WblA, a global regulator of antibiotic biosynthesis in Streptomyces

Abstract

Streptomyces species are soil-dwelling bacteria that produce vast numbers of pharmaceutically valuable secondary metabolites (SMs), such as antibiotics, immunosuppressants, antiviral, and anticancer drugs. On the other hand, the biosynthesis of most SMs remains very low due to tightly controlled regulatory networks. Both global and pathway-specific regulators are involved in the regulation of a specific SM biosynthesis in various Streptomyces species. Over the past few decades, many of these regulators have been identified and new ones are still being discovered. Among them, a global regulator of SM biosynthesis named WblA was identified in several Streptomyces species. The identification and understanding of the WblAs have greatly contributed to increasing the productivity of several Streptomyces SMs. This review summarizes the characteristics and applications on WblAs reported to date, which were found in various Streptomyces species and other actinobacteria.

Introduction

Streptomyces are Gram-positive high G + C filamentous soil bacteria with superior characteristics in producing a variety of secondary metabolites (SMs), including many pharmaceutically valuable compounds, such as antibiotics, as well as anticancer, antiviral, and immunosuppressant agents (Bérdy, 2005; Kinghorn et al., 2009; Mann, 2001; Sivalingam et al., 2019) . Streptomyces SMs are synthesized by a group of enzymes encoded by their corresponding biosynthetic gene cluster (BGC). They are typically under tight and complicated regulation at the transcriptional level (Liu et al., 2013). The biosynthesis of Streptomyces SM is regulated through multiple regulatory pathways induced by both nutritional and environmental stimuli (Lee et al., 2005; Sun et al., 2017; van Wezel & McDowall, 2011). Although various global regulatory systems present in most Streptomyces species can control both morphological differentiation and SM production, the SM biosynthetic gene sets are also subject to pathway-specific regulation by linked regulatory genes (van der Heul et al., 2018). Most of these pathway-specific regulatory genes are transcriptionally regulated by a range of global regulatory networks in most Streptomyces species (Liu et al., 2013; Xia et al., 2020).

Among the global regulatory proteins, the WhiB-like (Wbl) family of proteins, which are only present in Actinobacteria, such as Streptomyces, Corynebacteria, and Mycobacteria, are a major class (Bush, 2018, refer to this reference for comprehensive review on Wbls). Following the initial characterization of WhiB in S. coelicolor (Chater, 1972; Davis & Chater, 1992), multiple paralogs were identified in many other Streptomyces species (Soliveri et al., 2000). Genome sequencing revealed the prevalence of Wbl paralogs throughout the phylum. Fourteen Wbl proteins were identified in S. coelicolor, with 11 encoded on the chromosome, and 3 encoded on the large linear plasmid, SCP1 (Bentley et al., 2002, 2004). The WblA_sco is a WhiB4 ortholog, which is one of three Wbl-family members that regulate both differentiation and SM biosynthesis in S. coelicolor. In S. coeilcolor, a wblA_sco mutant exhibits a defect in sporulation, with some aerial hyphae failing to sporulate and appearing thinner compared to the wild type (Aínsa et al., 2000; Fowler-Goldsworthy et al., 2011).

A Global Antibiotic Downregulator, WblA_sco in S. coelicolor

The first biological function of WblA as an antibiotic downregulator was identified from somewhat unexpected experimental results. After the genome of S. coelicolor was sequenced nearly 20 years ago, Streptomyces interspecies DNA microarray analysis was applied to detect the global changes in mRNA abundance associated with the overproduction of the anticancer compound doxorubicin (DOX) in an S. peucetius overproducing industrial mutant (OIM) strain. S. coelicolor genome was the only available genome at the time to facilitate transcriptome analysis. The results showed that the wblA_sco gene was a pleiotropic downregulator of antibiotic biosynthesis in S. coelicolor (Fowler-Goldsworthy et al., 2011). Comparative transcriptome analyses of cultures of the wild-type and OIM mutant strains of S. peucetius using S. coelicolor cDNA microarrays which was the only available genome at the time identified more than 100 S. coelicolor potential candidate genes that showed at least a twofold change in transcription between the wild-type and OIM mutant strains. After further analysis of the growth phase-dependent transcription profiles of these potential candidate genes, 20 genes exhibiting particularly large transcriptional changes between the two strains were selected and overexpressed individually in S. coelicolor (Kang et al., 2007).

