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TetR family transcriptional regulators (TFRs) are widespread in actinomycetes, which exhibit diverse regulatory modes in antibiotic biosynthesis. Nitrogen regulators play vital roles in modulation of primary and secondary metabolism. However, crosstalk between TFR and nitrogen regulator has rarely been reported in actinomycetes. Herein, we demonstrated that a novel TFR, SACE_4839, was negatively correlated with erythromycin yield in Saccharopolyspora erythraea A226. SACE_4839 indirectly suppressed erythromycin synthetic gene eryAI and resistance gene ermE and directly inhibited its adjacent gene SACE_4838 encoding a homologue of nitrogen metabolite repression (NMR) regulator NmrA (herein named NmrR). The SACE_4839-binding sites within SACE_4839-nmrR intergenic region were identified. NmrR positively controlled erythromycin biosynthesis by indirectly stimulating eryAI and ermE and directly repressing SACE_4839. NmrR was found to affect growth viability under the nitrogen source supply. Furthermore, NmrR directly repressed glutamine and glutamate utilization-related genes SACE_1623, SACE_5070 and SACE_5979 but activated nitrate utilization-associated genes SACE_1163, SACE_4070 and SACE_4912 as well as nitrite utilization-associated genes SACE_1476 and SACE_4514. This is the first reported NmrA homolog for modulating antibiotic biosynthesis and nitrogen metabolism in actinomycetes. Moreover, combinatorial engineering of SACE_4839 and nmrR in the high-yield S. erythraea WB resulted in a 68.8% increase in erythromycin A production. This investigation deepens the understanding of complicated regulatory network for erythromycin biosynthesis. Key points: • SACE_4839 and NmrR had opposite contributions to erythromycin biosynthesis. • NmrR was first identified as a homolog of another nitrogen regulator NmrA. • Cross regulation between SACE_4839 and NmrR was revealed. [ABSTRACT FROM AUTHOR]

Validamycin A (VAL-A) is a CN aminocyclitol antibiotic produced by Streptomyces hygroscopicus var. jinggangensis 5008, which has been widely used as antifungal agent against rice sheath blight disease. VAL-A biosynthesis has been proven to be affected by γ-butyrolactone and temperature. Herein, we showed that GlnR, a global regulator in nitrogen metabolism, is specifically associated with valK-valA intergenic promoter region by DNA-affinity chromatography and MS-based protein identification. Subsequent EMSA and DNase I footprinting assays revealed two GlnR binding sites in this promoter region. Targeted disruption of glnR in S. hygroscopicus 5008 led to a significant increase in the transcription of VAL-A structural genes, albeit the VAL-A production was reduced by 80 % and the sporulation of the mutant was impaired. Compared with the wild-type 5008, site-specific mutagenesis of GlnR binding site I enhanced VAL-A production by 2.5-fold, whereas the mutation of GlnR binding site II resulted in a 50 % reduction of VAL-A yield. Moreover, tandem mutation of site I in the site II mutant led to a 66 % increase of VAL-A production. The result suggested that GlnR not only serves as an inhibitor by binding site I but also as an activator by binding site II for VAL-A biosynthesis. Furthermore, overexpression of glnR in the site I mutant JG45 improved VAL-A production for 41 % compared with the control strain containing the vector. Therefore, the obtained data illustrate a novel regulatory feature of the global regulator GlnR. GlnR is firstly proved to act simultaneously as an activator and a repressor in validamycin biosynthesis by binding to different loci within a promoter region of the gene cluster. [ABSTRACT FROM AUTHOR]

The Lrp and MarR families are two groups of transcriptional regulators widely distributed among prokaryotes. However, the hierarchical-regulatory relationship between the Lrp family and the MarR family remains unknown. Our previous study found that an Lrp (SACE_Lrp) from Saccharopolyspora erythraea indirectly repressed the biosynthesis of erythromycin. In this study, we characterized a novel MarR family protein (SACE_6745) from S. erythraea, which is controlled by SACE_Lrp and plays a direct regulatory role in erythromycin biosynthesis and export. SACE_Lrp directly regulated the expression of marR by specifically binding a precise site OM (5'-CTCCGGGAACCATT-3'). Gene disruption of marR increased the production of erythromycin by 45% in S. erythraea A226. We found that MarR has direct DNA-binding activity for the promoter regions of the erythromycin biosynthetic genes, as well as an ABC exporter SACE_2701-2702 which was genetically proved to be responsible for erythromycin efflux. Disruption of SACE_Lrp in industrial S. erythraea WB was an efficient strategy to enhance erythromycin production. Herein, we jointly engineered SACE_Lrp and its target MarR by deleting marR in WBΔSACE_Lrp, resulting in 20% increase in erythromycin yield in mutant WBΔLrpΔmarR compared to WBΔSACE_Lrp, and 39% to WB. Overall, our findings provide new insights into the hierarchical-regulatory relationship of Lrp and MarR proteins and new avenues for coordinating antibiotic biosynthesis and export by joint engineering regulators in actinomycetes. Key points: • The hierarchical-regulatory relationship between SACE_Lrp and MarR was identified. • MarR directly controlled the expression of erythromycin biosynthesis and export genes. • Joint engineering of SACE_Lrp-MarR regulatory element enhanced erythromycin production. [ABSTRACT FROM AUTHOR]

Leucine-responsive regulatory proteins (Lrps) are a family of transcription factors involved in diverse biological processes in bacteria. So far, molecular mechanism of Lrps for regulating antibiotics biosynthesis in actinomycetes remains largely unexplored. This study, for the first time in Streptomyces lincolnensis, identified an Lrp (named as SLCG_Lrp) associated with lincomycin production. SLCG_Lrp was validated to be a positive regulator for lincomycin biosynthesis by directly stimulating transcription of two structural genes (lmbA and lmbV), three resistance genes (lmrA, lmrB and lmrC), and a regulatory gene (lmbU) within the lincomycin biosynthetic gene (lin) cluster. SLCG_Lrp was transcriptionally self-inhibited and triggered the expression of its adjacent gene SLCG_3127 encoding a LysE superfamily protein. Further, the binding site of SLCG_Lrp in the intergenic region of SLCG_3127 and SLCG_Lrp was precisely identified. Inactivation of SLCG_3127 in S. lincolnensis resulted in yield improvement of lincomycin, which was caused by intracellular accumulation of proline and cysteine. Arginine and phenylalanine were identified as specific regulatory ligands, respectively, to reduce and promote DNA-binding affinity of SLCG_Lrp. We further found that SLCG_Lrp was directly repressed by SLCG_2919, the first identified transcription factor outside lin cluster for lincomycin production. Therefore, our findings revealed SLCG_Lrp-mediated transcriptional regulation of lincomycin biosynthesis. This study extends the understanding of molecular mechanisms underlying lincomycin biosynthetic regulation. [ABSTRACT FROM AUTHOR]