Characterization of Pathway-Specific Regulator NigR for High Yield Production of Nigericin in Streptomyces malaysiensis F913

Nigericin is a polyether antibiotic with potent antibacterial, antifungal, antimalarial and anticancer activity. NigR, the only regulator in the nigericin biosynthetic gene cluster in Streptomyces malaysiensis F913, was identified as a SARP family regulator. Disruption of nigR abolished nigericin biosynthesis, while complementation of nigR restored nigericin production, suggesting that NigR is an essential positive regulator for nigericin biosynthesis. Overexpression of nigR in Streptomyces malaysiensis led to significant increase in nigericin production compared to the wild-type strain. Nigericin production in the overexpression strain was found to reach 0.56 g/L, which may be the highest nigericin titer reported to date. Transcriptional analysis suggested that nigR is required for the transcription of structural genes in the nig gene cluster; quantitative RT-PCR analysis revealed that the expression of structural genes was upregulated in the nigR overexpression strain. Our study suggested that NigR acts in a positive manner to modulate nigericin production by activating transcription of structural genes and provides an effective strategy for scaling up nigericin production.


Introduction
Streptomyces are well known for their extraordinary capacity to produce bioactive secondary metabolites in industrial, agricultural and medical applications. Secondary metabolism in Streptomyces is regulated in a complex fashion in response to environmental manipulations. The regulation of secondary metabolite biosynthesis is governed by various regulators, including cluster-situated regulators (CSRs), pleiotropic regulators and global regulators [1]. Global regulators and pleiotropic regulators are scattered throughout the chromosome, affecting the production of multiple antibiotics and/or morphological development [2]. CSRs usually located within the antibiotic biosynthetic clusters and exert their regulatory functions via modulating the transcription of biosynthetic genes or other regulatory genes. Among these CSRs, Streptomyces antibiotic regulatory protein (SARP) family transcriptional regulators constitute the most frequently occurring transcriptional regulators, which serve mainly as activators in antibiotic biosynthesis [3]. ActII-ORF4, the founding member of this family, is an activator of actinorhodin production in model strain Streptomyces coelicolor [4]. Binding of ActII-ORF4 to upstream of actIV-orfA operon and actIV-orf1 operon activates their transcription [5]; the expressed structural genes then initiate the biosynthesis of actinorhodin. As with ActII-ORF4, RedD activated the production of undecylprodiginines in S. coelicolor [6]. SARP family regulators also function in many streptomycetes, such as SanG in Streptomyces ansochromgenes (nikkomycin cluster), CcaR in Streptomyces clavuligerus (cephamycin-clavulanic acid supercluster) and VlmI in Streptomyces viridifaciens (valanimycin cluster) [7][8][9]. Apart from directly activating the transcription of key structural genes, many other SARP family regulators function in different ways. Some pathways contain more than one SARP and form complex cascade regulation patterns. Representative members, include the PolY of polyoxin biosynthesis in Streptomyces cacaoi subsp. asoensis [10], which controls the biosynthesis of polyoxin indirectly through modulating the expression of polR, the activator of key structural genes such as polC and polB [11]. Though most SARP family regulators function as cluster-suited regulators which activate production of the cognate antibiotic biosynthesis cluster, examples of other roles have also been reported. The SARP regulator AfsR serves as a global regulator and appears to be present in all streptomycetes [12].
Polyether antibiotics are a unique class of ionophores with metal ion-binding ability and lipophilic characteristics [13]. One major worldwide commercial use of polyether antibiotics is to control coccidiosis in animal husbandry [14,15]. The first discovered carboxyl polyether antibiotic was nigericin (Figure 1a). It has the ability to selectively transport ions across cell membranes and is widely used as a research tool to disrupt intracellular H + and K + concentration [16,17]. Nigericin is reported to be a potent agent against a variety of multidrug-resistant strains of pathogens, including Paecilomyces variotii and Candida albicans [18]. In vivo and in vitro assays have also proved that nigericin exerts strong selective antitumor activities [19,20]. The entire gene cluster for nigericin biosynthesis was firstly cloned from Streptomyces sp. DSM4137; the biosynthetic pathway of nigericin was proposed based on analyses of intermediates accumulated in disruption mutants of several structural genes [21]. However, the regulation mechanism of nigericin biosynthesis is still unknown, rendering improvement of the production of nigericin production by genetic engineering problematic. Streptomyces malaysiensis F913 is a soil-borne actinomycete with high algicidal activity [22]. Whole genome scanning suggested that S. malaysiensis F913 has a nigericin biosynthetic gene cluster (nig gene cluster) which showed high synteny with the reported homologous gene cluster in Streptomyces sp. DSM413. In this paper, we determined the role of the only regulatory gene, nigR, in the nigericin biosynthesis gene cluster in S. malaysiensis F913. The results suggest that NigR positively regulated nigericin biosynthesis by activating the transcription of structural genes. Overexpression of nigR significantly improved nigericin production.