Among them, SCO3579, which was previously proposed to be a whiB-like putative transcription factor gene, was identified and later named wblA_sco in S. coelicolor (Soliveri et al., 2000). Although whiB is a developmental regulatory gene that was identified in S. coelicolor as being essential for the sporulation of aerial hyphae, the biological function of wblA_sco in SM regulation was not determined (Soliveri et al., 2000). The overexpression of wblA_sco inhibited the biosynthesis of actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA) in S. coelicolor. Moreover, transcripts encoded by pathway-specific activators of the three major S. coelicolor antibiotics (i.e. actIIORF4 for ACT, redD/Z for RED, and cdaR for CDA) were reduced in the wblA_sco-overexpressing S. coelicolor, implying that wblA_sco acts broadly to downregulate antibiotic biosynthesis in S. coelicolor (Kang et al., 2007).

During Streptomyces interspecies DNA microarray analysis, a previously-unidentified tetR family transcriptional regulatory gene (SCO1712), as well as a carbon flux regulating 6-phosphofructokinase gene (SCO5426), were also found to downregulate SM biosynthesis in the S. coelicolor wild-type strain as well as in a wblA_sco deletion mutant (Kim et al., 2011; Lee et al., 2010). The S. coelicolor triple knockout mutant (ΔwblA_sco, ΔSCO1712, and ΔSCO5426) showed the highest level of ACT production compared to any of the single and double knockout mutants in S. coelicolor, suggesting that wblA_sco along with other wblA_sco-independent regulatory and precursor pathway genes could be optimized synergistically for SM production in Streptomyces (Kim et al., 2011).

WblA Orthologs in Other Streptomyces Species

 

WblA_spe in S. peucetius

Subsequently, a wblA_sco ortholog named wblA_spe from S. peucetius was identified through the construction of a total genomic DNA library of the above-mentioned S. peucetius OIM and the screening using a wblA_sco gene probe. As expected, the production of both DXR and its precursor, daunorubicin (DNR) were improved through gene disruption of wblA_spe from S. peucetius OIM (Table 1). Moreover, several putative wblA_spe-dependent genes were also identified using interspecies DNA microarray analysis between the S. peucetius OIM and wblA_spe-disrupted S. peucetius OIM. Among the putative wblA_spe-dependent genes tested, a conserved hypothetical protein (SCO4967) further stimulated the production of DXR/DNR/aklavinone in the wblA_spe-disrupted S. peucetius OIM. These results suggest that sequential genetic manipulation of the wblA_spe and its dependent genes identified from comparative transcriptome analysis could provide an efficient and rational strategy for improving the titer of DXR/DNR in S. peucetius strain. This was the first example to improve the antibiotic-OIM strain using a microarray-driven reverse engineering approach in Streptomyces species (Noh et al., 2010).

Table 1.

