Overexpression of SAV111 in Streptomyces avermitilis increases avermectin production by binding to aveA1 gene promoter

doi:10.21203/rs.3.rs-3170967/v1

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Overexpression of SAV111 in Streptomyces avermitilis increases avermectin production by binding to aveA1 gene promoter

https://doi.org/10.21203/rs.3.rs-3170967/v1

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Background

Avermectin antibiotics from Streptomyces avermitilis are used widely in medicine and agriculture. The LuxR family transcription regulators modulate antibiotic biosynthesis in addition to regulating virulence factor expression, biofilm formation, and the hosts′ immune response. At present, there was no report about the regulation of LuxR family proteins on avermection production.

Results

We investigated the mechanism by which overexpression of SAV111, a LuxR family regulator, promoted avermectin production. Shaking flask fermentation of the SAV111 overexpression strain verified that SAV111 promotes avermectin biosynthesis, and indicated SAV111 stimulates cell growth. Streaking experiments showed earlier emergence of morphological differentiation of the SAV111 overexpression strain. Electrophoretic mobility shift assays indicated that SAV111 mainly affects avermectin production by binding to the promoter region of aveA1, a type I polyketide synthase gene in the avermectin biosynthesis pathway.

Conclusions

Results from this work showed that SAV111 promotes avermectin production, cell growth and morphological differentiation in S. avermitilis. Overexpression of SAV111 improves avermectin production mainly by promoting aveA1 transcription. Our findings will expand the regulation network of avermectin biosynthesis and provide a theoretical basis for constructing high-yield strains.

avermectin

Streptomyces avermitilis

LuxR-family regulator

SAV111

Streptomyces are filamentous gram-positive soil actinomycetes with a complex morphological differentiation cycle, including substrate hyphae formation, aerial hyphae formation, and spore chain formation. Streptomyces produce abundant secondary metabolites, such as antibiotics, immunosuppressive drugs, and anticancer drugs (Liu et al. 2018) that have high commercial and medical value. The biosynthesis of antibiotics is controlled by multi-level transcription regulation factors, including cluster-situated regulators and higher-level pleiotropic regulators. Some transcription factors in Streptomyces can simultaneously regulate morphological differentiation and antibiotic synthesis, and industrial strains with enhanced antibiotic production can be obtained by genetic manipulation of regulatory factors.

According to their structures and functions in bacteria, the transcription regulation factors can be divided into different families, such as LuxR (luminescence regulator), TetR (tetracycline repression), XRE (xenobiotic response element), SARP (Streptomyces antibiotic regulatory protein), MarR (multiple antibiotic resistance) and other families. The TetR family has the largest number of factors (Bhukya et al. 2017). The XRE family is the second most common and generally found in Streptomyces, Serratia marcescens, and Pseudomonas aeruginosa (Novichkov et al. 2013). The SARP family was the first to be identified, but it is found only in actinomycetes, mainly Streptomyces (Romero-Rodríguez et al. 2015). The MarR family is also common in Streptomyces, with approximately 50 MarR family factor genes are found in each genome (Liu et al. 2013).

A typical LuxR family factor has a C-terminal helix-turn-helix domain that binds DNA. Fifty-nine percent of LuxR proteins also have other domains, such as PAS-LuxR, REC-LuxR, CHD-LuxR, and LAL, and these domains are generally related to signal transduction. The LuxR family proteins regulate expression of pathogenic factors, biofilm formation, and hosts′ immune response (Fuqua et al. 2001). In Streptomyces, LuxR family regulatory proteins can affect antibiotic synthesis and may participate in intracellular signaling (Guerra et al. 2012). In the process of natural product synthesis, most LuxR family regulatory proteins act as positive regulators, whereas a few act as inhibitors. In S. hygrospinosus var. beijingensis, AniF promotes the synthesis of anisomycin, and overexpression of aniF in the wild type increased the yield of anisomycin by about 1.2 folds (Shen et al. 2019). LuxR family regulatory proteins can also affect the synthesis of macrolide antibiotics. Overexpression of pikD increased the yield of picomycin by about 1.8 folds in the wild type S. venezuelae (Mo and Yoon 2016). In the wild type S. rapamycinicus, overexpression of LAL gene rapH increased rapamycin production by 46.5% (He et al. 2022). In S. coelicolor, the LAL regulator SCO6993 inhibits the production of actinomycin and undecylprodigiosin (Tsevelkhoroloo et al. 2022).

