An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Streptomyces umbrella toxin particles block hyphal growth of competing species.

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions.

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Genomic Insights and Synthetic Biology Applications of Marine Actinomycete Streptomyces griseoincarnatus HNS054.

The marine bacterium Streptomyces sp. HNS054 shows promise as a platform for producing natural products. Isolated from a marine sponge, HNS054 possesses several desirable traits for bioengineering: rapid growth, salt tolerance, and compatibility with genetic tools. Its genome contains 21 potential biosynthetic gene clusters, offering a rich source of natural products. We successfully engineered HNS054 to increase the production of aborycin and actinorhodin by 4.5-fold and 1.2-fold, respectively, compared to S. coelicolor M1346 counterparts. With its unique features and amenability to genetic manipulation, HNS054 emerges as a promising candidate for developing novel marine-derived drugs and other valuable compounds.

Engineering Site 228 of Streptomyces coelicolor Laccase for Optimizing Catalytic Activity.

Malachite green (MG) poses a formidable threat to ecosystems and human health. Laccase emerges as a promising candidate for MG degradation, prompting an investigation into the catalytic activity modulation of a small laccase (SLAC) from Streptomyces coelicolor, with a focus on amino acid position 228. Through saturation mutagenesis, five mutants with a 50% increase in the specific activity were generated. Characterization revealed notable properties, Km of E228F was 8.8% of the wild type (WT), and E288T exhibited a 133% kcat compared to WT. Structural analyses indicated improved hydrophobicity and electrostatic potential on the mutants' surfaces, with the stable E228F-ABTS complex exhibiting reduced flexibility, possibly contributing to the observed decrease in turnover rate. Mutants demonstrated enhanced MG decolorization, particularly E228G. Site 228 acts as a crucial functional control switch, suggesting its potential role in SLAC engineering. This study provides insights into laccase modulation and offers promising avenues for enzymatic bioremediation applications.

Activation of zinc uptake regulator by zinc binding to three regulatory sites.

Zur is a Fur-family metalloregulator that is widely used to control zinc homeostasis in bacteria. In Streptomyces coelicolor, Zur (ScZur) acts as both a repressor for zinc uptake (znuA) gene and an activator for zinc exporter (zitB) gene. Previous structural studies revealed three zinc ions specifically bound per ScZur monomer; a structural one to allow dimeric architecture and two regulatory ones for DNA-binding activity. In this study, we present evidence that Zur contains a fourth specific zinc-binding site with a key histidine residue (H36), widely conserved among actinobacteria, for regulatory function. Biochemical, genetic, and calorimetric data revealed that H36 is critical for hexameric binding of Zur to the zitB zurbox and further binding to its upstream region required for full activation. A comprehensive thermodynamic model demonstrated that the DNA-binding affinity of Zur to both znuA and zitB zurboxes is remarkably enhanced upon saturation of all three regulatory zinc sites. The model also predicts that the strong coupling between zinc binding and DNA binding equilibria of Zur drives a biphasic activation of the zitB gene in response to a wide concentration change of zinc. Similar mechanisms may be pertinent to other metalloproteins, expanding their response spectrum through binding multiple regulatory metals.

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria.

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.

The ssgB gene is required for the early stages of sporangium formation in Actinoplanes missouriensis.

In Streptomyces, multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis, which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis. The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (ΔAmssgB) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB. Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) (ssgA-G) in the ΔAmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis. In contrast to the ΔAmssgB strain, the ftsZ-disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae. IMPORTANCE: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

Secretory production and characterization of a highly effective chitosanase from Streptomyces coelicolor A3(2) M145 in Pichia pastoris.

In this study, a glycoside hydrolase family 46 chitosanase from Streptomyces coelicolor A3(2) M145 was firstly cloned and expressed in Pichia pastoris GS115 (P. pastoris GS115). The recombinant enzyme (CsnA) showed maximal activity at pH 6.0 and 65°C. Both thermal stability and pH stability of CsnA expressed in P. pastoris GS115 were significantly increased compared with homologous expression in Streptomyces coelicolor A3(2). A stable chitosanase activity of 725.7 ± 9.58 U mL-1 was obtained in fed-batch fermentation. It's the highest level of CsnA from Streptomyces coelicolor expressed in P. pastoris so far. The hydrolytic process of CsnA showed a time-dependent manner. Chitosan oligosaccharides (COSs) generated by CsnA showed antifungal activity against Fusarium oxysporum sp. cucumerinum (F. oxysporum sp. cucumerinum). The secreted expression and hydrolytic performance make the enzyme a desirable biocatalyst for industrial controllable production of chitooligosaccharides with specific degree of polymerization, which have potential to control fungi that cause important crop diseases.

σE of Streptomyces coelicolor can function both as a direct activator or repressor of transcription.

σ factors are considered as positive regulators of gene expression. Here we reveal the opposite, inhibitory role of these proteins. We used a combination of molecular biology methods and computational modeling to analyze the regulatory activity of the extracytoplasmic σE factor from Streptomyces coelicolor. The direct activator/repressor function of σE was then explored by experimental analysis of selected promoter regions in vivo. Additionally, the σE interactome was defined. Taken together, the results characterize σE, its regulation, regulon, and suggest its direct inhibitory function (as a repressor) in gene expression, a phenomenon that may be common also to other σ factors and organisms.