WblA orthologs identified from various Streptomyces species

Gene	Strain	Size	Identity (with WblAsco)	Compounds	SM production in DIS mutant (fold)	References
WblAsco	S. coelicolor M145	112 aa	100%	Actinorhodin	Increased (5.6-fold)	Lee et al. (2010)
WblAspe	S. peucetius OIM-1	114 aa	95%	Doxorubicin, daunorubicin	Increased (1.7-fold)	Kang et al. (2007), Noh et al. (2010)
WblAtmc	Streptomyces sp. CK4412	130 aa	96%	Tautomycetin	Increased (3.2-fold)	Nah et al. (2012)
WblAgh	S. ghanaensis ATCC14672	128 aa	96%	Moenomycin A	Increased (2.3-fold)	Rabyk et al. (2011)
					Increased (2.5-fold)	Yan et al. (2020)
WblAsro	S. roseosporus NRRL15998	116 aa	90%	Daptomycin	Increased (1.5-fold)	Huang et al. (2017)
WblAsan	S. ansochromogenes 7100	112 aa	96%	Nikkomycin (major)	Abolished	Lu et al. (2015)
				Tylosin analogs (cryptic)	Activated
WblAso	S. somaliensis SCSIO ZH66	124 aa	85%	Violapyrone	Increased (NR)	Huang et al. (2016)
WblAsve	S. venezuelae ATCC15439	115 aa	90%	Pikromycin	Increased (3.5-fold)	Yan et al. (2016)
WblAscb	Streptomyces sp. CB03234	131 aa	93%	Tiancimycins	Increased (13.9-fold)	Nguyen et al. (2003)

SM, secondary metabolite; DIS, disruption; NR, not reported.

WblA_tmc in Streptomyces sp. CK4412

Tautomycetin (TMC) is a linear polyketide compound with a novel activated T cell-specific immunosuppressant and anticancer activities (Chae et al., 2004; Lee, Lee, et al., 2006; Niu et al., 2012). The whole-genome sequencing of the Streptomyces sp. CK4412 chromosome revealed the entire TMC (∼80-kb) BGC as well as the wblA_sco ortholog gene named wblA_tmc. The wblA_tmc from Streptomyces sp. CK4412 showed 96% amino acid identity compared to a previously known wblA_sco. (Figs. 1 & 2). The targeted gene disruption of wblA_tmc in Streptomyces sp. CK4412 caused an approximately threefold higher TMC production titer than that in the wild-type strain. Moreover, transcription analyses of the two TMC pathway-specific positive regulatory genes, tmcN and tmcT, located within its BGC showed that the only tmcT expression was strongly downregulated by wblA_tmc in Streptomyces sp. CK4412 (Nah et al., 2012). The tmcN expression was not affected by either deletion or overexpression of wblA_tmc in Streptomyces sp. CK4412 (Nah et al., 2012). These results suggest that the TMC BGC regulatory network is controlled by two pathway-specific positive regulators, WblA_tmc-dependent TmcT and WblA_tmc-independent TmcN, in Streptomyces sp. CK4412 (Nah et al., 2012).

Fig. 1.

WblAs phylogenetic tree among Streptomyces species. Phylogenetic tree was built using the Mega X software by neighbor joining test by Bootstrap. WblA_sco from S. coelicolor; wblA_spe from S. peucetius; wblA_tmc from Streptomyces sp. CK4412; wblA_gh from S. ghanaensis; wblA_sro from S. roseosporus; wblA_san from S. ansochromogenes; wblA_so from S. somaliensis; wblA_sve from S. venezuelae; wblA_scb from Streptomyces sp. CB03234; wblA_sav from S. avermitilis; and wblA_sgr from S. griseus.

Fig. 2.

Sequence alignments among the Streptomyces WblAs. Conserved (asterisk) and related (colon) amino acids were marked underneath. Gray box; cysteine conserved region.

WblA_gh in S. ghanaensis

Another wblA_sco ortholog named wblA_gh was identified from the whole-genome sequencing of the moenomycin producer, S. ghanaensis ATCC14672. A deletion of wblA_gh stimulated a more than twofold increase in moenomycins production along with inhibition of aerial mycelium sporulation. Moreover, the wblA_gh overexpression in S. ghanaensis ATCC14672 decreased the moenomycin production by 50%, implying that WblA_gh is a global antibiotic downregulator in S. ghanaensis ATCC14672. Since the moenomycin BGC in S. ghanaensis ATCC14672 did not contain any pathway-specific regulatory genes, the downstream target of WblA_gh is not known. Although the regulation of putative Streptomyces subtilisin inhibitor (SSI) named SSFG_01 620 was proposed to be linked to the WblA_gh deletion in S. ghanaensis ATCC14672, the detailed mechanism between WblA_gh and SSFG_01 620 needs to be further pursued (Rabyk et al., 2011).

