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Improvement of Wuyiencin Biosynthesis in Streptomyces wuyiensis CK-15 by Identification of a Key Regulator, WysR
Improvement of Wuyiencin Biosynthesis in Streptomyces wuyiensis CK-15 by Identification of a Key Regulator, WysR
Journal of Microbiology and Biotechnology. 2014. Dec, 24(12): 1644-1653
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 08, 2014
  • Accepted : August 06, 2014
  • Published : December 28, 2014
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About the Authors
Yanyan Liu
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China
Hojin Ryu
Department of Biology, Chungbuk National University, Cheongju 361-763, Republic of Korea
Beibei Ge
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China
Guohui Pan
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China
Lei Sun
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China
Kyungseok Park
Microbial Crop Health Lab, Division of Agricultural Microbiology, National Academy of Agricultural Science, RDA, Jeonju 560-500, Republic of Korea
zhangkecheng@sina.com
Kecheng Zhang
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China
zhangkecheng@sina.com

Abstract
Wuyiencin is produced by Streptomyces ahygroscopicus var. wuyiensis CK-15 and is widely used as an antifungal agent in agriculture. Analysis of wuyiencin biosynthetic gene clusters reveals wysR , a member of the LAL-family of transcriptional regulatory genes. WysR consists of an N-terminal PAS domain and a LuxR family C-terminal helix-turn-helix motif. However, the roles of WysR in wuyiencin biosynthesis are largely unknown. In this study, we showed that inactivation of WysR resulted in the complete loss of wuyiencin production, which could be restored by complementation with a single copy of WysR . Furthermore, we successfully increased wuyiencin production to a significantly higher level by overexpression of WysR in S. wuyiensis CK-15. Quantitative real-time RT-PCR analysis showed that WysR regulates wuyiencin biosynthesis by modulating other putative regulatory genes. Thus, WysR was identified as an activator of wuyiencin biosynthesis, and overexpression of WysR gene proved to be an effective strategy for improving wuyiencin production.
Keywords
Introduction
Streptomycetes are well known for their ability to produce a variety of secondary metabolites, which constitute an important source of small molecule drug leads. These secondary metabolites are commonly synthesized by dedicated biosynthetic enzymes, whose corresponding genes tend to be clustered in the chromosome. Biosynthesis of these secondary metabolites is tightly regulated through complex transcriptional programs [2] . The production of active natural products is linked to many environmental and physiological signals, which play important roles in the improvement of industrial strains [15 , 30] . The hierarchical structure of secondary metabolite regulation offers two distinct strategies for engineering: (i) manipulating global regulators to increase production of many secondary metabolites and (ii) targeting pathway-specific regulators to increase the titer of a particular compound of interest [5] . Pathway-specific regulatory genes can affect a single antibiotic biosynthetic pathway among a co-regulated antibiotic biosynthesis gene cluster [18] . Expression of the regulatory genes determining antibiotic production is controlled by complex regulatory mechanisms [2 , 15] .
Production of antibiotics can be improved through a better understanding of the mechanisms of regulatory proteins. Low titers produced by wild-type strains are a major problem, hindering industrial development and sales. Traditional strategies in the industry for improving the titer rely on iterative rounds of random mutagenesis and empirical screening [6] . However, the development of molecular microbiology and recombinant DNA technology has led to a number of strategies for rational strain improvement, one of them being “metabolic engineering” [12 , 17] . Most secondary metabolite pathways involve specific activator genes, and a commonly used strategy to improve natural metabolite production is the overexpression of pathway-specific activators.
Several LAL-family regulators have been identified in Streptomyces antibiotic gene clusters, including PikD from the pikromycin pathway in Streptomyces venezuelae [27] , RapH from the rapamycin pathway in Streptomyces hygroscopicus [16] , NysRI and NysRIII from the nystatin pathway in Streptomyces noursei [4] , and PimR from the pimaricin pathway in Streptomyces natalensis [1] . PAS–LuxR regulators, which are responsible for polyketide chain construction, sugar dehydration, and attachment, and the ABC transporters [11] share the same regulatory pattern in different polyene-producing strains. The expression of PAS–LuxR regulators, which are positive regulators and are functionally exchangeable [11] , constitutes a bottleneck in the biosynthesis of antifungals. For example, Aurj3M is a positive regulator of aureofuscin biosynthesis in Streptomyces aureofuscus [27] and PimM is a PAS domain-containing positive regulator of pimaricin biosynthesis in Streptomyces natalensis .