Antibiotics 2022, 11, x FOR PEER REVIEW 2 of 12 the production of undecylprodiginines in S. coelicolor [6]. SARP family regulators also function in many streptomycetes, such as SanG in Streptomyces ansochromgenes (nikkomycin cluster), CcaR in Streptomyces clavuligerus (cephamycin-clavulanic acid supercluster) and VlmI in Streptomyces viridifaciens (valanimycin cluster) [7][8][9]. Apart from directly activating the transcription of key structural genes, many other SARP family regulators function in different ways. Some pathways contain more than one SARP and form complex cascade regulation patterns. Representative members, include the PolY of polyoxin biosynthesis in Streptomyces cacaoi subsp. asoensis [10], which controls the biosynthesis of polyoxin indirectly through modulating the expression of polR, the activator of key structural genes such as polC and polB [11]. Though most SARP family regulators function as clustersuited regulators which activate production of the cognate antibiotic biosynthesis cluster, examples of other roles have also been reported. The SARP regulator AfsR serves as a global regulator and appears to be present in all streptomycetes [12]. Polyether antibiotics are a unique class of ionophores with metal ion-binding ability and lipophilic characteristics [13]. One major worldwide commercial use of polyether antibiotics is to control coccidiosis in animal husbandry [14,15]. The first discovered carboxyl polyether antibiotic was nigericin (Figure 1a). It has the ability to selectively transport ions across cell membranes and is widely used as a research tool to disrupt intracellular H + and K + concentration [16,17]. Nigericin is reported to be a potent agent against a variety of multidrug-resistant strains of pathogens, including Paecilomyces variotii and Candida albicans [18]. In vivo and in vitro assays have also proved that nigericin exerts strong selective antitumor activities [19,20]. The entire gene cluster for nigericin biosynthesis was firstly cloned from Streptomyces sp. DSM4137; the biosynthetic pathway of nigericin was proposed based on analyses of intermediates accumulated in disruption mutants of several structural genes [21]. However, the regulation mechanism of nigericin biosynthesis is still unknown, rendering improvement of the production of nigericin production by genetic engineering problematic. Streptomyces malaysiensis F913 is a soil-borne actinomycete with high algicidal activity [22]. Whole genome scanning suggested that S. malaysiensis F913 has a nigericin biosynthetic gene cluster (nig gene cluster) which showed high synteny with the reported homologous gene cluster in Streptomyces sp. DSM413. In this paper, we determined the role of the only regulatory gene, nigR, in the nigericin biosynthesis gene cluster in S. malaysiensis F913. The results suggest that NigR positively regulated nigericin biosynthesis by activating the transcription of structural genes. Overexpression of nigR significantly improved nigericin production.

NigR Encodes a Putative SARP Family Transcriptional Regulator
Assembly of the sequencing data of the S. malaysiensis F913 genome yielded 25 contigs, assembled into 6 scaffolds (GenBank accession number: LJIW00000000). The fine Antibiotics 2022, 11, 938 3 of 11 genome map of S. malaysiensis F913 was 11,608,319 bp in length without any plasmid, the chromosome encoded 9497 predicated genes, and the total length of the genes was 10,011,963 bp. The nig gene cluster was identified by searching the genome sequence of S. malaysiensis F913. As the nigericin biosynthesis gene cluster in S. malaysiensis F913 showed high synteny with the reported cluster in Streptomyces sp. DSM413, we used the same gene name as proposed for the original discovered cluster (Table S1). Among the 20 genes in the predicated nig gene cluster in Streptomyces malaysiensis F913, nigR was the only transcriptional regulator (Figure 1b). Sequence analysis of NigR was performed with BLAST (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 25 May 2022) and SMART (http://smart.embl-heidelberg.de (accessed on 25 May 2022). The results suggested that NigR is a SARP family regulator, with an N-terminal trans-Reg-C domain (amino acids 19-96) (Pfam no. PF00486) which may play a role in DNA binding, and a bacterial transcriptional activator domain (amino acids 103-248) (Pfam no. PF03704) located in the C-terminal (Figure 2a). NigR shows 63% identity to ChlF2 (AAZ77687.1), 58% identity to MonR1 (ANZ52472.1), 57% identity to NanR2 (AAP42854.1) and 50% identity to NanR1 (AAP42853.1) (Figure 2b). ChlF2 is a cluster-situated SARP family regulator which is essential for chlorothricin biosynthesis in Streptomyces antibioticus DSM 40725 [23]. MonR1 positively regulated monensin biosynthesis in Streptomyces cinnamonensis ST021 [24]. Both NanR1 and NanR2 are transcriptional activators of polyketide genes in the nanchangmycin biosynthesis gene cluster in Streptomyces nanchangensis NS3226 [25]. A rare TTA codon was located in codon 22 in NigR, suggesting that the translation of nigR may rely on the bldA gene [26].