WblA_sro in S. roseosporus

The wblA_sco ortholog gene named wblA_sro was also identified in the whole genome sequencing of the daptomycin producer S. roseosporus. Three types of strains, the wblA_sro disruption strain, the complemented strain, and the overexpression strains, were generated to determine if it could affect the production of SMs as well as morphogenesis. The disruption mutant of wblA_sro enhanced daptomycin production by more than 50% as well as blocked aerial mycelium sporulation. In contrast, overexpression of wblA_sro resulted in a significant decrease in the daptomycin production titer. As expected, the transcription of the key daptomycin positive regulatory genes atrA, dptR2, and dptR3, and the structural gene, dptE, were increased remarkably in the wblA_sro disruption mutant. These results suggest that wblA_sro plays a key downregulatory role in controlling daptomycin biosynthesis (Huang et al., 2017).

WblA_san in S. ansochromogenes

Another wblA_sco ortholog named wblA_san was found in S. ansochromogenes 7100 sequencing analysis. The wblA_san disruption mutant of S. ansochromogenes 7100 failed to sporulate as well as to produce nikkomycin, a major SM produced by S. ansochromogenes 7100 during fermentation. Moreover, two novel 16-membered tylosin-like macrolides were observed only in a fermentation broth of ΔwblA_san strain. These two compounds, which had different functional groups at the C6 position comparing with tylosin, exhibited similar antibacterial activities against several Gram-positive bacteria including Staphylococcus aureus and Bacillus cereus. Interestingly, however, these two compounds displayed much higher activity against S. pneumoniae than tylosin, suggesting that wblA ortholog disruption approach could activate cryptic compounds hidden in Streptomyces species, and the compounds identified by the ΔwblA_san in S. ansochromogenes might be used to broaden the application of tylosin (Lu et al., 2015).

WblA_so in S. somaliensis

A wblA_sco ortholog named wblA_so was found in deep sea-derived S. somaliensis SCSIO ZH66 through the whole-genome sequencing analysis. To activate cryptic BGCs in S. somaliensis SCSIO ZH66, the wblA_so from S. somaliensis SCSIO ZH66 was inactivated. Noticeable changes in SM production from the S. somaliensis ΔwblA_so mutant were observed and the α-pyrone compound named violapyrone B (VLP B) was isolated. The VLP BGC consisted of a type III polyketide synthase (PKS) gene vioA and a pathway-specific positive regulatory gene vioB. The inactivation of vioB further led to the isolation of another four VLPs analogs, one novel SM and two improved anti-MRSA (methicillin-resistant S. aureus, MRSA) SMs compared to VLP B. Transcriptional analysis showed that wblA_so seemed to regulate the expression levels of whi genes and wbl genes by different degrees, suggesting an intertwined regulatory mechanism of wblA_so in SM biosynthesis as well as in morphological differentiation from S. somaliensis SCSIO ZH66. The wblA_so inactivation-driven VLPs identification results imply that the wblA_so orthologs would be effective targets for the activation of cryptic BGCs in marine-derived Streptomyces strains (Huang et al., 2016).

WblA_sve in S. venezuelae

S. venezuelae ATCC15439 is a versatile producer for various macrolide antibiotics. The 12-membered and 14-membered ring macrolides are biosynthesized by pikromycin BGC (Lee, Park, et al., 2006; Oh & Kang, 2012; Xue et al., 1998; Zhang & Sherman, 2001). Because of low levels of pikromycin production, genetic engineering for titer improvement have been developed (Maharjan et al., 2008; Pyeon et al., 2017; Yi et al., 2018). The wblA ortholog gene named wblA_sve was also found with a high degree of amino acid identity (90% with WblA_sco) from S. venezuelae ATCC15439. Sporulation was blocked by a disruption of wblA_sve in S. venezuelae ATCC15439. The production of pikromycin was increased by 3.5-fold in S. venezuelaeΔwblA_sve and decreased by 2.5-fold in the wblA_sve overexpressing strain (Woo et al., 2014), suggesting that the WblA_sve controls both morphological differentiation and pikromycin production in S. venezuelae.