Wuyiencin is produced by Streptomyces ahygroscopicus var. wuyiensis , which was isolated from the natural soil habitat of Wuyi Mountain in China. As a nucleoside antibiotic, wuyiencin contains a cytidine skeleton [31] . It is highly water-soluble and acts as a pollution-free biological fungicide characterized by a high efficiency, broad spectrum, and low toxicity compared with that observed in other chemical pesticides. Wuyiencin has been widely used as an industrially produced biopesticide for the control of various fungal diseases of vegetable and field crops. Over the past 20 years, extensive research has focused on the regulatory mechanism of wuyiencin in terms of biological prevention and biosynthesis [3 , 23] , and to evaluate its antifungal activity. In our work on wuyiencin biosynthesis, we partially sequenced the wuyiencin biosynthetic cluster from S. wuyiensis CK-15. This sequencing information allowed the identification of putative regulatory elements of wuyiencin biosynthesis, including the wysR gene. The present study was undertaken in order to understand whether alteration of the regulatory gene wysR can improve the production of wuyiencin. Cloning, sequencing, and detailed characterization of this novel member of the PAS-LuxR family of regulators from S. wuyiensis CK-15 demonstrated its role as a transcriptional activator for wuyiencin biosynthesis in this bacterium. The purpose of this study is to improve industrial wuyiencin production and application.
Materials and Methods
- Bacterial Strains, Plasmids, and Culture Conditions
S. wuyiensis CK-15 strains (China General Microbiological Culture Collection Center, CGMCC No. 0703) were grown at 28℃ on Mannitol-Soybean (MS) agar [10 , 11] or in yeast extract malt extract (YEME) liquid medium [11] . Soluble fermentation medium was made of 2 g soybean flour, 2 g glucose, 3 g corn starch, 300 mg CaCO 3 , 400 mg (NH 4 ) 2 SO 4 , and H 2 O making up the final volume to 100 ml. Escherichia coli strains were grown in Luria-Bertani (LB) liquid broth or on LB agar. E. coli strain DH5α and pSETC [14] (kindly provided by Prof. Yang Keqian) were used for cloning and expression according to standard molecular biology procedures [11 , 20] . pKC1139 and pSET152 plasmids [3] (kindly provided by Prof. Yang Keqian) were used as the disruption and complementation vectors, respectively. pSETC contains the constitutive and strong expression promoter P SF14 on the genome-integrating plasmid pSET152. E. coli ET12567/pUZ8002 was used for gene conjugation from E. coli to S. wuyiensis CK-15 [28] . Rhodotorula rubra [29] was incubated in potato-dextrose-agar (PDA) medium at 28℃ overnight, and was used as an indicator strain for the wuyiencin bioassay experiments. Apramycin and gentamicin were used at 50 µg/ml in LB medium as and when required.
- DNA Manipulation and Sequence Analysis
Isolation of genomic DNA from Streptomycetes and plasmid DNA from E. coli and other DNA manipulations were carried out using standard protocols [11] . The enzymes and chemicals used in this study were purchased from Tiangen Biotech Co., Ltd (Beijing) and TaKaRa Biotechnology Co., Ltd (Dalian). Restriction enzymes and molecular biology reagents were used according to the recommendations of suppliers (NEB, MBI Fermentas, Promega). Total DNA was extracted using the Streptomyces genomic DNA purification kit (Tiangen). Standard primers were used for DNA sequencing according to the dideoxynucleotide chain termination method. The DNA sequence was determined using an automated DNA sequence analyzer. Comparisons of nucleotides and protein sequences were performed using the programs DNAMAN, BLAST, FASTA, and ClustalW.
