master (ROX; Roche, USA) on an instrument of Roche LightCycler 480 (Roche, USA).

Electrophoretic mobility shift assay (EMSA)

Electrophoretic mobility shift assays (EMSAs) were performed using the Roche DIG Gel Shift Kit (2nd Generation) as previously described (Luo et al. 2014). DNA probes containing the promoter regions were amplified by PCR, labeled with digoxigenin, and incubated with varying concentrations of purified GST-SAV111 protein. Each 20 µL binding reaction was disaggregated on a non-denaturing polyacrylamide gel. DNA was transferred to a positively charged nylon membrane via electroblotting. The membrane was treated and detected using a luminescent substrate. Autoradiography was performed using X-ray film.

The response of SAV111 to alkaline, osmotic, oxidative, detergent and EDTA stress

To detect the stress factors that elicit responses from SAV111 in S. avermitilis, gradient dilution spore suspensions of the SAV111 overexpression strain OESAV111 and the WT strain were spotted onto YMS solid medium containing 3.5% NaCl (osmotic stress), 0.03% sodium dodecyl sulfate (SDS detergent), 0.0004% NaOH (pH 10), H₂O₂ (oxidative stress), and ethylenediaminetetraacetic acid (EDTA), respectively, for cultivation.

Results

Overexpression of SAV111 Enhances Avermectin Biosynthesis

The SAV111 gene encodes a LuxR family transcriptional regulator characterized by a C-terminal HTH DNA-binding domain. Despite this structural information, the biological function of SAV111 protein remains uncharacterized. To elucidate its functional role, we performed RT-qPCR analysis to quantify SAV111 expression levels in wild-type S. avermitilis. Notably, SAV111 expression was undetectable on day 2 and day 6 during the fermentation process (Fig. S1). Based on this observation, we hypothesized that SAV111 deletion would not result in phenotypic alterations compared to the wild-type strain. Consequently, we focused on constructing a SAV111 overexpression strain, designated as WT/pSET152-111. HPLC analysis revealed a significant 37% increase in avermectin production in the overexpression strain (Fig. 1), demonstrating that SAV111 positively regulates avermectin biosynthesis.

Fig. 1.

Conserved domain of SAV111 (A) and avermectin production in wild-type and SAV111 overexpression strain (µg avermectin per millilitre of culture) (B). Error bars: Standard deviation from three independent biological replicate experiments. Statistical significance was analyzed by Student’s t-test. *P < 0.05; **, P < 0.01; ***, P < 0.001. NS: not significant

To determine whether the enhanced avermectin production was associated with changes in cellular biomass, we conducted comparative growth analysis between the wild-type and SAV111 overexpression strains. Fermentation data indicated that the overexpression strain exhibited higher dry cell weight measurements compared to the wild-type (Fig. 2B). Simultaneously, HPLC analysis of the fermentation products revealed that the avermectin yield of the overexpression strain was significantly higher than that of the wild-type strain. (Fig. 2A). Furthermore, when normalized to biomass, the avermectin yield per unit was significantly greater in the overexpression strain (Fig. 2C). These findings suggest that SAV111 not only functions as a positive regulator of avermectin biosynthesis pathway but also enhances the growth capacity. The increased avermectin production in the overexpression strain can be partially attributed to the elevated biomass accumulation.

Fig. 2.

Avermectin yield (A), Growth curve (B) and avermectin yield curve per mg dry cell weight (C) of WT and SAV111 overexpression strain. Error bars: Standard deviation from three independent biological replicate experiments. Statistical significance was analyzed by Student’s t-test. *P < 0.05; **, P < 0.01; ***, P < 0.001. NS: not significant

SAV111 up-regulates transcription of aveA1 and aveR

To elucidate the mechanism underlying avermectin overproduction by SAV111, we analyzed mycelial RNA samples collected at days 2 and 6 of FM-II cultivation. Quantitative transcriptional profiling revealed significantly elevated expression levels of both aveR (the pathway-specific positive regulatory gene for avermectin biosynthesis) and aveA1 (the structural gene encoding avermectin polyketide synthase) in the overexpression strain compared to wild-type at both time points (Fig. 3). This transcriptional upregulation correlated with the observed enhancement of avermectin production, demonstrating that SAV111 modulates avermectin biosynthesis through transcriptional regulation.