WblA_scb in Streptomyces sp. CB03234

Streptomyces sp. CB03234 is a native producer of both ten-membered enediyne tiancimycins (TNMs) and diterpernoid tiancilactones (TNLs) (Dong et al., 2018; Yan et al., 2016). Among the TNMs discovered, both TNM-A and TNM-D showed superior cytotoxic activities against several cancer cell lines (Yan et al., 2016, 2018). Comparative transcription analysis between a wild-type and a streptomycin-induced ribosome engineered TNMs high-producer CB03234-S exhibited that the wblA_scb transcription level was relatively higher than those of other wbl genes (Zhang et al., 2020). To overcome the low titer issue of these compounds, the wblA_sco ortholog named wblA_scb (tentatively named here) was disrupted via homologous recombination, resulting in 13.9- and 1.7-fold increases of TNM-A in CB3234S and CB3234-S, respectively (Zhang et al., 2020). The production of TNLs, a group of main fermentation metabolites in CB03234, was also affected by the deletion of wblA_scb. Although the wblA_scb in CB03234 deletion could improve the titers of TNMs significantly, the sporeless bald phenotypes could lead to the CB03234ΔwblA_scb unsuitable for the scaled-up TNMs production. Since the existence of wblA_scb is necessary for a spore formation but undesirable for TNMs overproduction, the NitR-ε-caprolactam (CPL) based inducible expression system for wblA_scb was constructed in the CB03234ΔwblA_scb. This cell factory system successfully maintained the overproduction of TNMs without any additional processes and recovered the normal development of sporulation upon induction (Zhang et al., 2020).

WblA Orthologs as SM Upregulators

Although most WblAs stated above negatively regulate SM biosynthesis in Streptomyces species, a couple of WblA orthologs such as WblA_ch in S. chattanoogensis L10 and WblA_sxi in S. xiamenensis 318 have been reported to act as SM upregulators in their hosts (Bu et al., 2019; Yu et al., 2014). The wblA_sxi overexpression under the Streptomyces constitutive promoter ermE*p stimulated the production of two tetramate macrolactams, ikarugamycin and capsimycin B in S. xiamenensis 318 (Bu et al., 2020). Similarly, the WblA_ch was proved to function as an activator of natamycin biosynthesis in S. chattanoogensis L10. The wblA_ch disruption downregulated natamycin production, while the wblA_ch overexpressed stimulated the natamycin yield by ∼30% (Yu et al., 2014). These findings suggest that some wblA orthologs are positively involved in the regulation of SM biosynthesis in some Streptomyces species.

Putative Regulatory Mechanism of WblA in Streptomyces

 

AdpA Binding to the wblA Promoter Region

Although many reports support that WblA is a general downregulator of SM biosynthesis in Streptomyces species, its regulatory target and mechanism are unclear. Although WblA has been proposed to be a transcriptional factor binding to a specific promoter of the target gene, there is no experimental evidence showing direct binding between WblA and the target promoter sequence (Bush, 2018). On the other hand, some studies on how the wblA gene expression is controlled have been reported. The upstream region of wblA in S. coelicolor was predicted to contain several putative AdpA binding motifs (Lee et al., 2013; Nguyen et al., 2003; Wolanski et al., 2011). AdpA is a key regulator controlling various processes involved in Streptomyces morphological differentiation and SM biosynthesis (Higo et al., 2012; Ohnishi et al., 2005). AdpA_sc was shown to bind specifically the wblA_sco upstream binding motifs through an electrophoretic mobility shift assay (EMSA), even though AdpA_sc failed to bind to the mutated wblA upstream motif (Lee et al., 2013). Moreover, an AdpA_sco disruption mutant showed increased wblA_sc transcription in S. coelicolor, suggesting that AdpA_sc negatively regulates wblA_sco transcription in S. coelicolor (Fig. 3, Lee et al., 2013).