NigR Encodes a Putative SARP Family Transcriptional Regulator
Assembly of the sequencing data of the S. malaysiensis F913 genome yielded 25 contigs, assembled into 6 scaffolds (GenBank accession number: LJIW00000000). The fine genome map of S. malaysiensis F913 was 11,608,319 bp in length without any plasmid, the chromosome encoded 9497 predicated genes, and the total length of the genes was 10,011,963 bp. The nig gene cluster was identified by searching the genome sequence of S. malaysiensis F913. As the nigericin biosynthesis gene cluster in S. malaysiensis F913 showed high synteny with the reported cluster in Streptomyces sp. DSM413, we used the same gene name as proposed for the original discovered cluster (Table S1). Among the 20 genes in the predicated nig gene cluster in Streptomyces malaysiensis F913, nigR was the only transcriptional regulator (Figure 1b). Sequence analysis of NigR was performed with BLAST (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 25 May 2022) and SMART (http://smart.embl-heidelberg.de (accessed on 25 May 2022). The results suggested that NigR is a SARP family regulator, with an N-terminal trans-Reg-C domain (amino acids 19-96) (Pfam no. PF00486) which may play a role in DNA binding, and a bacterial transcriptional activator domain (amino acids 103-248) (Pfam no. PF03704) located in the Cterminal ( Figure 2a). NigR shows 63% identity to ChlF2 (AAZ77687.1), 58% identity to MonR1 (ANZ52472.1), 57% identity to NanR2 (AAP42854.1) and 50% identity to NanR1 (AAP42853.1) (Figure 2b). ChlF2 is a cluster-situated SARP family regulator which is essential for chlorothricin biosynthesis in Streptomyces antibioticus DSM 40725 [23]. MonR1 positively regulated monensin biosynthesis in Streptomyces cinnamonensis ST021 [24]. Both NanR1 and NanR2 are transcriptional activators of polyketide genes in the nanchangmycin biosynthesis gene cluster in Streptomyces nanchangensis NS3226 [25]. A rare TTA codon was located in codon 22 in NigR, suggesting that the translation of nigR may rely on the bldA gene [26].

Disruption of NigR Abolished Nigericin Production
To determine the role of nigR in nigericin biosynthesis, a nigR disruption mutant was constructed via double-crossover recombination. The resulting mutant strain (DM01R) was tested for nigericin production. Unlike the S. malaysiensis F913 wild-type strain (WT), nigericin production was abolished in the nigR disruption mutant (Figure 3). To prove that the increase in nigericin production was attributable to the effect nigR disruption, complementation strain DM01Rc was constructed by integrating a copy of nigR with native promoters into the chromosome of DM01R by pSET152. The results show that nigericin production was restored in the complementary strain, and further confirmed that NigR is a key activator of nigericin biosynthesis in S. malaysiensis F913 (Figure 3). The DM01R strain showed patterns of growth and morphology identical to those of the wild-type strain on MS or Gause's medium, indicating that nigR has no significant effect on the growth and differentiation of S. malaysiensis F913. Amino acid alignment of NigR and its orthologs. Identical or similar residues in all sequences are highlighted in black and gray, respectively. Examples of orthologs include: ChlF2 for chlorothricin from S. antibioticus, MonR1 for monensin from S. cinnamonensis, NanR1 and NanR2 for nanchangmycin from S. nanchangensis.

