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Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.

Additional file 1: Figure S1. Degradation of leucine and isovalerate via the Liu-pathway as described for P. aeruginosa PAO1 [29]. Figure S2. Genetic organization of the caprazamycin biosynthetic gene cluster. Genes putatively involved in colour coding according to McErlean et al. [17]. Figure S3. Extracted ion chromatograms of S. coelicolor M1154 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S4. Extracted ion chromatograms of S. coelicolor M1154/cpzLK09 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S5. Extracted ion chromatograms of S. coelicolor M1154/cpzDB04 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S6. Genetic organization of clusters encoding for Liu-pathway from P. aeruginosa PAO1, caprazamycin wildtype producer S. sp. MK730-62F2 and the heterologous caprazamycin producer S. coelicolor M1154. Additional genes in Streptomyces strains are shown transparent. Table shows genes from P. aeruginosa PAO1 and their proposed function along with homologue genes found in S. sp. MK730-62F2 and S. coelicolor M1154. Values in brackets indicate % identities/similarities (n.s. no significant similarities). Figure S7. Distribution of liu clusters in Actinobacteria and Streptomyces. A: Cblaster detected 4837 similar clusters containing at least three genes homolog to the query sequence sco2775-sco2779 from S. coelicolor M1154. We discovered that 1.431 out of 31.598 (4.5%) Actinobacteria assemblies and 1.344 out of 2.574 (52.2%) Streptomyces assemblies and listed in the GTDB contain a liu cluster with homologues of all five query genes. B: Cblaster detected 5009 similar clusters containing at least three genes homolog to the query sequence sco2774-sco2779 from S. coelicolor M1154. We discovered that 1.246 out of 31.598 (3.9%) Actinobacteria assemblies and 1.241 out of 2.574 (48.2%) Streptomyces assemblies and listed in the GTDB contain a liu cluster with homologues of all six query genes. A homologue of sco2774 could only be found in 1.376 out of 5009 (27,5%) of the Actinobacteria clusters detected by cblaster, whereas the majority of detected Streptomyces cluster contain this gene with 1.343 out of 1.652 sequences (81,3%). Figure S8. Extracted ion chromatograms of S. coelicolor M1154Δsco2776-sco2779/cpzLK09 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S9. Extracted ion chromatograms of S. coelicolor M1154Δsco2776-sco2779/cpzDB04 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S10. Total ion chromatograms of S. coelicolor M1154Δsco2776-sco2779/cpzDB04 and S. coelicolor M1154Δsco2776-sco2779/cpzLK09. MS2-fragmentation patterns of peak 1 (hydoxyacylcaprazol E, Rt 12.9 min) and peak 2 (caprazamycin aglycon E, Rt 13.8 min) are shown together with corresponding fragmentation schemes. Figure S11. Extracted ion chromatograms of S. coelicolor M1154Δsco2779/cpzLK09 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S12. Extracted ion chromatograms of S. coelicolor M1154Δsco2779/cpzDB04 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S13. Extracted ion chromatograms of S. coelicolor M1154Δsco2776/cpzLK09 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z of 830.5. Figure S14. Extracted ion chromatograms of S. coelicolor M1154Δsco2776/cpzDB04 (three individual mutants). Masses are shown for caprazamycin aglycons E/F with m/z of 930.5, caprazamycin aglycons C/D/G with m/z of 944.5, caprazamycin aglycons A/B with m/z of 958.5 and the respective hydroxyacylcaprazols E/F with m/z of 802.5, hydroxyacylcaprazols C/D/G with m/z of 816.5 and hydroxyacylcaprazols A/B with m/z

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound’s purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Streptomyces coelicolor M145 was shown to be able to grow in the presence of high concentrations of polyamines, such as putrescine, cadaverine, spermidine, or spermine, as a sole nitrogen source. However, hardly anything is known about polyamine utilization and its regulation in streptomycetes. In this study, we demonstrated that only one of the three proteins annotated as glutamine synthetase-like protein, GlnA3 (SCO6962), was involved in the catabolism of polyamines. Transcriptional analysis revealed that the expression of glnA3 was strongly induced by exogenous polyamines and repressed in the presence of ammonium. The ΔglnA3 mutant was shown to be unable to grow on defined Evans agar supplemented with putrescine, cadaverine, spermidine, and spermine as sole nitrogen source. HPLC analysis demonstrated that the ΔglnA3 mutant accumulated polyamines intracellularly, but was unable to degrade them. In a rich complex medium supplemented with a mixture of the four different polyamines, the ΔglnA3 mutant grew poorly showing abnormal mycelium morphology and decreased life span in comparison to the parental strain. These observations indicated that the accumulation of polyamines was toxic for the cell. An in silico analysis of the GlnA3 protein model suggested that it might act as a gamma-glutamylpolyamine synthetase catalyzing the first step of polyamine degradation. GlnA3-catalyzed glutamylation of putrescine was confirmed in an enzymatic in vitro assay and the GlnA3 reaction product, gamma-glutamylputrescine, was detected by HPLC/ESI-MS. In this work, the first step of polyamine utilization in S. coelicolor has been elucidated and the putative polyamine utilization pathway has been deduced based on the sequence similarity and transcriptional analysis of homologous genes expressed in the presence of polyamines.