Discovery of a Bacterial Hydrazine Transferase That Constructs the N-Aminolactam Pharmacophore in Albofungin Biosynthesis.

Structural motifs containing nitrogen-nitrogen (N-N) bonds are prevalent in a large number of clinical drugs and bioactive natural products. Hydrazine (N2H4) serves as a widely utilized building block for the preparation of these N-N-containing molecules in organic synthesis. Despite its common use in chemical processes, no enzyme has been identified to catalyze the incorporation of free hydrazine in natural product biosynthesis. Here, we report that a hydrazine transferase catalyzes the condensation of N2H4 and an aromatic polyketide pathway intermediate, leading to the formation of a rare N-aminolactam pharmacophore in the biosynthesis of broad-spectrum antibiotic albofungin. These results expand the current knowledge on the biosynthetic mechanism for natural products with N-N units and should facilitate future development of biocatalysts for the production of N-N-containing chemicals.

SVEN_5003 is a Major Developmental Regulator in Streptomyces venezuelae.

Many regulatory genes that affect cellular development in Streptomyces, such as the canonical bld genes, have already been identified. However, in this study, we identified sven_5003 in Streptomyces venezuelae as a major new developmental regulatory gene, the deletion of which leads to a bald phenotype, typical of bld mutants, under multiple growth conditions. Our data indicated that disruption of sven_5003 also has a differential impact on the production of the two antibiotics jadomycin and chloramphenicol. Enhanced production of jadomycin but reduced production of chloramphenicol were detected in our sven_5003 mutant strain (S. venezuelae D5003). RNA-Seq analysis indicated that SVEN_5003 impacts expression of hundreds of genes, including genes involved in development, primary and secondary metabolism, and genes of unknown function, a finding confirmed by real-time PCR analysis. Transcriptional analysis indicated that sven_5003 is an auto-regulatory gene, repressing its own expression. Despite the evidence indicating that SVEN_5003 is a regulatory factor, a putative DNA-binding domain was not predicted from its primary amino acid sequence, implying an unknown regulatory mechanism by SVEN_5003. Our findings revealed that SVEN_5003 is a pleiotropic regulator with a critical role in morphological development in S. venezuelae.

Exploring the specialized metabolome of the plant pathogen Streptomyces sp. 11-1-2.

Streptomyces bacteria are notable for producing chemically diverse specialized metabolites that exhibit various bioactivities and mediate interactions with different organisms. Streptomyces sp. 11-1-2 is a plant pathogen that produces nigericin and geldanamycin, both of which display toxic effects against various plants. Here, the 'One Strain Many Compounds' approach was used to characterize the metabolic potential of Streptomyces sp. 11-1-2. Organic extracts were prepared from 11-1-2 cultures grown on six different agar media, and the extracts were tested in antimicrobial and plant bioassays and were subjected to untargeted metabolomics and molecular networking. Most extracts displayed strong bioactivity against Gram-positive bacteria and yeast, and they exhibited phytotoxic activity against potato tuber tissue and radish seedlings. Several known specialized metabolites, including musacin D, galbonolide B, guanidylfungin A, meridamycins and elaiophylin, were predicted to be present in the extracts along with closely related compounds with unknown structure and bioactivity. Targeted detection confirmed the presence of elaiophylin in the extracts, and bioassays using pure elaiophylin revealed that it enhances the phytotoxic effects of geldanamycin and nigericin on potato tuber tissue. Overall, this study reveals novel insights into the specialized metabolites that may mediate interactions between Streptomyces sp. 11-1-2 and other bacteria and eukaryotic organisms.

Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins.

BACKGROUND: Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines. RESULTS: In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10. CONCLUSIONS: Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Chalkophomycin Biosynthesis Revealing Unique Enzyme Architecture for a Hybrid Nonribosomal Peptide Synthetase and Polyketide Synthase.

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.

Integrative Genomics and Bioactivity-Guided Isolation of Novel Antimicrobial Compounds from Streptomyces sp. KN37 in Agricultural Applications.