Fig. 3.

Transcriptionanalysis of aveA1 (A) and aveR (B) in SAV111 overexpression and wild-type strains. Error bars: Standard deviation from three independent biological replicate experiments. Statistical significance was analyzed by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. NS: not significant

SAV111 binds to aveA1 gene promoter

Electrophoretic mobility shift assays (EMSAs) were conducted using purified GST-SAV111 recombinant protein to investigate direct regulatory interactions. DNA probes spanning the promoter regions of aveR (aveRp) and aveA1 (aveA1p) were designed (Fig. 4A). EMSA results demonstrated specific binding of GST-SAV111 to aveA1p, evidenced by multiple shifted bands, while no binding occurred with aveRp or the constitutive control hrdB (Fig. 4B). Binding specificity was validated through competitive displacement using 100-fold molar excess of unlabeled aveA1p. Negative controls with GST alone showed no probe retardation, even at 0.8 µM concentrations (Fig. 4B). These data establish that SAV111 directly promote aveA1 transcription through promoter binding, while its effect on aveR expression occurs indirectly. The enhanced avermectin production in the overexpression strain primarily stems from transcriptional activation of aveA1.

Fig. 4.

Electrophoretic mobility shift assays of SAV111 binding to aveA1p and aveRp. Each lane contained 0.15 nM labeled probe, and 15 nM unlabeled probe was used in competition assays. GST protein and probe hrdB were used as negative controls. Arrow: free probes. Bracket: SAV111-DNA complex

SAV111 accelerates morphological development in S. avermitilis

Developmental progression was compared between strains using streak cultures on YMS medium and minimal medium over 7 days. The SAV111 overexpression strain initiated aerial hyphae formation by day 2 and a developmental milestone not observed in wild-type until later stages on YMS (Fig. 5A). Sporulation commenced in the overexpression strain by day 4, preceding wild-type sporogenesis. Similarly, aerial hyphae emergence occurred by day 3 in the overexpression strain, contrasting with delayed differentiation in wild-type on MM (Fig. 5B), These findings demonstrate that SAV111 overexpression accelerates the morphological differentiation cascade in S. avermitilis.

Fig. 5.

Aerial mycelium and spore-mass production by the WT and SAV111 overexpression strains grown on YMS (A) and MM (B) agar plates

Discussion

Transcriptional profiling demonstrated undetectable SAV111 expression on day 2 and day 6 in wild-type (WT) S. avermitilis during fermentation (Fig. S1). Bioinformatic analysis identified three tandem GC-rich direct repeats within the proximal upstream regulatory region of SAV111. We hypothesize that these motifs serve as binding platforms for transcriptional repressors mediating SAV111 silencing. In the high-yielding industrial strain 76-02-e, elevated SAV111 expression may result from mutational disruption of the upstream regulatory region, preventing repressor binding.

Integrated RT-qPCR and EMSA data demonstrate that SAV111 mediates avermectin biosynthesis through direct promoter binding of aveA1. LuxR-family transcriptional regulators in Streptomyces typically function as pathway-specific controllers within antibiotic biosynthetic gene clusters (BGCs), though some exhibit pleiotropic regulatory roles (Zhang et al. 2022). Global transcriptional regulators positioned outside BGCs that modulate antibiotic production via structural gene activation are well-documented in S. avermitilis (Lu et al. 2020, 2021; Wang et al. 2022). This direct structural gene regulation may facilitate precise metabolic responses to environmental or physiological cues, bypassing hierarchical signaling through pathway-specific regulators.

