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(-)-Geosmin has high demand in perfumes and cosmetic products for its earthy congenial aroma. The current production of (-)-geosmin is either by distillation of sun-baked soil or by inefficient chemical synthesis because of the presence of multiple chiral centers. Fermentation processes are not viable as the titers of the Streptomyces sp. based processes are low. This work presents an alternative route by the heterologous synthesis of (-)-geosmin in Saccharomyces cerevisiae. The enzyme involved is the bifunctional geosmin synthase that catalyzes the conversion of farnesyl diphosphate to germacradienol and germacradienol to geosmin. This study evaluated the activity of many orthologs of geosmin synthase when expressed heterologously in S. cerevisiae. When the well-characterized CAB41566 from Streptomyces coelicolor origin was tested, germacradienol and germacrene D were detected but no geosmin. Bioinformatic analysis based on high/low identities to N-terminal and C-terminal domains of CAB41566 was carried out to identify different orthologs of geosmin synthase proteins from different bacterial and fungal origins. ADO68918 of Stigmatella aurantiaca origin showed the best activity among the tested orthologs, not only in terms of geosmin production but also an order of magnitude higher total abundance of the products of geosmin synthase as compared to CAB41566. This study successfully demonstrated the production of (-)-geosmin in S. cerevisiae and offers an alternative, sustainable and environment-friendly approach to producing (-)-geosmin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Streptomyces are important soil bacteria used for bioremediation of metal-contaminated soils, however, it is still unknown how metal-selective Streptomyces are and which mechanisms are involved during their capture. In this work, we exposed S. coelicolor spores to environmentally relevant concentrations (0.1, 1, 10, 100 μM) of Ce, U and Cd in solid medium for one week to investigate the uptake behaviour of hyphae in the newly formed spores. Additionally, metal adsorption onto the spores was explored by incubating inactive, ungerminated spores for one day in aqueous metal solution. The spore-washing treatment was key to distinguishing between strongly spore-associated (e.g. incorporation; Tris-EDTA buffer) and weakly spore-associated metals (Tris buffer alone minus Tris-EDTA). Single cell (sc) ICP-MS was used to quantify metal-associated content in individual spores. Our results revealed element-specific adsorption onto inactive spores showing that out of the total metal exposure, both strongly (Ce: 58%; U: 54%; Cd: 28%) and weakly (Ce: 12%; U: 1%; Cd: 18%) adsorbed metals occur. However, scICP-MS showed that from metal-amended solid medium, only Ce and U were strongly spore-associated (averages 0.040 and 0.062 fg spore -1 for 10 μM exposures, respectively) while Cd was below the limit of detection (< 0.006 fg spore -1 ). We propose that hyphae only metabolically interact with Ce in a controlled manner but uncontrolled with U, as 66-73% Ce and only 2-4% U were inherited from adsorbed content. We conclude that Streptomyces spore-metal interaction starts with a relevant adsorption step of Ce, U and Cd as presented for aqueous conditions. If spores start to germinate, hyphae are capable of effectively encapsulating Ce and U, but not Cd. This study brings light into the still unknown field of metal interactions with Streptomyces and applied understanding for more efficient and metal-specific use of Streptomyces in bioremediation of metal-polluted soils., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Steffen Hellmann reports financial support was provided by German Research Foundation (DFG SPP, 2005), Opus Fluidum Futurum (project number 313773090, grant no. SCHA, 18547-1). Paula Garcia-Cancela reports financial support was provided by Government of Principado de Asturias (Severo Ochoa scholarship for predoctoral studies). Sergio Alonso-Fernandez reports financial support was provided by Consejeria de Ciencia, Innovacion y Universidad del Principado de Asturias (Severo Ochoa predoctoral grant (grant no. PA-20-PFBP19-006)). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

Combined-cultures involving mycolic acid-containing bacteria (MACB) can stimulate secondary metabolite (SM) production in actinomycetes. In a prior investigation, we screened Streptomyces coelicolor JCM4020 mutants with diminished production of SMs, specifically undecylprodigiosin (RED), which was enhanced by introducing the MACB Tsukamurella pulmonis TP-B0596. In this study, we conducted mutational analysis that pinpointed the sco1842 gene, which we assigned the gene name ccr1 (combined-culture related regulatory protein no. 1), as a crucial factor in the deficient phenotype observed in the production of various major SMs in S. coelicolor A3(2). Notably, the Ccr1 (SCO1842) homolog was found to be highly conserved throughout the Streptomyces genome. Although Ccr1 lacked conserved motifs, in-depth examination revealed the presence of a helix-turn-helix (HTH) motif in the N-terminal region and a helicase C-terminal domain (HCTD) motif in the C- terminal region in some of its homologs. Ccr1 was predicted to be a nucleoid- associated protein (NAP), and its impact on gene transcription was validated by RNA-seq analysis that revealed genome-wide variations. Furthermore, RT- qPCR demonstrated that ccr1 was transcriptionally activated in combined- culture with T. pulmonis, which indicated that Ccr1 is involved in the response to bacterial interaction. We then investigated Streptomyces nigrescens HEK616 in combined-culture, and the knockout mutant of the ccr1 homolog displayed reduced production of streptoaminals and 5aTHQs. This finding reveals that the Ccr1 homolog in Streptomyces species is associated with SM production. Our study elucidates the existence of a new family of NAP-like proteins that evolved in Streptomyces species and play a pivotal role in SM production. [ABSTRACT FROM AUTHOR]

Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. Actinoplanes sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50_0507 is a homolog of the developmental and osmotic-stress-regulating Streptomyces coelicolor σHSc. Therefore, the protein encoded by ACSP50_0507 was named σHAs. Here, an Actinoplanes sp. SE50/110 expression strain for the alternative sigma factor gene ACSP50_0507 (sigHAs) achieved a two-fold increased acarbose yield with acarbose production extending into the stationary growth phase. Transcriptome sequencing revealed upregulation of acarbose biosynthesis genes during growth and at the late stationary growth phase. Genes that are transcriptionally activated by σHAs frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its S. coelicolor homolog. The DNA-binding motif of σHAs was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σHAs to the upstream sequence of a strongly upregulated gene. Autoregulation of σHAs was observed, and binding to its own gene promoter region was also confirmed. [ABSTRACT FROM AUTHOR]

Background: Streptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential. Results: We engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host. Conclusion: Our engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces. [ABSTRACT FROM AUTHOR]

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the −35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β′ zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription. Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. This study combines cryo-EM structures with biochemical assays to explain how the Streptomyces coelicolor SARP AfsR activates transcription. [ABSTRACT FROM AUTHOR]