Disruption of NigR Abolished Nigericin Production
To determine the role of nigR in nigericin biosynthesis, a nigR disruption mutant was constructed via double-crossover recombination. The resulting mutant strain (DM01R) was tested for nigericin production. Unlike the S. malaysiensis F913 wild-type strain (WT), nigericin production was abolished in the nigR disruption mutant (Figure 3). To prove that the increase in nigericin production was attributable to the effect nigR disruption, complementation strain DM01Rc was constructed by integrating a copy of nigR with native promoters into the chromosome of DM01R by pSET152. The results show that nigericin production was restored in the complementary strain, and further confirmed that NigR is a key activator of nigericin biosynthesis in S. malaysiensis F913 (Figure 3). The DM01R strain showed patterns of growth and morphology identical to those of the wildtype strain on MS or Gause's medium, indicating that nigR has no significant effect on the growth and differentiation of S. malaysiensis F913.

Gene Expression Analysis in the Wild-Type and Strain DM01R
In order to determine the regulation mechanism and potential targets of NigR, reverse transcription PCR (RT-PCR) tests were performed to detect the transcription of the gene in the nig gene cluster. RT-PCR products of the all genes in the nig gene cluster were detected in the S. malaysiensis F913 strain after 27 cycles of PCR, while no band could be observed in the DM01R strain, indicating that these genes are probably regulated by NigR ( Figure 4). A primer pair designed to amplify the cDNA of hrdB, encoding an essential sigma factor in S. malaysiensis (accession no. PNG92438.1), was used as internal control. The results suggest that NigR is essential for the expression of structural genes; the promoters of structural genes are potential targets of NigR. A consensus sequence of "CGWWWCCG" was identified in the promoter of orf9, intergenic region of nigD-C and nigAI-nigE ( Figure S1).

Gene Expression Analysis in the Wild-Type and Strain DM01R
In order to determine the regulation mechanism and potential targets of NigR, reverse transcription PCR (RT-PCR) tests were performed to detect the transcription of the gene in the nig gene cluster. RT-PCR products of the all genes in the nig gene cluster were detected in the S. malaysiensis F913 strain after 27 cycles of PCR, while no band could be observed in the DM01R strain, indicating that these genes are probably regulated by NigR (Figure 4). A primer pair designed to amplify the cDNA of hrdB, encoding an essential sigma factor in S. malaysiensis (accession no. PNG92438.1), was used as internal control. The results suggest that NigR is essential for the expression of structural genes; the promoters of structural genes are potential targets of NigR. A consensus sequence of "CGWWWCCG" was identified in the promoter of orf9, intergenic region of nigD-C and nigAI-nigE ( Figure S1).

Overexpression of NigR Significantly Enhanced Production of Nigericin
In general, overexpression of a pathway-specific positive regulator will lead to increased production of the corresponding antibiotics. To examine the effects of nigR overexpression in S. malaysiensis F913, we constructed nigR overexpression vectors pSET152::P hrdB R and pSET152::P kasO R. pSET152, pSET152::P hrdB R and pSET152::P kasO R were introduced into S. malaysiensis F913 to generate F913-pSET152, F913-hrdBR and F913-kasOR, respectively. Nigericin production was increased in both F913-hrdBR and F913-kasOR strains compared to F913 and F913-pSET152 controls. Compared to the F913 strain, nigericin production rose by 68% in F913-hrdBR and 54% in F913-kasOR at day five (Figure 5a). The biomass of S. malaysiensis F913, F913-pSET152, F913-hrdBR and F913-kasOR were determined to exclude the effect of growth on antibiotic production. The results showed that F913, F913-pSET152, F913-hrdBR and F913-kasOR showed comparable growth rates and final dry weights (Figure 5b), indicating that the increase in nigericin production could be attributed to the effects of nigR overexpression.

Gene Expression Analysis in the Wild-Type and Overexpression Strains
To determine the effects of nigR overexpression on nigericin biosynthetic genes, qRT PCR analysis was performed to assess the transcription of genes involved in the nig gene cluster. Transcriptional analyses showed that the expression levels of nigR, nigAVII, nigD nigCII, nigAI and nigE increased dramatically in nigR overexpression strains F913-hrdBR and F913-kasOR, compared with the unmodified wild-type strain ( Figure 6). In addition the expression of nigR also increased significantly in the F913-hrdBR and F913-kasOR strains. These results further proved that the enhanced production of nigericin in the over expression strains can be attributed to increased expression of nigR and subsequent acti vation of nigericin biosynthetic genes.

