Milbemycins, a group of 16-membered macrolide antibiotics, are used widely as insecticides and anthelmintics. Previously, a limited understanding of the transcriptional regulation of milbemycin biosynthesis has hampered efforts to enhance antibiotic production by engineering of regulatory genes. Here, a novel ArpA/AfsA-type system, SbbR/SbbA (SBI_08928/SBI_08929), has been identified to be involved in regulating milbemycin biosynthesis in the industrial strain S. bingchenggensis BC04. Inactivation of sbbR in BC04 resulted in markedly decreased production of milbemycin, while deletion of sbbA enhanced milbemycin production. Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting studies showed that SbbR has a specific DNA-binding activity for the promoters of milR (the cluster-situated activator gene for milbemycin production) and the bidirectionally organized genes sbbR and sbbA. Transcriptional analysis suggested that SbbR directly activates the transcription of milR, while represses its own transcription and that of sbbA. Moreover, 11 novel targets of SbbR were additionally found, including seven regulatory genes located in secondary metabolite biosynthetic gene clusters (e.g. sbi_08420, sbi_08432, sbi_09158, sbi_00827, sbi_01376, sbi_09325 and sig24sbh) and four well-known global regulatory genes (e.g. glnRsbh, wblAsbh, atrAsbh and mtrA/Bsbh). These data suggest that SbbR is not only a direct activator of milbemycin production, but also a pleiotropic regulator that controls the expression of other cluster-situated regulatory genes and global regulatory genes. Overall, this study reveals the upper-layer regulatory system that controls milbemycin biosynthesis, which will not only expand our understanding of the complex regulation in milbemycin biosynthesis, but also provide a basis for an approach to improve milbemycin production via genetic manipulation of SbbR/SbbA system.

Flowering is a crucial stage for plant reproductive success; therefore, the regulation of plant flowering has been widely researched. Although multiple well-defined endogenous and exogenous flowering regulators have been reported, new ones are constantly being discovered. Here, we confirm that a novel plant growth regulator guvermectin (GV) induces early flowering in Arabidopsis. Interestingly, our genetic experiments newly demonstrated that WRKY41 and its homolog WRKY53 were involved in GV-accelerated flowering as positive flowering regulators. Overexpression of WRKY41 or WRKY53 resulted in an early flowering phenotype compared to the wild type (WT). In contrast, the w41/w53 double mutants showed a delay in GV-accelerated flowering. Gene expression analysis showed that flowering regulatory genes SOC1 and LFY were upregulated in GV-treated WT, 35S:WRKY41, and 35S:WRKY53 plants, but both declined in w41/w53 mutants with or without GV treatment. Meanwhile, biochemical assays confirmed that SOC1 and LFY were both direct targets of WRKY41 and WRKY53. Furthermore, the early flowering phenotype of 35S:WRKY41 lines was abolished in the soc1 or lfy background. Together, our results suggest that GV plays a function in promoting flowering, which was co-mediated by WRKY41 and WRKY53 acting as new flowering regulators by directly activating the transcription of SOC1 and LFY in Arabidopsis.

Streptomyces can produce numerous antibiotics and many other bioactive compounds. Recently, the molecular mechanisms of transcriptional regulators in control of antibiotic production by influencing the expression of biosynthetic gene clusters (BGCs) have been extensively studied. However, for regulators that affect both antibiotic production and cell growth, the way to influence antibiotic production may be diverse, but related studies are limited. Here, based on time-course transcriptome analysis, a four-component system, SbrH1-R, consisting of the two-component system SbrKR (SBI_03479/3478) and two hypothetical proteins SbrH1 (SBI_03481) and SbrH2 (SBI_03480) potentially related with the biosynthesis of milbemycins was identified in Streptomyces bingchenggensis BC-101-4. Deletion of sbrH1-R resulted in weakened cell growth but a 110% increase of milbemycin production compared with that in BC-101-4. Comparative transcriptome analyses of the sbrH1-R mutant and BC-101-4 revealed that SbrH1-R not only indirectly represses milbemycin BGC expression, but also inhibits milbemycin production by modulating expression levels of genes related to precursor supply and antibiotic efflux. Further genetic experiments identified several new targets, including five precursor supply-associated reactions/pathways (e.g., the reaction from pyruvate to acetyl-CoA, the reaction from acetyl-CoA to citrate, the fatty acid β-oxidation process, and the branched chain amino acid and phenylalanine acid degradation pathways) and a milbemycin exporter system (MilEX2) that can be engineered for milbemycin overproduction. These results shed new light on the understanding of regulation of milbemycin biosynthesis and provide useful targets for future metabolic engineering of the native host to improve milbemycin production.

Streptomyces can produce a wealth of pharmaceutically valuable antibiotics and other bioactive compounds. Production of most antibiotics is generally low due to the rigorously controlled regulatory networks, in which global/pleiotropic and cluster-situated regulatory proteins coordinate with various intra- and extracellular signals. Thus, mining new antibiotic regulatory proteins, particularly the ones that are widespread, is essential for understanding the regulation of antibiotic biosynthesis. Here, in the biopesticide milbemycin producing strain Streptomyces bingchenggensis, a novel global/pleiotropic regulatory protein, SspH, a single domain protein containing only the HATPase domain, was identified as being involved in controlling antibiotic biosynthesis. The sspH overexpression inhibited milbemycin production by repressing the expression of milbemycin biosynthetic genes. The sspH overexpression also differentially influenced the expression of various antibiotic biosynthetic core genes. Site-directed mutagenesis revealed that the HATPase domain was essential for SspH’s function, and mutation of the conserved amino acid residues N54A and D84A led to the loss of SspH function. Moreover, cross-overexpression experiments showed that SspH and its orthologs, SCO1241 from Streptomyces coelicolor and SAVERM_07097 from Streptomyces avermitilis, shared identical functionality, and all exerted a positive effect on actinorhodin production but a negative effect on avermectin production, indicating that SspH-mediated differential control of antibiotic biosynthesis may be widespread in Streptomyces. This study extended our understanding of the regulatory network of antibiotic biosynthesis and provided effective targets for future antibiotic discovery and overproduction.

Guvermectin, a purine nucleoside natural product produced by the genus Streptomyces, has recently been registered as a new biopesticide to boost rice yield. Despite its economic and agricultural significance, the regulatory mechanisms of guvermectin biosynthesis remain essentially unknown, hindering industrial production and widespread agricultural application. Here, we examined the roles of two LacI family regulators, gvmR and gvmR2, located within and adjacent to the guvermectin biosynthesis cluster, respectively, in guvermectin production in Streptomyces caniferus NEAU6. GvmR activated the expression of the guvermectin cluster by binding to the promoters of gvmR, gvmA, and O1, while GvmR2 repressed the guvermectin cluster via competitive binding to promoters containing GvmR-binding sites, specifically, a 14-bp palindromic sequences: 5′-RTCATWCGYATGAY-3′ (R = G/A, W = A/T, Y = T/C). Moreover, GvmR indirectly activates the expression of gvmR2 while GvmR2 feedback inhibits gvmR transcription, suggesting a functional interaction between the two regulators for coordinating guvermectin production. Overexpression of gvmR via the T7 RNA polymerase-T7 promoter system in the gvmR2 mutant significantly elevated guvermectin production by 125 % (from 631 mg L⁻¹ to 1422 mg L⁻¹), compared to the parental strain NEAU6. This suggested that combinatorial manipulation of gvmR and gvmR2 is useful for improving guvermectin production. These findings enrich our knowledge of the regulatory network for guvermectin biosynthesis, and offer key targets and effective strategies for high-titer guvermectin production.