SAV111 exhibits dual regulatory functionality: (1) direct transcriptional activation of the aveA1 structural gene and (2) indirect modulation of the pathway-specific regulator aveR through unidentified intermediaries. This multi-tiered control mechanism enables cascaded amplification of avermectin biosynthesis. The regulatory complexity underscores the sophisticated transcriptional architecture governing secondary metabolism in Streptomyces, where critical biosynthetic pathways are coordinately controlled through both direct and network-level regulatory inputs.

Streptomyces experience a complex life cycle, which begin to form aerial hyphae after sensing the signal of nutrient deficiency during growth. Besides, the initiation of secondary metabolism in Streptomyces is usually correlated with development, suggesting that the two processes are potentially regulated by common protein which may also respond to other physiological changes such as growth rate, imbalanced metabolism and environmental stress (Bibb 2005; van Wezel and McDowall 2011). In the present work, the SAV111 overexpression strain exhibited accelerated growth, produced more avermectin and formed aerial hyphae and spores earlier than WT strain. It is possible that SAV111 interacts with other regulators and responds to some physiological signals to crossregulate avermectin production and morphological development. Concurrently, we observed that SAV111 can upregulate the expression of sig25 and smrA genes (Fig. S1). In S. avermitilis, Sig25 is the ECF-sigma factor (Extracytoplasmic function), SmrA is the response regulator of SmrAB, and they were all shown to regulate avermectin production (Luo et al. 2014). These findings led us to hypothesize that SAV111 expression might be triggered under specific conditions. Growth of SAV111 overexpression strain was not significantly different from that of wild-type strain on YMS plates following treatment with NaCl, H₂O₂, alkali, EDTA, or SDS separately (Fig. S2), which suggest that SAV111 expression was not induced by these environmental stresses. In future experiments, we will continue to explore different environmental conditions and expand our detection parameters to focus on avermectin yield and growth performance.

Morphological development is usually accompanied by increased secondary metabolism in Streptomyces. In the SAV111 overexpression strain, earlier aerial hyphae development was noted on day 2 on YMS plate (Fig. 5A). At the same time, we can observe the clearly increased avermectin yield per unit (mg/dry cell weight) from day 3 (Fig. 2C), indicating SAV111 can crossregulate avermectin production and aerial hyphae formation. The SAV111 overexpression strain also displayed earlier sporulation (Fig. 5A). amfC encodes aerial mycelium-associated regulator in S. coelicolor and Streptomyces griseus, and it was recently reported as the cognate anti-sigma factor of σ^WhiG, which encodes the sporulation-specific sigma factor. whiB, ssgB and ftsZ expression are required for the initiation of sporulation in S. coelicolor. The results of this experiment provided supplementary evidence for the validity of the microarray data. By day 6, S. avermitilis entered the growth stationary phase with autolysis. We hypothesized that the 1.29-fold downregulation of SAV111 in high-yield strains appeared because genes involved in growth and development were no longer required in large quantities during the stable growth period. Additionally, the promoter region of the SAV111 gene contained a segment with high GC content. We hypothesized that the high GC region may recruit more regulatory proteins to suppress transcription on day 6, consist with our transcriptional analysis in wild-type strains that SAV111 expression was extremely low, nearly completely repressed. On this basis, we speculated that certain regulatory proteins in high-yield strains inhibited SAV111 expression on day 6 in high-producing avermectin strain 76-02-e and wild-type respectively. Further extensive studies are needed to explore how the development-related SAV111 target genes contribute to the earlier differentiation phenotype.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Jianya Zhu and Shuai Luo designed the research and performed experiments; Hucheng Zhang, Yaqin Wen, Xiaojie Wang, and Lina Deng contributed study materials; Liang Chen and Ming Yang revised the manuscript. All authors reviewed the manuscript.

Funding

This work was supported by Beijing Polytechnic project (2024 × 016-KXZ).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval and consent to participate

Not applicable. This article does not contain human and animal experiments.

Consent for publication

All the authors agree to publish the paper.