Fig. 3.

Regulatory network of WblA controlled by AdpA_sc in S. coelicolor. ACT, actinorhodin; RED, undecylprodigiocin; CDA, calcium-dependent antibiotic.

BldD_sgh Binding to the wblA_gh Promoter Region

Cyclic dimeric 3′–5′ guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria (Römling et al., 2013). In Streptomyces, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating the activity of the pleiotropic regulator BldD (Tschowri et al., 2014). Previous work on the transcriptional analysis of the BldD-targeted genes in non-Streptomyces actinobacterium Actinoplanes missouriensis showed that BldD repressed 12 genes, including a wblA ortholog (Mouri et al., 2017). The deletion of bldD_gh led to an increase in wblA_gh expression. While the bldD_gh-deleted mutant of S. ghanaensis showed strongly reduced moenomycin A (MmA) production, the double mutant S. ghanaensis ΔbldD_ghΔwblA_gh restored the capacity to produce MmA. This suggests that WblA_gh plays a crucial role in the regulation of MmA biosynthesis and that BldD_gh controls the expression of wblA_gh. EMSA proved the direct control of wblA_gh transcription by BldD_gh. Increasing concentrations of BldD_gh resulted in the formation of one nucleoprotein complex with the wblA_gh promoter through EMSA. Moreover, binding of BldD_gh to the wblA_gh promoter was improved significantly in the presence of increasing concentrations of c-di-GMP. The specificity of this interaction is shown by the ability of the unlabeled wblA_gh promoter to compete with the radiolabeled one for BldD_gh (Makitrynskyy et al., 2020). This suggests that the high expression of wblA is mediated by the deletion of bldD, which leads to the repression of antibiotic synthesis (Yan et al., 2020).

Oxidative Stress Response by WblA

In addition to the Streptomyces SM regulation, WblA was also suggested to be involved in the oxidative stress response (Kim et al., 2012). Since WhcA, a WblA ortholog in C. glutamicum, was found to play a negative role in the oxidative stress response, wblA_sco was speculated to have a similar role in S. coelicolor. A wblA_sco-deletion mutant showed a less sensitive response to oxidative stress induced by the diamide present in the solid plate culture. Comparative real-time qRT-PCR analysis showed that the transcription levels of oxidative stress-related genes, including sodF, sodF2, sodN, trxB, and trxB2, were higher in the wblA_sco-deletion mutant than the wild type, both in the absence and presence of oxidative stress. Moreover, expression of these target genes in the S. coelicolor wild type was stimulated only in the presence of oxidative stress, suggesting that WblA_sco might play a negative role in the oxidative stress response of S. coelicolor, similar to that found in C. glutamicum WhcA (Kim et al., 2012).

Because WhcA was confirmed to interact with dioxygenase-encoding SpiA (stress protein interacting with WhcA) in C. glutamicum, a SpiA ortholog in S. coelicolor SCO2553 protein (named SpiA_sco) was also proposed to interact with WblA in S. coelicolor. Using heterologous expression in Escherichia coli and in vitro pull-down assays, WblA_sco was confirmed to bind to the SpiA_sco, which was influenced by oxidants, such as diamide. These observations suggest that the interaction between WblA_sco and SpiA_sco is not only specific but also modulated by the redox status of the cell. Moreover, a spiA_sco-disruption mutant exhibited a less sensitive response to the oxidative stress induced by the diamide present in solid plate culture. Real-time qRT-PCR analysis also showed that the transcription levels of oxidative stress response genes were higher in the spiA_sco-deletion mutant than in wild-type S. coelicolor. These results show that SpiA_sco negatively regulates WblA during the oxidative stress responses in S. coelicolor (Kim et al., 2013).