Actinomycetes have long been recognized as an important source of antibacterial natural products. In recent years, actinomycetes in extreme environments have become one of the main research directions. Streptomyces sp. KN37 was isolated from the cold region of Kanas in Xinjiang. It demonstrated potent antimicrobial activity, but the primary active compounds remained unclear. Therefore, we aimed to combine genomics with traditional isolation methods to obtain bioactive compounds from the strain KN37. Whole-genome sequencing and KEGG enrichment analysis indicated that KN37 possesses the potential for synthesizing secondary metabolites, and 41 biosynthetic gene clusters were predicted, some of which showed high similarity to known gene clusters responsible for the biosynthesis of antimicrobial antibiotics. The traditional isolation methods and activity-guided fractionation were employed to isolate and purify seven compounds with strong bioactivity from the fermentation broth of the strain KN37. These compounds were identified as 4-(Diethylamino)salicylaldehyde (1), 4-Nitrosodiphenylamine (2), N-(2,4-Dimethylphenyl)formamide (3), 4-Nitrocatechol (4), Methylsuccinic acid (5), Phenyllactic acid (6) and 5,6-Dimethylbenzimidazole (7). Moreover, 4-(Diethylamino)salicylaldehyde exhibited the most potent inhibitory effect against Rhizoctonia solani, with an EC50 value of 14.487 mg/L, while 4-Nitrosodiphenylamine showed great antibacterial activity against Erwinia amylovora, with an EC50 value of 5.715 mg/L. This study successfully isolated several highly active antimicrobial compounds from the metabolites of the strain KN37, which could contribute as scaffolds for subsequent chemical synthesis. On the other hand, the newly predicted antibiotic-like substances have not yet been isolated, but they still hold significant research value. They are instructive in the study of active natural product biosynthetic pathways, activation of silent gene clusters, and engineering bacteria construction.

Biodegradation of atrazine and nicosulfuron by Streptomyces nigra LM01: Performance, degradative pathway, and possible genes involved.

Microbial herbicide degradation is an efficient bioremediation method. In this study, a strain of Streptomyces nigra, LM01, which efficiently degrades atrazine and nicosulfuron, was isolated from a corn field using a direct isolation method. The degradation effects of the identified strain on two herbicides were investigated and optimized using an artificial neural network. The maximum degradation rates of S. nigra LM01 were 58.09 % and 42.97 % for atrazine and nicosulfuron, respectively. The degradation rate of atrazine in the soil reached 67.94 % when the concentration was 108 CFU/g after 5 d and was less effective than that of nicosulfuron. Whole genome sequencing of strain LM01 helped elucidate the possible degradation pathways of atrazine and nicosulfuron. The protein sequences of strain LM01 were aligned with the sequences of the degraded proteins of the two herbicides by using the National Center for Biotechnology Information platform. The sequence (GE005358, GE001556, GE004212, GE005218, GE004846, GE002487) with the highest query cover was retained and docked with the small-molecule ligands of the herbicides. The results revealed a binding energy of - 6.23 kcal/mol between GE005358 and the atrazine ligand and - 6.66 kcal/mol between GE002487 and the nicosulfuron ligand.

Antioxidant and Antibacterial Properties of 1,3-Dihydroxy-,2', 2'-Dimethylpyrano-(5,6)-Xanthone from Streptomyces sp. SU84.

<b>Background and Objective:</b> The SU84 was isolated from the rhizosphere of <i>Curcuma longa</i> and identified to be <i>Streptomyces</i> sp. via analysis of its 16S rDNA sequence, chemotaxonomy and morphology. This study aimed to isolate major compounds from the extract culture of strain SU84 and evaluate their antibacterial activity. <b>Materials and Methods:</b> The TLC and silica gel column chromatography were used to purify major compounds, elucidate 1,3-dihydroxy-,2',2'-dimethylpyrano-(5,6)-xanthone (compound <b>1</b>) and lupeol (compound <b>2</b>) using mass spectrometry and nuclear magnetic resonance. One new chemical, compound <b>1</b>, was first isolated from microbial sources. Antibacterial, antioxidant and cytotoxic properties of these compounds were carried out. <b>Results:</b> Various bioassays showed that compound <b>1</b> displayed antibacterial property against Gram-positive bacteria, with a minimum inhibitory concentration of 8-32 µg/mL and minimum bactericidal concentration of 32-128 µg/mL. In addition, the purified compounds were tested against normal cell lines using tetrazolium assay. The results did not show cytotoxic property against L929 and Vero cells, with IC₅₀ values of >512.00 µg/mL. Compounds <b>1</b> and <b>2</b> have also antioxidant properties, with IC₅₀ values of 16.67±7.48 and 38.86±8.45 µg/mL, respectively. <b>Conclusion:</b> The findings suggested that compounds of <i>Streptomyces</i> sp. SU84 displayed antibacterial and antioxidant properties without cytotoxic activity. Extensive studies of compound <b>1</b> may be useful for the advancement of improved methods for avoidance, control and management of bacterial infections and metabolic-related free radical contribution.