Avermectin, oligomycin, and filipin are three intensively studied antibiotics in S. avermitilis. Avermectin, a 16-membered ring macrolide compound, is an economically important, potent anthelmintic and insecticidal drug widely utilized in human medicine, animal husbandry, and agriculture. Oligomycin is a 26-membered ring macrolide compound that has inhibitory activity against a variety of pathogenic fungi and is also used in research as an ATP synthase inhibitor. filipin is a pentanemacrolide used clinically for research and diagnosis of C type Niemann Pick's disease and as an inhibitor of the "raft"/"caveolin" pathway in mammalian cells. Currently, there is only one functional PAS-LuxR protein, PteF was reported in S.avermitilis. PteF positively regulates the production of filipin, oligomycin (Vicente et al. 2015), and formation of spores (Vicente et al. 2014).

By multiple mutagenesis, we obtained a strain of S. avermitilis, 76-02-e, that had high avermectin yield. By analyzing the differential expressed genes between wild type S. avermitilis ATCC31267 and 76-02-e, we obtained several genes with more than 2 folds difference in expression level. SAV111 encodes a LuxR family transcription factor, and its expression level increases significantly at day 6 of fermentation process in S. avermitilis 76-02-e, which suggested that SAV111 could affect avermectin synthesis. In this work, we characterized SAV111 regulation of avermectin biosynthesis. Our findings will expand the regulation network of avermectin biosynthesis and provide a theoretical basis for constructing high-yield strains.

Bacterial strains, plasmids, and growth conditions

Culture conditions for S. avermitilis and Escherichia coli were as described previously (Guo et al. 2018). YMS, MM, and RM14 agar were used for observation of S. avermitilis phenotype. For routine avermectin production, fermentation medium FM-I was used. For growth analysis, liquid fermentation medium FM-II was used.

Construction of SAV111 overexpression strain

To construct SAV111 overexpression strain, the SAV111 gene was amplified by PCR with primers 5'-CGGGATCCCCGCTTCCGTGCTGCGCC-3' and 5'-CCCAAGCTTTCACAGGCCCAGGGAGAT-3' with genomic DNA of wild-type S. avermitis ATCC31267 as template. The SAV111 gene was digested by BamHI and HindIII and ligated downstream of the erythromycin resistance gene promoter ermE*p in plasmid pJL117. The fused ermE*p and SAV111 fragment was ligated into BglII-digested pSET152 to generate pSET152-111, which was transformed into S. avermitilis ATCC31267.After screening and identification, SAV111 overexpression strain was obtained.

Fermentation of S. avermitilis and analysis of avermectin

Fermentation of wild type S. avermitilis and SAV111 overexpression strains was performed as described previously (Luo et al. 2014). The fermentation broth (1.0 mL) was extracted with 4.0 mL methanol for 30 min and centrifuged at 4,000×g for 10 min. The supernatant was applied directly to an HPLC system (Model 600; Waters, Milford, CT, USA) with a C18 column (10 µm, 4.6×150 mm) with the conditions of methanol: water (90: 10) as mobile phase at 40℃, 1.0 ml/min flow rate, and injection volume 20 µL. Avermectins were detected by UV absorption at 246 nm, using authentic samples of avermectin B1 as internal standards.

SAV111 heterogeneous expression in E. coli and purification

The fragment containing 330-bp SAV111 gene coding sequence (110 amino acids) was amplified by PCR with primers 5'-CGGGATCCTTGTCCTCTGCTTCCGAG-3' and 5'-CCCAAGCTTTCACAGGCCCAGGGAGAT-3'. The PCR product was digested with BamHI/HindIII (TaKaRa) and inserted into expression vector pGEX-4T-1 to generate the N-terminal GST tagged SAV111 protein expression vector pGEX-111, which was verified by DNA sequencing. The pGEX-111 plasmid was introduced into E.coli BL21 (DE3), and recombinant GST-SAV111 protein was induced with 0.2 mM isopropyl-β-D-thiogalactoside for 8 h at 16°C. The E. coli cells were collected, washed, and disrupted in STE buffer (10.0 mM Tris-HCl, 1.0 mM EDTA, 150.0 mM NaCl, pH 8.0) by sonication on ice. The lysate was centrifuged, and the supernatant containing soluble GST-SAV111 was loaded onto aglutathione-Sepharose column (Tiangen). After extensive washing with PBS buffer (11.3mM NaH₂PO₄, 38.7 mM Na₂HPO₄, 150.0 mM NaCl, pH 7.4), the GST-SAV111 protein was eluted with buffer (pH8.0) containing 10.0 mM Tris-HCl, 1.0 mM EDTA, 150.0 mM NaCl, 20.0 mM glutathione. Concentrations of purified protein were determined by Bradford assay, and protein was stored at -80°C.