WblA Orthologs in Other Actinobacteria

The Gram-positive rare actinomycete Pseudonocardia autotrophica KCTC9441 was previously identified in the novel di-sugar-containing polyene compound producer, which was called NPP (Nystatin-like Pseudonocardia Polyene) (Kim et al., 2009). By whole-genome sequencing, a WblA ortholog was isolated and identified from P. autotrophica. WblA_pau showed 49% amino acid identity with various Streptomyces WblAs and 39% amino acid identity with a WblA ortholog, WhcA from C. glutamicum (Kim et al., 2014). Although no significant differences in NPP production were observed in the heterologous expression of wblA_sco, a disruption of wblA_pau resulted in an approximately 80% increase in NPP production (Kim et al., 2014). These results suggest that the biological significance of wblA_pau might be similar to a previously known wblA from various Streptomyces strains, even though the amino acid identity was relatively low.

Corynebactirum rarely produces antibiotics and other SMs, there are no reports on WhcA-driven SM regulation. On the other hand, WhcA and other WhiB-like proteins appear to play key roles in the regulation of stress-related processes. WhcA appears to regulate negatively the genes involved in response to oxidative stress (Choi et al., 2009). WhcA was reported to interact directly with its partner, SpiA (Stress protein encoding a dioxygenase/oxidoreductase interacting with WhcA). This WhcA-SpiA interaction was confirmed experimentally to be disrupted in the presence of the oxidant diamide (Park et al., 2012). As stated above, this mechanism appears to be conserved in S. coelicolor, in which a SpiA_sco was found to bind to WblA_sco and downregulate the WblA-dependent oxidative stress response (Kim et al., 2013).

There is also a WblA ortholog named WhiB4_mtb in Mycobacterium tuberculosis (Bush, 2018). The genome of M. smegmatis MC² 155, a model strain to study M. tuberculosis, has been sequenced (Mohan et al., 2015). The M. smegmatis MC² 155 and M. tuberculosis share significant similarities in their genome. The in silico analysis using antiSMASH 5.0 predicted presence of 18 SM BGCs in MC² 155. However, little is known on relation between SM regulation and WhiB4mtb. A major role of WhiB4_mtb in Mycobacteria is believed to regulate redox-sensing and its homeostasis. A deletion of whiB4_mtb leads to the hyper-induction of antioxidants, increased resistance to oxidative stress in vitro, and enhanced survival in macrophages (Alam et al., 2007; Chawla et al., 2012). WhiB4_mtb also contains an O₂- and NO-sensitive [4Fe–4S] cluster and the WhiB4_mtb [4Fe–4S] cluster appears to be more sensitive to O₂ than that other reported Wbls (Crack et al., 2009; Kudhair et al., 2017; Singh et al., 2007).

Concluding remarks

WblAs are highly homologous global SM regulators present in most Streptomyces species as well as its closely related actinobacteria, of which the detailed mechanism needs to be further elucidated. Although WblAs typically regulate the SM BGC expression at the transcriptional level by controlling the target pathway-specific regulatory gene expression, there was no direct evidence showing direct binding between WblA and the target promoter sequence. The WblA is believed to be controlled by another global regulator such as AdpA and also involved in morphological differentiation and oxidative stress response through its iron-sulfur cluster. Considering the prevalence of WblAs and its conserved regulatory roles, the strategy for selective manipulation of WblAs should provide an efficient approach to improve the SM titer and discover cryptic SMs in actinobacteria.

Funding

This study was supported by the National Research Foundation of Korea (No. 2021R1A2C2012203). H.J. Nah was partially supported by the Basic Science Research Program through the National Research Foundation of Korea (No. 2018R1A6A3A01013335).

Conflict of Interest

The authors declare no conflict of interest.