- Construction of the wysR Deletion Mutant
The wysR deletion mutant was constructed using the polymerase chain reaction (PCR)-targeting system [8] . Primers were designed for amplification of the flanking regions of the wysR gene. Primers YF and YR were used to amplify the upstream region (1,046 bp) of wysR ( Table 1 ). Another set of primers, KF and KR, was used to amplify the downstream region (1,291 bp) ( Table 1 ). The PCR product of the upstream region was inserted at the Hin dIII/ Xba I site of the vector pKC1139, which contains a temperature-sensitive origin of replication, to create pKCLW. The PCR product of the downstream region was then introduced into the Xba I/ Bam HI site of pKCLW to create pKC-W. The gentamicin resistance gene, amplified by the primers Gm-F and Gm-R ( Table 1 ), was inserted at the Xba I site between the two inserted flanking regions to make the complete pKC-WG. The resulting mutated cosmid was first electrotransformed into the non-methylating E. coli ET12567 strain with plasmid pUZ8002 and then transferred into S. wuyiensis CK-15 by intergeneric conjugation. After incubation at 30℃ for 16 h, each plate was overlaid with 1 ml of sterile water containing apramycin at a final concentration of 50 mg/ml and nalidixic acid at a final concentration of 25 mg/ml. Incubation continued at 34℃ until conjugants appeared. Double-crossover recombinants were selected by apramycin sensitivity (AmS) and gentamicin resistance (GmR), followed by PCR confirmation with the primers TF and TR ( Table 1 ).
Primers used in this study.
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Primers used in this study.
- Complementation of the wysR Mutant
For complementation studies, the wysR gene with its upstream region was PCR-amplified from S. wuyiensis CK-15 genomic DNA using the primers Com-F (with an Xba I restriction site engineered into the 3’ end) and Com-F (with an Bam HI restriction site engineered into the 3’ end) ( Table 1 ). The 973 bp amplification product was cloned into the pEASY-T1 simple vector, resulting in the construct PwysR . The PwysR fragment was excised from this plasmid as an Xba I/ Bam HI fragment and cloned into the corresponding restriction sites of pSET152 [3] . The resulting plasmid pSET wysR was first electrotransformed into non-methylating E. coli ET12567 with plasmid pUZ8002 and then transferred into the S. wuyiensis CK-15 wysR mutant by intergeneric conjugation. The complementation strain is designated as S. wuyiensis comR. Apramycin-resistant transconjugants were confirmed by PCR, and the phenotype of the resulting complementation strain was analyzed on MS medium with 50 mg/ml apramycin.
- Overexpression of wysR in S. wuyiensis CK-15 Strains
Primers OF and OR ( Table 1 ) containing Bam HI and Spe I restriction sites were used to amplify the wysR gene with 50 bp of its upstream region from the genomic library of S. wuyiensis CK-15. The PCR fragment was cloned into the pEASY-T1 simple vector (TransGen Biotech) to generate plasmid pEASY-R. After digestion with Bam HI and Spe I, the wysR -containing fragment from pEASY-R was ligated into the corresponding sites in the expression vector pSETC, containing the constitutive and strong expression promoter P SF14 on the genome-integrating plasmid pSET152. This resulted in the recombinant plasmid pSTEC-R, which was introduced into wild-type S. wuyiensis CK-15 via E. coli–Streptomyces conjugation. The overexpressing strain is designated as S. wuyiensis ooR . The empty vector pSETC was also introduced into wild-type S. wuyiensis CK-15 via E. coli–Streptomyces conjugation. Apramycin-resistant transconjugants were confirmed by PCR with the primers Am-F and Am-R ( Table 1 ), and the phenotype of the resulting overexpression strain was analyzed on MS medium with 50 mg/ml apramycin.
- Assay of Wuyiencin Production
Fresh ripe seed slants were inoculated with spores (1.0 × 10 6 ) in 50 ml of culture medium and kept for incubation at 28℃ for 20 h. The seeds were placed in 100 ml of 10% fermentation medium and incubated at 28℃ at 220 rpm for 64 h, after which the fermentation broth was collected by filtration. The antibacterial effect of the fermentation broth against Rhodotorula rubra was tested by measuring the diameter of bacteriostasis circles. The Agilent 1100 high-performance liquid chromatography (HPLC) system was used for the determination of wuyiencin in the fermentation broth. Analytical conditions included the use of a SB-AQ column (4.6 mm × 250 mm i.d., 5 µm) at a temperature of 25℃. The mobile phase was 1.4 g/l trichloroacetic acid with a flow rate of 1 ml/min and detection wavelength of 254 nm [26] . The retention time for wuyiencin was 21 min.