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

The stringent response is strongly activated in the antibiotic producing strain, Streptomyces coelicolor.

S. lividans and S. coelicolor are phylogenetically closely related strains with different abilities to produce the same specialized metabolites. Previous studies revealed that the strong antibiotic producer, S. coelicolor, had a lower ability to assimilate nitrogen and phosphate than the weak producer, Streptomyces lividans, and this resulted into a lower growth rate. A comparative proteomic dataset was used to establish the consequences of these nutritional stresses on the abundance of proteins of the translational apparatus of these strains, grown in low and high phosphate availability. Our study revealed that most proteins of the translational apparatus were less abundant in S. coelicolor than in S. lividans whereas it was the opposite for ET-Tu 3 and a TrmA-like methyltransferase. The expression of the latter being known to be under the positive control of the stringent response whereas that of the other ribosomal proteins is under its negative control, this indicated the occurrence of a strong activation of the stringent response in S. coelicolor. Furthermore, in S. lividans, ribosomal proteins were more abundant in phosphate proficiency than in phosphate limitation suggesting that a limitation in phosphate, that was also shown to trigger RelA expression, contributes to the induction of the stringent response.

High-intensity green light potentially activates the actinorhodin biosynthetic pathway in Streptomyces coelicolor A3(2).

The development of practices that enhance the potential of actinomycetes as major antibiotic producers is a challenge in discovering new secondary metabolites. Light, an essential external stimulus for most microorganisms, could be exploited to manipulate their physiological processes. However, the effects of monochromatic green light on the production of secondary metabolites in actinomycetes have not yet been reported. In this paper, we report a novel and simple method that uses high-intensity monochromatic green light to potentially induce the production of cryptic secondary metabolites in the model actinomycete Streptomyces coelicolor A3(2). Using actinorhodin (ACT), a blue-pigmented antibiotic, and undecylprodigiosin (RED), a red-pigmented antibiotic, as indicators, we found that irradiation with high-intensity monochromatic green light-emitting diodes promoted sporulation, significantly decreased RED production, and increased ACT production. Semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses revealed, for the first time, that stimulation with green light accelerated the expression of ActII-ORF4, a pathway-specific regulator of ACT biosynthesis in S. coelicolor A3(2). This approach of stimulating secondary metabolite biosynthesis pathways in actinomycetes by irradiation with high-intensity monochromatic green light is expected to facilitate the discovery of cryptic antibiotics that are not typically produced under conventional dark culture conditions. However, the effective intensity and duration of irradiation with green light that are required to activate these metabolite pathways may vary markedly among actinomycetes.

Construction of Streptomyces coelicolor A3(2) mutants that exclusively produce NA4/NA6 intermediates of agarose metabolism through mutation induction.

NA4/NA6, an intermediate degradation product of ß-agarase, is a high value-added product with anticancer, anti-obesity, and anti-diabetic effects. Therefore, a method that enables the efficient production of NA4/NA6 would be useful from economic and medical perspectives. In this study, we aimed to generate a Streptomyces coelicolor A3(2) mutant M22-2C43 that produces NA4/NA6 as a final product; this method serves as a more efficient alternative to the enzymatic conversion of ß-agarase for the generation of these products. The M22-2C43 strain was generated through two rounds of mutagenesis and screening for increased ß-agarase activity and effective production of NA4/NA6. We assembled the complete genomes of two mutants, M22 and M22-2C43, which were identified following a two-round screening. Large and small genetic changes were found in these two mutants, including the loss of two plasmids present in wild-type S. coelicolor A3(2) and chromosome circularization of mutant M22-2C43. These findings suggest that mutant M22-2C43 can produce NA4/NA6 as a degradation product due to functional inactivation of the dagB gene through a point mutation (G474A), ultimately preventing further degradation of NA4/NA6 to NA2. To our knowledge, this is the first report of a microbial strain that can effectively produce NA4/NA6 as the main degradation product of ß-agarase, opening the door for the use of this species for the large-scale production of this valuable product.

Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations.

The key to type 1 copper (T1Cu) function lies in the fine tuning of the CuII/I reduction potential (E°'T1Cu ) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°'T1Cu in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°'T1Cu of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°'T1Cu . While minimal and small E°'T1Cu increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°'T1Cu increase. Moreover, the influence of these mutations on E°'T1Cu is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°'T1Cu of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (kcat /KM ) relative to WT-SLAC.

Systems-wide analysis of the ROK-family regulatory gene rokL6 and its role in the control of glucosamine toxicity in Streptomyces coelicolor.

IMPORTANCE: Central metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate or glucosamine 6-phosphate is lethal to many bacteria, a yet unresolved phenomenon referred to as "aminosugar sensitivity." We made use of this concept by selecting for suppressors in genes related to glucosamine toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and major facilitator superfamily transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents the toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 shed new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.

Heterologous overproduction of oviedomycin by refactoring biosynthetic gene cluster and metabolic engineering of host strain Streptomyces coelicolor.