Gene Expression Analysis in the Wild-Type and Overexpression Strains
To determine the effects of nigR overexpression on nigericin biosynthetic genes, qRT-PCR analysis was performed to assess the transcription of genes involved in the nig gene cluster. Transcriptional analyses showed that the expression levels of nigR, nigAVII, nigD, nigCII, nigAI and nigE increased dramatically in nigR overexpression strains F913-hrdBR and F913-kasOR, compared with the unmodified wild-type strain ( Figure 6). In addition, the expression of nigR also increased significantly in the F913-hrdBR and F913-kasOR strains. These results further proved that the enhanced production of nigericin in the

Discussion
Nigericin has a broad spectrum of biological activities. Recent research has focused on its anticancer activities [19,27]. Previous studies have analyzed the detailed mechanism of nigericin biosynthesis; however, little is known about the regulation of nigericin biosynthesis. In this study, a SARP family activator, NigR-the only regulator in the nigericin biosynthetic gene cluster-was identified. Sequence analysis indicated that NigR has an N-terminal DNA-binding domain and an adjacent transcriptional activation domain. The presence of TTA codons in coding regions suggests that the translation of NigR may be regulated by bldA. RT-PCR analysis revealed that NigR is essential for the expression of structural genes, suggesting that these genes are the potential targets of NigR. Unfortunately, all attempts to overexpress and purify recombinant NigR proteins failed, and we were unable to perform gel mobility shift assays to identify the direct target genes of NigR. In a future study, we will try GUS (β-glucuronidase) assays to detect the interaction of NigR with promoters of target genes [28].
Combinational mutation and culture condition optimization have been carried out to improve nigericin production; however, titer improvement of nigericin production by overexpressing regulators has not been reported [29,30]. After decades of extensive research about the regulation of the production of secondary metabolites in actinomycetes, much is known particularly about Streptomyces species. Secondary metabolite titers can be influenced by global regulatory networks and pathway-specific regulation, which makes these regulators perfect targets for the rational engineering of overproduction strains. Overexpression of positive regulatory genes is a well-established strategy for strain improvement. By multiplying copy numbers of sanG, nikkomycin production was increased significantly [31]. In Streptomyces fradiae, insertion of a second copy of the positive regulator tylR under control of the strong constitutive ermE* promoter caused a 50% improvement in tylosin production [26]. The ermE* promoter from Saccharopolyspora erythraeus is Figure 6. qRT-PCR analysis of selected genes in nigericin biosynthesis. The expression level of samples collected from the F913 strain at fermentation day 2 was assigned a value of 1; the expression levels of nigR, nigAVII, nigD, nigCII, nigAI and nigE in in all samples are presented relative to that of samples collected from the F913 strain at fermentation day 2. Error bars show standard deviations from three independent experiments. A Student's t-test was used to determine the p-values. Significant difference: * p < 0.05, ** p < 0.01, *** p < 0.001.