- Isolation of Total RNA
S. wuyiensis CK-15 wild-type, mutant, and overexpression strains were grown for 36 h in soluble fermentation medium. The cultures were harvested by centrifugation at 2,000 × g for 10 min and immediately frozen by immersion in liquid nitrogen. Frozen mycelia were then broken by grinding in a mortar, and frozen Trizol lysate (Tiangen) was added. RNeasy mini spin columns were used for RNA isolation according to the manufacturer’s instructions (Promega). Preparations of total RNA were treated with DNase I (Promega) to eliminate possible chromosomal DNA contamination. The RNA concentration and purity were determined by measuring the ratio of OD 260 /OD 280 , and an equal amount of RNA from each strain was used for subsequent reverse transcription PCR (RT-PCR).
- Transcriptional Analysis by Real-Time qRT-PCR
Transcription was studied using the Superscript one-step RT-PCR system with Platinum Taq DNA polymerase (Saibaisheng). Fifty nanograms of total RNA was used as the template. The conditions were as follows: first-strand cDNA synthesis, 37℃ for 1 h followed by 95℃ for 5 min; 30 cycles of 95℃ for 30 sec, 59–62℃ (depending of the set of primers used) for 30 sec, and 72℃ for 1 min. Primers (13 to 31 mers, Table 1 ) were designed to generate PCR products. HrdB was amplified as the internal control. The qPCRs were performed according to the protocol of the Maxima SYBR Green/ROX qPCR Master Mix in the Applied Biosystems 7500 Fast Real-Time PCR system. Transcripts were analyzed from the four representative genes of the wuyiencin biosynthetic gene cluster after 30 PCR cycles. All the quantitative real-time PCR assays were carried out in triplicate for each culture and repeated three times with RNA isolated from independent cultures.
- Nucleotide Sequence Accession Number
The sequences reported have been deposited in the GenBank database (Accession No. KF667489).
Results
- Sequence Analysis of the wysR Gene
Computer-assisted analysis of the wysR gene product (193 amino acids with a calculated molecular mass of 20,867 Da) showed high sequence identity (84.29%) with protein ORF4 of S. noursei ATCC11455, which is a putative regulatory protein of 210 amino acid residues present within the nystatin biosynthesis gene cluster. WysR contains an N-terminal PAS domain and a C-terminal helix-turnhelix (HTH) motif of the LuxR family. Further analysis using NCBI BLASTp showed that the protein is homologous with the other putative transcriptional regulators FscRI (60% identity, FR008/Candicidin; [5] ), ScnRII (66.49% identity, natamycin; [7] ), and PimM (67.5% identity, pimaricin; [1] ). Each of these transcriptional regulators has a PAS sensor-binding domain at the N terminus (SMART 00091 [9 , 25] ) as well as an HTH motif of the LuxR type at the C terminus (SMART 00421; Fig. 1 ).
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Amino acid sequence alignments of Streptomyces LAL family transcriptional regulators. Conserved and homologous amino acids are highlighted.