RNA preparation and RT-qPCR analyses

The mycelia from S. avermitilis culture grown in FM-II for two days (exponential phase) and six days (stationary phase) were ground into powder in liquid nitrogen. Total RNA extraction of the powders was performed with TRIzol (Tiangen, China). The quality and quantity of RNA were assessed using a NanoVue™ Plus spectrophotometer and confirmed by agarose gel electrophoresis. To eliminate chromosomal DNA contamination, RNA samples were treated with DNase I and tested by PCR to confirm the absence of chromosomal DNA. The treated RNA sample (2 µg) was reverse transcribed using M-MLV (RNasefree, Promega), random hexamers (25 µM), and a dNTP mixture (10 mM each). Using the obtained cDNA as template, RT-qPCR analysis was performed to determine the transcription levels of aveR (primers 5'-CCGCGACTTCCTCACCG-3', 5'-GGACTCGCTCAGCAG-3') and aveA1 (primers 5'-ACGCTTCCGACGTCTTCCG-3', 5'-TTGTCCTCGGTCCACGGAG-3'). The 16S rRNA gene (primers 5'-AGCGGAGCATGTGGCTTAAT-3', 5'-ACGTATTCACCGCAGCAATG-3') was used as internal control. The experiments were performed using FastStart Universal SYBR Green Master (ROX; Roche, USA) with analysis by Roche Light Cycler 480 (Applied Biosystems, Swiss) using optical-grade 96-well plates. Template cDNA, 10 µL SYBR Green Master, and forward and reverse primers (each 300 nM) were mixed in each reaction of 20 µL. The PCR protocol consisted of 95°C for 10 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s with a single fluorescence measurement.

Electrophoretic mobility shift assays

Electrophoretic mobility shift assays (EMSA) were performed according to the description in Roche DIG Gel Shift Kit (2nd Generation) (Luo et al. 2014). The EMSA DNA probes harboring promoter regions of tested genes were obtained by PCR using corresponding primers (data not shown), labeled with non-radioactive digoxigenin (DIG) at the 3’end, and incubated individually with various amounts of GST-SAV111 in binding reactions. To verify specificity of probe-protein interaction, a ~ 100-fold excess of unlabeled aveA1p or aveRp probe or non-specific probe hrdB was added to the reaction mixture. Each lane was configured with a 25 µL mixed system and separated by electrophoresis in 5% non-denaturing polyacrylamide gels. After electrophoresis, DNA was transferred by electroblotting to a positively charged nylon film. After drying and UV crosslinking, DNA was detected and labeled with a luminescent substrate and autoradiographed with X-ray film.

Overexpression of SAV111 promotes avermectin production

The SAV111 gene contains 330 nucleotides (nt) and encodes a LuxR family transcription factor with a C-terminal conserved helix-turn-helix DNA-binding motif and the function of SAV111 is still unknown. To investigate the role of SAV111, we used RT-qPCR to measure the expression level of SAV111 gene in wild-type S. avermitilis and SAV111 was not expressed during fermentation process (data not shown). We speculated that there was no difference in phenotype between SAV111-deleted strain and wild-type strain. Therefore, we only constructed SAV111 overexpression strain, WT/pSET152-111. Avermectin production was increased by about 37% in WT/pSET152-111 as measured by HPLC (Fig. 1), which indicated that SAV111 can promote avermectin synthesis.

To investigate whether SAV111 promoted synthesis of avermectin resulted from altered biomass, we measured the cell growth and yield of WT and SAV111 overexpression strain. During fermentation process, the values of dry cell weight of SAV111 overexpression strain were higher than that of WT (Fig. 2A) and the avermectin production per mg dry cell weight of SAV111 overexpression strain was also greater than that of WT (Fig. 2B). Thus, SAV111 not only promoted avermectin biosynthesis, but also stimulated growth of S. avermitilis. The avermectin overproduction in SAV111 overexpression strain was due partly to an increase in biomass.

SAV111 positively regulates transcription of aveR and aveA1

To investigate the causes of avermectin overproduction in SAV111 overexpression strain, we extracted RNAs of mycelia grown in FM-II for two and six days. The transcription levels of aveR (avermectin synthesis pathway-specific regulator gene) and aveA1 (avermectin structural gene) for SAV111 overexpression strain were significantly higher than that for WT at days 2 and 6 (Fig. 3). These results were consistent with the phenotype that avermectin production for SAV111 overexpression strain was higher than that for WT, and we concluded that SAV111 promotes avermectin synthesis at the transcription level.

SAV111 binds to aveA1 gene promoter

To determine whether SAV111 directly regulates the transcription of aveR and aveA1 by binding to their promoter regions, we performed EMSAs with GST-SAV111 protein expressed in E. coli. The aveR and aveA1 promoter regions were labeled as probes aveRp and aveA1p (Fig. 4). GST-SAV111 did not bind to aveRp or hrdB, but it bound specifically to aveA1p and generated several clearly retarded bands (Fig. 4). Binding specificity was confirmed by competition experiments with a∼100-fold excess of unlabeled aveA1p probe. GST did not retard probe aveA1p, even at high concentration (0.8 µM). These findings indicated that the positive regulatory effect of SAV111 on aveR expression was indirect, and that SAV111 directly regulates aveA1 by binding to the promoter region. Overexpression of SAV111 improves avermectin production mainly by promoting aveA1 transcription.