Genome-guided discovery of two undescribed 6,6-spiroketal polyketides and stereochemical correction of bafilomycins P and Q from the marine-derived Streptomyces sp. SCSIO 66814.

Bafilomycins are macrocyclic polyketides with intriguing structures and therapeutic value. Genomic analysis of Streptomyces sp. SCSIO 66814 revealed a type I polyketide synthase biosynthetic gene cluster (BGC), namely blm, which encoded bafilomycins and featured rich post-modification genes. The One strain many compounds (OSMAC) strategy led to the discovery of six compounds related to the blm BGC from the strain, including two previously undescribed 6,6-spiroketal polyketides, streptospirodienoic acids D (1) and E (2), and four known bafilomycins, bafilomycins P (3), Q (4), D (5), and G (6). The structures of 1 and 2 were determined by extensive spectroscopic analysis, quantum calculation, and biosynthetic analysis. Additionally, the absolute configurations of the 6/5/5 tricyclic ring moiety containing six consecutive chiral carbons in the putative structures of 3 and 4 were corrected through NOE analysis, DP4+ calculation, and single-crystal X-ray diffraction data. Bioinformatic analysis uncovered a plausible biosynthetic pathway for compounds 1-6, indicating that both streptospirodienoic acids and bafilomycins were derived from the same blm BGC. Additionally, sequence analysis revealed that the KR domains of module 2 from blm BGC was B1-type, further supporting the configurations of 1-4. Notably, compounds 3 and 4 displayed significant cytotoxic activities against A-549 human non-small cell lung cancer cells and HCT-116 human colon cancer cells.

Discovery of a Novel Chromone Enantiomer and the Precursors of Nonactic Acid from the Coral-Reef-Derived Streptomyces sp. SCSIO 66814.

Three pairs of enantiomers (1-3)-the new 12R-aloesol (1a) and two new fatty acids (2 and 3)-and one new natural product (4) together three known compounds (5-7) were isolated from a coral-reef-derived Streptomyces sp. SCSIO 66814. Their structures were determined through extensive spectroscopic analysis, chiral analysis, and single-crystal X-ray diffraction data. Compounds 2 and 3 were presumed to be intermediates for further generating homononactic acid (5) and nonactic acid, and the latter two molecules were able to act as precursors to form macrotetrolides with remarkable biological activity. The isolation of related precursors, compounds 2-5, provided more evidence to support the proposal of a plausible biosynthetic pathway for nonactic acid and its homologs. Additionally, (+)-1 exhibited a weak activity against DPPH radicals.

The Discovery of Weddellamycin, a Tricyclic Polyene Macrolactam Antibiotic from an Antarctic Deep-Sea-Derived Streptomyces sp. DSS69, by Heterologous Expression.

Polyene macrolactams are a special group of natural products with great diversity, unique structural features, and a wide range of biological activities. Herein, a cryptic gene cluster for the biosynthesis of putative macrolactams was disclosed from a sponge-associated bacterium, Streptomyces sp. DSS69, by genome mining. Cloning and heterologous expression of the whole biosynthetic gene cluster led to the discovery of weddellamycin, a polyene macrolactam bearing a 23/5/6 ring skeleton. A negative regulator, WdlO, and two positive regulators, WdlA and WdlB, involved in the regulation of weddellamycin production were unraveled. The fermentation titer of weddellamycin was significantly improved by overexpression of wdlA and wdlB and deletion of wdlO. Notably, weddellamycin showed remarkable antibacterial activity against various Gram-positive bacteria including MRSA, with MIC values of 0.10-0.83 µg/mL, and antifungal activity against Candida albicans, with an MIC value of 3.33 µg/mL. Weddellamycin also displayed cytotoxicity against several cancer cell lines, with IC50 values ranging from 2.07 to 11.50 µM.