- Inactivation of wysR Blocks Wuyiencin Biosynthesis
To test whether wysR was involved in the wuyiencin biosynthesis pathway, we performed gene inactivation experiments. A disruption vector, pKC-WG, was constructed and introduced into S. wuyiensis CK-15 by intergeneric conjugation. The wysR gene was disrupted by a homologous recombination method in which a gentamicin-resistant gene replaced the disrupted gene ( Fig. 2 A). Finally, the wysR deletion mutant ( S. wuyiensis wysR ) was constructed using a PCR-targeting system and selected based on resistance to apramycin and sensitivity to gentamicin. Because the resistant gene is 400 bp longer than the target gene, a 1.1 kb band was amplified from genomic DNA samples isolated from S. wuyiensis CK-15, whereas a band of 1.5 kb was observed from S. wuyiensis wysR ( Fig. 2 B). The growth rate of S. wuyiensis wysR was compared with that of the wild-type strain to assess growth phenotypes. Whereas the ∆ wysR mutant showed no obvious morphological changes when grown on solid MS media, a retardation of growth was observed in S. wuyiensis wysR compared with the wild-type strain S. wuyiensis CK-15 ( Fig. 2 C). Additionally, the color of ∆ wysR spores was lighter than that of the wild-type strains. Next, we investigated the wuyiencin production. Fermentation broths from wild-type and ∆ wysR strains were purified by filtration and analyzed for the presence of wuyiencin. The HPLC spectrum resulting from the wysR strain was devoid of wuyiencin ( Figs. 2 D, 2 E, and 2 F). Furthermore, crude extracts of S. wuyiensis wysR could not inhibit the growth of R. rubra ( Fig. 2 G). This result indicated that the wysR gene is essential for wuyiencin biosynthesis in S. wuyiensis CK-15.
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Inactivation of wysR blocks wuyiencin biosynthesis. (A) Gene replacement of wysR in S. wuyiensis CK-15. (B) Confirmation of the constructed ∆wysR mutant by PCR. Lanes: M, 200 bp DNA ladder; 1, PCR verification with primers TF and TR using S. wuyiensis CK-15 genomic DNA as the template; 2, PCR verification with primers TF and TR of S. wuyiensiswysR genomic DNA as the template. (C) Phenotypes of 6-day-old S. wuyiensis CK-15 and S. wuyiensiswysR. (D) HPLC analysis of standard wuyiencin. (E) HPLC analysis of wuyiencin production levels in S. wuyiensis CK-15. (F) HPLC analysis of wuyiencin production levels in S. wuyiensiswysR. (G) Antibacterial effect of S. wuyiensis CK-15 and S. wuyiensiswysR.
- Trans-Complementation of the wysR Mutant
In order to exclude the possibility that the abolition of wuyiencin production was due to polar effects on downstream genes or a random mutation in another locus rather than the disruption of wysR , complementation of the deletion mutant with the wysR gene was carried out. A DNA fragment containing wysR along with the putative promoter region was inserted into the integrative vector pSET152 [3] and designated as pSET wysR . pSET wysR was then transferred from E. coli ET12567 (pUZ8002) to S. wuyiensis wysR . The apramycin-resistant transconjugants were confirmed using PCR. The expected 0.7 kb band was observed only in S. wuyiensis comR (a complementation strain) ( Fig. 3 A, lane 2). Furthermore, no notable visual difference in phenotype was observed between S. wuyiensis comR and S. wuyiensis CK-15 ( Fig. 3 B). Introduction of pSET wysR restored wuyiencin biosynthesis to the wild-type level. HPLC analysis indicated that the production of wuyiencin from the complementation strain was equivalent to that of the wild-type strain ( Figs. 2 E and 3 C). Consistently, as shown in Fig. 3 D, the inhibitory effects of S. wuyiensis extracts against R. rubra were not different from the complementation strain. Based on the results of HPLC analysis of the components in the fermentation liquid and inhibitory ability against R. rubra , we concluded that the introduction of wysR into S. wuyiensis wysR restored wuyiencin biosynthesis to wild-type levels. These results strongly support the biological function of wysR in wuyiencin biosynthesis by indicating that the abolition of wuyiencin production in S. wuyiensis wysR was due to wysR deletion.
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Effect of wysR complementation on wuyiencin production. (A) Confirmation of the complementation strain S. wuyiensis comR by PCR. Lanes: M, 1Kb Plus DNA Ladder; 1, PCR verification with primers Am-F and Am-R using S. wuyiensis CK-15 genomic DNA as the template; 2, PCR verification with primers Am-F and Am-R using S. wuyiensis comR genomic DNA as the template; 3, PCR verification with primers Am-F and Am-R using S. wuyiensis ooR genomic DNA as the template. (B) Phenotypes of 6-day-old S. wuyiensis CK-15 and S. wuyiensis comR cultured on an MS plate. (C) HPLC analysis of wuyiencin production levels in S. wuyiensis comR. (D) Antibacterial effect of S. wuyiensis CK-15 and S. wuyiensis comR.