SAV111 promotes morphological differentiation in S. avermitilis

To investigate whether SAV111 affects morphological differentiation in S. avermitilis, we observed the morphological differentiation and growth status of SAV111 overexpression strain and WT on solid YMS sporulation medium and minimum medium (MM) for seven days by streaking experiment. On YMS medium (Fig. 5A), SAV111 overexpression strain began to form aerial hyphae on day 2, whereas WT did not form aerial hyphae. SAV111 overexpression strain began to form spores on day 4, while WT did not produce spores on day 4. On MM (Fig. 5B), SAV111 overexpression strain began to form aerial hyphae on day 3 and WT did not. Thus, SAV111 overexpression strain differentiated earlier than that of WT, and SAV111 promoted the morphological differentiation in S. avermitilis.

Transcription analysis revealed that SAV111 was not expressed in WT during fermentation process. After finding three GC-rich direct repeat sequences in the upstream sequence close to the SAV111 gene (Fig. 6), we speculated that some transcription regulation factors bind to these direct repeat sequences to silence the transcription of SAV111. In the high-yield S. avermitilis strain 76-02-e, increased expression of SAV111 may have been due to the mutation of the upstream sequence of SAV111, which blocked association of transcription inhibition factors to that region so that SAV111 was transcribed successfully.

From RT-qPCR and EMSA experiments, we found that SAV111 promotes avermectin production by binding to aveA1 promoter regions. In Streptomyces, LuxR family transcription regulatory genes often exist in the antibiotic biosynthetic gene clusters (BGC) to act as pathway-specific regulators, and some regulators even have additional regulatory functions. The phenomenon that a peiotropic regulatory gene outside a BGC which contains pathway-specific regulatory gene affects antibiotic production mainly through direct regulation of structural gene transcription such as SAV111 have been reported more in S. avermitilis, and less in other Streptomyces. In S. avermitilis, the regulators SAV742 (Sun et al. 2016), HspR (Lu et al. 2021), SoxR (Wang et al. 2022), and ROK7B7 (Lu et al. 2020) all regulate the synthesis of avermectin by controlling transcription of avermectin structural genes. In S. coelicolor, the two-component regulatory system response protein AfsQ1 promotes the synthesis of coelimycin P2 by directly activating the transcription of structural gene cpkABC of yellow pigment coelimycin P2 (Chen et al. 2016). Direct regulation of structural genes instead of pathway-specific regulatory genes may be conducive to a rapid and accurate response to extracellular/intracellular signals to regulate secondary metabolism. SAV111 not only directly regulates the structural gene aveA1, but also indirectly affects the transcription of the pathway-specific regulatory gene aveR through other unknown pathway to promote avermectin biosynthesis in a cascade manner. Therefore, SAV111 can regulate the biosynthesis of avermectin in various ways, which also reflects the accuracy and complexity of regulation of secondary metabolism in Streptomyces.

Consistent with our hypotheses, overexpreesion of SAV111 can also improve avermectin production in wild type S. avermitilis. However, we found that SAV111 also promotes cell growth. With the measure of avermectin production per mg dry cell weight of the two S. avermitilis strains, we confirmed that the avermectin overproduction in SAV111 overexpression strain not only resulted from increased biomass. Moreover, SAV111 positively regulated aveA1 transcription by binding to the promoter region, thus leading to the promotion of avermectin yield. SAV111 is the first LuxR family regulator shown to display control of avermectin biosynthesis in S. avermitilis, which will extend the limited knowledge of the regulation of LuxR family protein in physiological metabolism in S. avermitilis and lead to more effective strategies for increasing avermectin production.

Acknowledgments

The authors thank AiMi Academic Services (www.aimieditor.com) for English language editing and review services.

Authors' contributions

SL and JZ designed the research and performed experiments, HZ, HZ, XW, RZ, XW and LL contributed study materials, LD and JZ wrote the manuscript. The authors read and approved the final manuscript.

Funding

This work was supported by Beijing Polytechnic project (2022X002-KXD) and R&D Program of Beijing Municipal Education Commission (2022Z019-KWY).

Availability of data and materials

The datasets are available from the first author.

Ethics 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.

Competing interests

The authors declare that they have no competing interests.

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You are reading this latest preprint version

Overexpression of SAV111 in Streptomyces avermitilis increases avermectin production by binding to aveA1 gene promoter

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Material and methods

Bacterial strains, plasmids, and growth conditions

RNA preparation and RT-qPCR analyses

Electrophoretic mobility shift assays

Results

Discussion

Conclusions

Declarations

References

Status:

Version 1