Engineering Streptomyces sp. CPCC 204095 for the targeted high-level production of isatropolone A by elucidating its pathway-specific regulatory mechanism.

BACKGROUND: Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. RESULTS: Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield-980.8 mg/L of isatropolone A-achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. CONCLUSION: In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.

Isolation of Streptomyces inhibiting multiple-phytopathogenic fungi and characterization of lucensomycin biosynthetic gene cluster.

Soil microorganisms with diverse bioactive compounds such as Streptomyces are appreciated as valuable resources for the discovery of eco-friendly fungicides. This study isolated a novel Streptomyces from soil samples collected in the organic green tea fields in South Korea. The isolation process involved antifungal activity screening around 2400 culture extracts, revealing a strain designated as S. collinus Inha504 with remarkable antifungal activity against diverse phytopathogenic fungi. S. collinus Inha504 not only inhibited seven phytopathogenic fungi including Fusarium oxysporum and Aspergillus niger in bioassays and but also showed a control effect against F. oxysporum infected red pepper, strawberry, and tomato in the in vivo pot test. Genome mining of S. collinus Inha504 revealed the presence of the biosynthetic gene cluster (BGC) in the chromosome encoding a polyene macrolide which is highly homologous to the lucensomycin (LCM), a compound known for effective in crop disease control. Through genetic confirmation and bioassays, the antifungal activity of S. collinus Inha504 was attributed to the presence of LCM BGC in the chromosome. These results could serve as an effective strategy to select novel Streptomyces strains with valuable biological activity through bioassay-based screening and identify biosynthetic gene clusters responsible for the metabolites using genome mining approach.

Metabolic Comparison and Molecular Networking of Antimicrobials in Streptomyces Species.

Streptomyces are well-known for producing bioactive secondary metabolites, with numerous antimicrobials essential to fight against infectious diseases. Globally, multidrug-resistant (MDR) microorganisms significantly challenge human and veterinary diseases. To tackle this issue, there is an urgent need for alternative antimicrobials. In the search for potent agents, we have isolated four Streptomyces species PC1, BT1, BT2, and BT3 from soils collected from various geographical regions of the Himalayan country Nepal, which were then identified based on morphology and 16S rRNA gene sequencing. The relationship of soil microbes with different Streptomyces species has been shown in phylogenetic trees. Antimicrobial potency of isolates was carried out against Staphylococcus aureus American Type Culture Collection (ATCC) 43300, Shigella sonnei ATCC 25931, Salmonella typhi ATCC 14028, Klebsiella pneumoniae ATCC 700603, and Escherichia coli ATCC 25922. Among them, Streptomyces species PC1 showed the highest zone of inhibition against tested pathogens. Furthermore, ethyl acetate extracts of shake flask fermentation of these Streptomyces strains were subjected to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis for their metabolic comparison and Global Natural Products Social Molecular Networking (GNPS) web-based molecular networking. We found very similar metabolite composition in four strains, despite their geographical variation. In addition, we have identified thirty-seven metabolites using LC-MS/MS analysis, with the majority belonging to the diketopiperazine class. Among these, to the best of our knowledge, four metabolites, namely cyclo-(Ile-Ser), 2-n-hexyl-5-n-propylresorcinol, 3-[(6-methylpyrazin-2-yl) methyl]-1H-indole, and cyclo-(d-Leu-l-Trp), were detected for the first time in Streptomyces species. Besides these, other 23 metabolites including surfactin B, surfactin C, surfactin D, and valinomycin were identified with the help of GNPS-based molecular networking.

Enhancement of acyl-CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes.

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 µg/mL - 4.36-fold higher than WT value (48.9 µg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 µg/mL. Overexpression of fatty acid ß-oxidation pathway genes in the strain further increased B1a titer to 452.8 µg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 µg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.