- Improvement of Wuyiencin Production by Overexpression of wysR in S. wuyiensis CK-15
To test the overexpression effects of wysR on wuyiencin production, we next cloned the wysR gene next to the P SF14 promoter in a Streptomyces integrative expression vector, pSETC, to produce pSTEC-R. pSTEC-R was then transferred from E. coli ET12567 (pUZ8002) to S. wuyiensis CK-15 (referred to as S. wuyiensis ooR ). The apramycin-resistant transconjugants were confirmed by PCR ( Fig. 3 A, lane 3). At the same time, the empty vector pSETC was also introduced into S. wuyiensis CK-15 as a negative control ( S. wuyiensis pSETC ). The levels of sporulation of S. wuyiensis ooR exconjugants on the plate and the wild-type strain showed no significant phenotypic differences ( Fig. 4 A). Furthermore, wuyiencin production by S. wuyiensis pSETC did not change ( Figs. 4 B and 2 E). However, HPLC analysis indicated that the production of wuyiencin from the overexpression strain increased by almost 3-fold of that in the wild-type strain ( Figs. 4 C and 2 E).
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Improvement of wuyiencin production by overexpression of wysR in S. wuyiensis CK-15. (A) Phenotypes of 6-day-old S. wuyiensis CK-15 and S. wuyiensis ooR. (B) HPLC analysis of wuyiencin production levels in S. wuyiensis pSETC. (C) HPLC analysis of wuyiencin production levels in S. wuyiensis ooR. (D) Antibacterial effect of S. wuyiensis pSETC and S. wuyiensis ooR. (E) Bacteriostasis diameter of S. wuyiensis CK-15 and S. wuyiensis ooR after 10 days.
The bacteriostasis diameter was also measured to confirm the increase in wuyiencin production. The wildtype and recombinant strains were both cultivated for 5 days. Culture filtrate was subsequently used to compare the biological activity of the two strains. Fig. 4 D shows an increased inhibition of R. rubra growth by the recombinant strain. Furthermore, we also found that the bacteriostasis diameter of the wild-type strain decreased gradually, whereas the recombinant strain maintained the original bacteriostasis diameter ( Fig. 4 E). Thus, it can be concluded that the enhanced production of wuyiencin is strictly due to the overexpression of the wysR gene.
- Transcriptional Control of Wuyiencin Production
S. wuyiensis wysR ( wysR defective mutant) was deficient in wuyiencin production, whereas wysR overexpression resulted in increased production. To determine the effects of wysR on wuyiencin biosynthetic genes, we performed real-time qRT-PCR analysis. The wuyiencin biosynthetic gene wysE , which encodes a putative thioesterase in the biosynthetic gene cluster of S. wuyiensis CK-15, was subjected to transcriptional analysis. The relative level of the transcripts of two other putative regulatory genes, wysRI and wysRIII , were also analyzed in this study. The wysRI gene displays 85% end-to-end identity to NysRI, a well-characterized pathway-specific transcriptional activator for streptomycin biosynthesis in S. noursei ATCC11455 [16] . wysRIII shared high sequence identity (>40% along the entire gene) to the DeoR family of transcriptional regulators. Wild-type, S. wuyiensis wysR , and S. wuyiensis ooR strains were harvested at the peak of wuyiencin production in the stationary phase and used to prepare total RNA for RT-PCR analysis. As displayed in Fig. 5 , wysRIII and wysE transcripts were negligible in the ∆ wysR mutant, whereas they were detected in the wild-type strain. However, their expression levels were highly up-regulated in the overexpression strains ( Fig. 5 ). On the other hand, the transcription levels of wysRI were higher in the ∆ wysR mutant strain than in the wild-type strain, whereas S. wuyiensis ooR had lower transcription levels of wysRI ( Fig. 5 ). These results indicate that wysR directly or indirectly controls the expression of other putative regulators in the same wuyiencin biosynthetic gene cluster. These qRT-PCR results suggest that WysR is involved in the transcriptional control of late biosynthetic enzymes for wuyiencin production, and may affect the transcription of early wuyiencin polyketide moiety biosynthetic genes.
