Data_Sheet_1_SbbR/SbbA, an Important ArpA/AfsA-Like System, Regulates Milbemycin Production in Streptomyces bingchenggensis.docx

<p>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 sig24_sbh) and four well-known global regulatory genes (e.g., glnR_sbh, wblA_sbh, atrA_sbh, and mtrA/B_sbh). 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.</p

Substitution of a Single Amino Acid Reverses the Regiospecificity of the Baeyer–Villiger Monooxygenase PntE in the Biosynthesis of the Antibiotic Pentalenolactone

In the biosynthesis of pentalenolactone (<b>1</b>), PenE and PntE, orthologous proteins from <i>Streptomyces exfoliatus</i> and <i>S. arenae</i>, respectively, catalyze the flavin-dependent Baeyer–Villiger oxidation of 1-deoxy-11-oxopentalenic acid (<b>4</b>) to the lactone pentalenolactone D (<b>5</b>), in which the less-substituted methylene carbon has migrated. By contrast, the paralogous PtlE enzyme from <i>S. avermitilis</i> catalyzes the oxidation of <b>4</b> to neopentalenolactone D (<b>6</b>), in which the more substituted methane substitution has undergone migration. We report the design and analysis of 13 single and multiple mutants of PntE mutants to identify the key amino acids that contribute to the regiospecificity of these two classes of Baeyer–Villiger monooxygenases. The L185S mutation in PntE reversed the observed regiospecificity of PntE such that all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of <b>4</b> to <b>6</b> as the major product. The recombinant PntE mutant harboring R484L exhibited reduced regiospecificity, generating a mixture of lactones containing more than 17% of <b>6</b>. These in vitro results were corroborated by analysis of the complementation of the <i>S. avermitilis ΔptlED</i> double deletion mutant with <i>pntE</i> mutants, such that <i>pntE</i> mutants harboring L185S produced <b>6</b> as the major product, whereas complemention of the <i>ΔptlED</i> deletion mutant with <i>pntE</i> mutants carrying the R484L mutation gave <b>6</b> as more than 33% of the total lactone product mixture

Novel Plant Growth Regulator Guvermectin from Plant Growth-Promoting Rhizobacteria Boosts Biomass and Grain Yield in Rice

Food is a fundamental human right, and global food security is threatened by crop production. Plant growth regulators (PGRs) play an essential role in improving crop yield and quality, and this study reports on a novel PGR, termed guvermectin (GV), isolated from plant growth-promoting rhizobacteria, which can promote root and coleoptile growth, tillering, and early maturing in rice. GV is a nucleoside analogue like cytokinin (CK), but it was found that GV significantly promoted root and hypocotyl growth, which is different from the function of CK in Arabidopsis. The Arabidopsis CK receptor triple mutant ahk2-2 ahk3-3 cre1-12 still showed a GV response. Moreover, GV led different growth-promoting traits from auxin, gibberellin (GA), and brassinosteroid (BR) in Arabidopsis and rice. The results from a four-year field trial involving 28 rice varieties showed that seed-soaking treatment with GV increased the yields by 6.2 to 19.6%, outperforming the 4.0 to 10.8% for CK, 1.6 to 16.9% for BR, and 2.2 to 7.1% for GA-auxin-BR mixture. Transcriptome analysis demonstrated that GV induced different transcriptome patterns from CK, auxin, BR, and GA, and SAUR genes may regulate GV-mediated plant growth and development. This study suggests that GV represents a novel PGR with a unique signal perception and transduction pathway in plants