Discussion
Nigericin has a broad spectrum of biological activities. Recent research has focused on its anticancer activities [19,27]. Previous studies have analyzed the detailed mechanism of nigericin biosynthesis; however, little is known about the regulation of nigericin biosynthesis. In this study, a SARP family activator, NigR-the only regulator in the nigericin biosynthetic gene cluster-was identified. Sequence analysis indicated that NigR has an N-terminal DNA-binding domain and an adjacent transcriptional activation domain. The presence of TTA codons in coding regions suggests that the translation of NigR may be regulated by bldA. RT-PCR analysis revealed that NigR is essential for the expression of structural genes, suggesting that these genes are the potential targets of NigR. Unfortunately, all attempts to overexpress and purify recombinant NigR proteins failed, and we were unable to perform gel mobility shift assays to identify the direct target genes of NigR. In a future study, we will try GUS (β-glucuronidase) assays to detect the interaction of NigR with promoters of target genes [28].
Combinational mutation and culture condition optimization have been carried out to improve nigericin production; however, titer improvement of nigericin production by overexpressing regulators has not been reported [29,30]. After decades of extensive research about the regulation of the production of secondary metabolites in actinomycetes, much is known particularly about Streptomyces species. Secondary metabolite titers can be influenced by global regulatory networks and pathway-specific regulation, which makes these regulators perfect targets for the rational engineering of overproduction strains. Overexpression of positive regulatory genes is a well-established strategy for strain improvement. By multiplying copy numbers of sanG, nikkomycin production was increased significantly [31]. In Streptomyces fradiae, insertion of a second copy of the positive regulator tylR under control of the strong constitutive ermE* promoter caused a 50% improvement in tylosin production [26]. The ermE* promoter from Saccharopolyspora erythraeus is the most widely used strong constitutive promoter; other optional tools, such as SF14p, are also available [32]. In recent years, strong and constitutive promoters such as kasO* promoter and hrdB promoter, have been developed, and have shown better performance in some Streptomyces species [33,34]. In this work we used kasO* promoter and hrdB promoter as cis regulatory elements to drive nigR expression. The results suggest that both promoters can upregulate nigericin expression and the F913-hrdBR strain showed higher nigericin production. The mean production of nigericin in the F913-hrdBR strain was 0.56 g/L. To our knowledge, this is the highest nigericin titer reported to date, representing a new step for scaling-up the production of nigericin. Promoter strength varied in different Streptomyces strains, implying that more promoters need to be screened to choose the most suitable one for strain improvement in S. malaysiensis F913. Secondary metabolites are synthesized from precursors through multistep biosynthetic pathways with the cells. Medium and fermentation process optimization are also required to further increase the production of nigericin in engineered overexpression strains.

Construction of NigR Mutants
To construct nigR disruption vectors, a 2329 bps PCR product from primer NigRdm-upF/R (Table S3) was amplified and named as the upstream arm, which contained partial N-terminal nigR and adjacent flanking sequences. A 2585 bps PCR product from primer NigRdm-dnF/R (Table S3) was amplified and named as the downstream arm, which contained partial C-terminal nigR and adjacent flanking sequences. The upstream arm was digested with HindIII and XbaI; the downstream arm was digested with XbaI and BamHI. A kanamycin-resistance cassette was amplified from pUC119::neo by PCR with primer kanF/R, and digested with XbaI. Three resulting DNA fragments were inserted into the HindIII and BamHI digested pSET152 to generate pSET152sv::nigR::neoR in which a 625 bp fragment of nigR was replaced by the kanamycin-resistance gene. As the ΦC31 integrase in pSET152 contained two HindIII sites and was digested by HindIII, the integrase coding region in pSET152sv::nigR::neoR was incomplete and this vector became a suicide vector. After restriction digestion analysis and PCR verification, pSET152sv::nigR::neoR was transformed into E. coli ET12567/pUZ8002 and conjugally transferred to S. malaysiensis F913 by intergeneric conjugation. After growing for 5 days at 28 • C, the colonies were replicated on MS agar plates containing kanamycin. The double cross-over exconjugants were apramycin sensitive and kanamycin resistant and these strains were then verified by PCR.

Complementation and Overexpression of NigR
The coding region of nigR with its native promoter was amplified from the genomic DNA of S. malaysiensis F913 with primer pair nigRNP-F/R to generate nigRNP (Table S3). NigRNP was digested with XbaI/EcoRI, then ligated with XbaI/EcoRI digested pSET152 to generate complementary vector pSET152::nigRc. The pSET152::nigRc was introduced into the DM01R strain to generate the DM01Rc strain. For construction of nigR overexpression vectors, the coding region of nigR was amplified from the genomic DNA of S. malaysiensis F913 with primer pair nigR-CDSF/R (Table S3) and digested with NdeI/EcoRI to gener- ate DNA fragment NigR-CDS. hrdB promoter was amplified from the genomic DNA of S. malaysiensis F913 with primer pair PhrdBF/R (Table S3) and digested with NdeI/XbaI to generate DNA fragment PhradB. NigR-CDS and PhrdB were ligated into XbaI/EcoRI digested pSET152 to generate pSET152::P hrdB R. To construct kasO* promoter, oligonucleotides PkasOF and PkasOR (Table S3) were mixed in a tube and heated to 95 • C for 5 min; the tube together with its content was then incubated into a beaker containing 200 mL water at 95 • C and cooled down slowly to room temperature to anneal these two oligonucleotides to PkasO, a double-strand DNA fragment with sticky ends on both sides. NigR-CDS and PkasO were ligated into XbaI/EcoRI digested pSET152 to generate pSET152::P kasO R. After restriction digestion analysis and PCR verification, pSET152::P hrdB R and pSET152::P kasO R were transformed into E. coli ET12567/pUZ8002 and conjugally transferred to S. malaysiensis F913 to generate F913-hrdBR and F913-kasOR, respectively.