Development of a group II intron-based genetic manipulation tool for Streptomyces.

The availability of an alternative and efficient genetic editing technology is critical for fundamental research and strain improvement engineering of Streptomyces species, which are prolific producers of complex secondary metabolites with significant pharmaceutical activities. The mobile group II introns are retrotransposons that employ activities of catalytic intron RNAs and intron-encoded reverse transcriptase to precisely insert into DNA target sites through a mechanism known as retrohoming. We here developed a group II intron-based gene editing tool to achieve precise chromosomal gene insertion in Streptomyces. Moreover, by repressing the potential competition of RecA-dependent homologous recombination, we enhanced site-specific insertion efficiency of this tool to 2.38%. Subsequently, we demonstrated the application of this tool by screening and characterizing the secondary metabolite biosynthetic gene cluster (BGC) responsible for synthesizing the red pigment in Streptomyces roseosporus. Accompanied with identifying and inactivating this BGC, we observed that the impair of this cluster promoted cell growth and daptomycin production. Additionally, we applied this tool to activate silent jadomycin BGC in Streptomyces venezuelae. Overall, this work demonstrates the potential of this method as an alternative tool for genetic engineering and cryptic natural product mining in Streptomyces species.

Isolation, characterisation and description of the roseoflavin producer Streptomyces berlinensis sp. nov.

The Gram-positive bacteria Streptomyces davaonensis and Streptomyces cinnabarinus have been the only organisms known to produce roseoflavin, a riboflavin (vitamin B2) derived red antibiotic. Using a selective growth medium and a phenotypic screening, we were able to isolate a novel roseoflavin producer from a German soil sample. The isolation procedure was repeated twice, that is, the same strain could be isolated from the same location in Berlin 6 months and 12 months after its first isolation. Whole genome sequencing of the novel roseoflavin producer revealed an unusual chromosomal arrangement and the deposited genome sequence of the new isolate (G + C content of 71.47%) contains 897 genes per inverted terminal repeat, 6190 genes in the core and 107 genes located on an illegitimate terminal end. We identified the roseoflavin biosynthetic genes rosA, rosB and rosC and an unusually high number of riboflavin biosynthetic genes. Overexpression of rosA, rosB and rosC in Escherichia coli and enzyme assays confirmed their predicted functions in roseoflavin biosynthesis. A full taxonomic analysis revealed that the isolate represents a previously unknown Streptomyces species and we propose the name Streptomyces berlinensis sp. nov. for this roseoflavin producer.

Exogenous Streptomyces spp. enhance the drought resistance of naked oat (Avena nuda) seedlings by augmenting both the osmoregulation mechanisms and antioxidant capacities.

Drought is a major obstacle to the development of naked oat industry. This work investigated mechanisms by which exogenous Streptomyces albidoflavus T4 and Streptomyces rochei D74 improved drought tolerance in naked oat (Avena nuda ) seedlings. Results showed that in the seed germination experiment, germination rate, radicle and hypocotyl length of naked oat seeds treated with the fermentation filtrate of T4 or D74 under PEG induced drought stress increased significantly. In the hydroponic experiment, the shoot and root dry weights of oat seedlings increased significantly when treated with the T4 or D74 fermentation filtrate under the 15% PEG induced drought stress (S15). Simultaneously, the T4 treatment also significantly increased the surface area, volume, the number of tips and the root activity of oat seedlings. Both T4 and D74 treatments elicited significant increases in proline and soluble sugar contents, as well as the catalase and peroxidase activities in oat seedlings. The results of comprehensive drought resistance capacity (CDRC) calculation of oat plants showed that the drought resistance of oat seedlings under the T4 treatment was better than that under the D74 treatment, and the effect was better under higher drought stress (S15). Findings of this study may provide a novel and effective approach for enhancing plant defenses against drought stress.

Characterization of a pleiotropic regulator MtrA in Streptomyces avermitilis controlling avermectin production and morphological differentiation.

BACKGROUND: The macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav_5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated. RESULTS: In this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis. CONCLUSIONS: In this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators.

Application of Cas12j for Streptomyces Editing.

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.