BACKGROUND: Oviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds. RESULTS: Oviedomycin BGC was captured from the genome of Streptomyces antibioticus by a newly constructed plasmid, pCBA, and conjugated into the heterologous strain, S. coelicolor M1152. To increase the production of oviedomycin, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized in an in vitro setting to refactor the native promoters within the ovm BGC. The target promoters of refactoring were selected based on examination of factors such as transcription levels and metabolite profiling. Furthermore, genome-scale metabolic simulation was applied to find overexpression targets that could enhance the biosynthesis of precursors or cofactors related to oviedomycin production. The combined approach led to a significant increase in oviedomycin production, reaching up to 670 mg/L, which is the highest titer reported to date. This demonstrates the potential of the approach undertaken in this study. CONCLUSIONS: The metabolic engineering approach used in this study led to the successful production of a valuable polyketide, oviedomycin, via BGC cloning, promoter refactoring, and gene manipulation of host metabolism aided by genome-scale metabolic simulation. This approach can be also useful for the efficient production of other secondary molecules encoded by 'silent' BGCs.

Mycothiol maintains the homeostasis and signalling of nitric oxide in Streptomyces coelicolor A3(2) M145.

BACKGROUND: Previous studies have revealed a nitric oxide (NO) metabolic cycle in which NO, nitrate (NO3-), and nitrite (NO2-) circulate. The NO produced in this cycle serves as a signalling molecule that regulates actinorhodin (ACT) production via the DevS/DevR NO-dependent two-component system (TCS) in Streptomyces coelicolor A3(2) M145. However, the mechanisms involved in the regulation of NO signalling in S. coelicolor have not yet been elucidated. Mycothiol (MSH), a thiol molecule produced by Actinomyces, is involved in the defence mechanisms against oxidative stress. Therefore, this study focused on the correlation between intracellular NO and MSH levels. RESULTS: To investigate the interaction of MSH with endogenously produced NO, we generated an S. coelicolor A3(2) strain deficient in MSH biosynthesis. This mutant strain exhibited a decrease in low-molecular-weight S-nitrosothiols and intracellular NO levels during culture compared to those of the wild-type strain. Moreover, the mutant strain exhibited reduced activity of the DevS/DevR TCS, a regulator of NO homeostasis and ACT production, from the early stage of culture, along with a decrease in ACT production compared to those of the wild-type strain. CONCLUSIONS: This study suggests that MSH maintains intracellular NO homeostasis by forming S-nitrosomycothiol, which induces NO signalling. Finally, we propose a metabolic model in which MSH from endogenously produced NO facilitates the maintenance of both NO homeostasis and signalling in S. coelicolor A3(2) M145.

LcbR1, a newly identified GntR family regulator, represses lincomycin biosynthesis in Streptomyces lincolnensis.

The Actinomycetes Streptomyces lincolnensis is the producer of lincosamide-type antibiotic lincomycin, a widely utilized drug against Gram-positive bacteria and protozoans. In this work, through gene knockout, complementation, and overexpression experiments, we identified LcbR1 (SLINC_1595), a GntR family transcriptional regulator, as a repressor for lincomycin biosynthesis. Deletion of lcbR1 boosted lincomycin production by 3.8-fold, without obvious change in morphological development or cellular growth. The homologues of LcbR1 are widely distributed in Streptomyces. Heterologous expression of SCO1410 from Streptomyces coelicolor resulted in the reduction of lincomycin yield, implying that the function of LcbR1 is conserved across different species. Alignment among sequences upstream of lcbR1 and their homologues revealed a conserved 16-bp palindrome (-TTGAACGATCCTTCAA-), which was further proven to be the recognition motif of LcbR1 by electrophoretic mobility shift assays (EMSAs). Via this motif, LcbR1 suppressed the transcription of lcbR1 and SLINC_1596 sharing the same bi-directional promoter. SLINC_1596, one important target of LcbR1, exerted a positive effect on lincomycin production. As detected by quantitative real-time PCR (qRT-PCR) analyses, the expressions of all selected structural (lmbA, lmbC, lmbJ, lmbV, and lmbW), resistance (lmrA and lmrB) and regulatory genes (lmrC and lmbU) from lincomycin biosynthesis cluster were upregulated in deletion strain ΔlcbR1 at 48 h of fermentation, while the mRNA amounts of bldD, glnR, ramR, SLCG_Lrp, and SLCG_2919, previously characterized as the regulators on lincomycin production, were decreased in strain ΔlcbR1, although the regulatory effects of LcbR1 on the above differential expression genes seemed to be indirect. Besides, indicated by EMSAs, the expression of lcbR1 might be regulated by GlnR, SLCG_Lrp, and SLCG_2919, which shows the complexity of the regulatory network on lincomycin biosynthesis. KEY POINTS: • LcbR1 is a novel and conservative GntR family regulator regulating lincomycin production. • LcbR1 modulates the expressions of lcbR1 and SLINC_1596 through a palindromic motif. • GlnR, SLCG_Lrp, and SLCG_2919 can control the expression of lcbR1.