Production and Analysis of Nigericin
For nigericin production, spore suspensions were inoculated into liquid Gause's medium and incubated for 168 h at 28 • C before sampling. For analysis of nigericin, culture supernatants were extracted twice with an equal volume of ethyl acetate, and mycelia were extracted with 50 mL of methanol. Combined extracts were dried by removing the solvent under reduced pressure at 50 • C. The dried pellet was then dissolved in 1 mL methanol. High-performance liquid chromatography (HPLC) analysis was performed on an Agilent 1260 HPLC system and ZORBAX SB-C18 column (5 µm, 4.6 × 250 mm) (Agilent Technologies, Inc., Santa Clara, CA, USA). HPLC conditions were as follows: a linear gradient of MeOH/H 2 O from 80:20 to 100:0 over 15 min, 100% MeOH for 5.5 min and 20% MeOH + 80% H 2 O for 6 min, flow rate = 1 mL/min, UV = 305 nm. For determination of cell dry weight, mycelium from 20 mL cell cultures were collected and dried at 65 • C for 16 h to constant weight. All experiments are carried out in triplicate. Significance was analyzed by means of a Student's t-test (GraphPad Prism 6).

RNA Extraction, RT-PCR and qRT-PCR
The primers used in RT-PCR and qRT-PCR are listed in Table S3. RNA extraction, genomic DNA removal, cDNA Synthesis and reverse-transcription PCR (RT-PCR) were the same as described [11]. For the RT-PCR experiment, total RNAs were extracted from the S. malaysiensis F913 and DM01R strains after inoculation for 96 h and used as templates for analysis of gene expression. For qRT-PCR analysis, total RNAs were isolated from mycelium of F913, F913-hrdBR and F913-kasOR strains grown in Gause's medium at various fermentation times (48, 72, 96, 120 and 144 h). The qRT-PCR was performed by a LightCycle 96 instrument (Roche, Zurich, Switzerland). All experiments were carried out in triplicate. The relative expression levels of all samples were calculated using the 2 −∆∆CT method [35]. All qRT-PCR values were normalized to the endogenous control hrdB (a housekeeping sigma factor in Streptomyces). Significance tests were calculated by means of a Student's t-test (GraphPad Prism 6).

Genome Sequencing, Assembly and Annotation
The genomic DNA of S. malaysiensis F913 was extracted, followed by RNase treatment. The draft genome sequence of S. malaysiensis F913 was achieved using Illumina Hiseq2000 sequencing system at NovoGene Bioinformatics Institute in Beijing, China. The pairedend reads generated by the Illumina sequencer were assembled by using SOAPdenovo 1.05 [36]. Functional gene annotation was based on Rapid Annotation using Subsystem Technology (RAST) [37]. The putative secondary metabolites' biosynthetic gene clusters were further specifically identified and categorized using antibiotics and a Secondary Metabolite Analysis Shell [38].

Nucleotide Sequence Accession Number
The nucleotide sequences of nig gene clusters determined in this study have been submitted to the GenBank database under accession number ON664936.

Conclusions
NigR, the only regulator in the nigericin biosynthetic gene cluster in S. malaysiensis F913, was identified as a SARP family regulator in this study. Disruption of nigR resulted in loss of nigericin production, while complementation of nigR restored nigericin production. The targets of NgrR might be structural genes in the nig gene cluster, given the knockout of ngrR abolishes the transcription of all biosynthesis genes. Nigericin overproducing strains were constructed through overexpression of nigR. The maximum production of nigericin was achieved in the F913-hrdBR strain at about 0.56 g/L, which was about 1.5-fold higher than S. malaysiensis F913. These results extended our understanding about the regulatory mechanism of nigericin biosynthesis, and have also provided a promising strategy for scaling up the production of nigericin.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antibiotics11070938/s1. Figure S1: Nucleotide sequence and consensus sequence in the promoter regions of target genes; Table S1: Deduced functions of genes in the nigericin biosynthetic gene cluster; Table S2: Strains and plasmid used in this work; Table S3: Primers used in this work. References [11,22,39,40] are cited in the supplementary materials.