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Real-time qRT-PCR analysis of wysR in S. wuyiensis CK-15, S. wuyiensiswysR, and S. wuyiensis ooR. All RNA samples were isolated from 36 h cultures. Data are presented as the averages of three independent experiments conducted in triplicate. HrdB transcription was monitored and used as the internal control. Error bars show standard deviations.
Discussion
This is the first report of the identification and characterization of the transcriptional activator gene wysR required for wuyiencin biosynthesis in S. wuyiensis CK-15. We have shown that overexpression of wysR caused an increase in wuyiencin production, whereas the deletion of wysR decreased production, thus supporting the positive effect of wysR on this biosynthetic pathway. Biosynthetic gene clusters in Streptomyces often contain at least one putative regulatory gene whose product has a pathwayspecific mode of action that positively affects the production of specific secondary metabolites [21 , 22] . Our data implicate wysR as a key regulatory gene for wuyiencin production.
Sequencing of the S. wuyiensis CK-15 wuyiencin gene cluster revealed the presence of a gene, wysR , which could play a role as a regulator for wuyiencin biosynthesis. Computer-assisted analysis of the WysR protein showed that it has an N-terminal region strikingly similar to PAS sensory domains [9 , 24 , 25] and a C-terminal region with a LuxR-type HTH motif for DNA binding. The presence of a PAS-like domain within WysR indicates that this protein could contribute to energy levels in the cell [18] , and the HTH motif suggests the ability of WysR to bind to DNA [23] and thus regulate the expression of wuyiencin genes. The absence of wuyiencin production upon disruption of the wysR gene by removal of the HTH domain clearly indicates that WysR is an activator of wuyiencin biosynthesis. Among WysR orthologs, only NysRIV and PimM have been characterized by gene disruption and in vivo complementation experiments [1 , 21] . According to these previous studies, NysRIV and PimM are likely to control the expression of nystatin and pimaricin biosynthetic genes directly. Our results indicate that the control of wuyiencin biosynthesis exerted by WysR takes place through the specific transcriptional modulation of key enzymes encoding genes for wuyiencin construction.
Based on the results of RT-PCR analyses, it is conceivable that wysR regulates wuyiencin production by activating other genes, including wysRIII and wysE , which are involved in wuyiencin biosynthesis. Stimulation of wuyiencin production suggests that the expression of the early genes in the wuyiencin biosynthetic pathway is activated by wysR . The abundant spore formation and higher wuyiencin production upon wysR overexpression was also consistent with the elevated transcriptional level of wysE and other genes involved in the early stages of biosynthesis.
Transformants containing the amplified wysR gene within the pSETC plasmid showed an approximately 3-fold increase in wuyiencin production during fermentation. The effects of various environmental factors on the wysR mutant ( S. wuyiensis wysR ) and overexpression strains were also examined under various growth conditions. Although growth and morphological analyses revealed no differences between the knockout mutant and wild-type strain, a distinct increase in wuyiencin production was observed in S. wuyiensis ooR .
As a useful biopesticide to control plant fungal diseases in agricultural fields, wuyiencin faces the challenge of cost reduction during industrial production. A novel breeding method is the crux of the matter. This work indicates that wysR regulates wuyiencin biosynthesis in S. wuyiensis CK-15, suggesting possible alternatives to increase wuyiencin production, such as the overexpression of wysR and relevant genes in the same regulatory pathway. Although wuyiencin biosynthesis is controlled by numerous genes and affected by multiple environmental factors, the results of this study provide the first step in understanding the complicated regulatory mechanism governing wuyiencin production in S. wuyiensis CK-15. At the same time, the study lays the foundation for developing new molecular breeding methods for wuyiencin-producing strains in the future.
Acknowledgements
This work was supported by a grant from the National Natural Science Foundation (31371985, 31301710, 31240051) and Special Fund for Agro-Scientific Research in the Public Interest in China (201103002, 201303025). K.P. and H.R. were supported by a grant from the Research Program for Agricultural Science & Technology Development (PJ008652), RDA, the National Research Foundation (NRF-2013R1A1A1076010) from the Korean Ministry of Science, ICT and Future Planning.
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