Ψ-Footprinting approach for the identification of protein synthesis inhibitor producers.

Today, one of the biggest challenges in antibiotic research is a targeted prioritization of natural compound producer strains and an efficient dereplication process to avoid undesired rediscovery of already known substances. Thereby, genome sequence-driven mining strategies are often superior to wet-lab experiments because they are generally faster and less resource-intensive. In the current study, we report on the development of a novel in silico screening approach to evaluate the genetic potential of bacterial strains to produce protein synthesis inhibitors (PSI), which was termed the protein synthesis inhibitor ('psi') target gene footprinting approach = Ψ-footprinting. The strategy is based on the occurrence of protein synthesis associated self-resistance genes in genome sequences of natural compound producers. The screening approach was applied to 406 genome sequences of actinomycetes strains from the DSMZ strain collection, resulting in the prioritization of 15 potential PSI producer strains. For twelve of them, extract samples showed protein synthesis inhibitory properties in in vitro transcription/translation assays. For four strains, namely Saccharopolyspora flava DSM 44771, Micromonospora aurantiaca DSM 43813, Nocardioides albertanoniae DSM 25218, and Geodermatophilus nigrescens DSM 45408, the protein synthesis inhibitory substance amicoumacin was identified by HPLC-MS analysis, which proved the functionality of the in silico screening approach.

Bioreporters for direct mode of action-informed screening of antibiotic producer strains.

A big challenge in natural product research of today is rapid dereplication of already known substances, to free capacities for the exploration of new agents. Prompt information on bioactivities and mode of action (MOA) speeds up the lead discovery process and is required for rational compound optimization. Here, we present a bioreporter approach as a versatile strategy for combined bioactivity- and MOA-informed primary screening for antimicrobials. The approach is suitable for directly probing producer strains grown on agar, without need for initial compound enrichment or purification, and works along the entire purification pipeline with culture supernatants, extracts, fractions, and pure substances. The technology allows for MOA-informed purification to selectively prioritize activities of interest. In combination with high-resolution mass spectrometry, the biosensor panel is an efficient and sensitive tool for compound deconvolution. Concomitant information on the affected metabolic pathway enables the selection of appropriate follow-up assays to elucidate the molecular target.

Investigation of the Autoregulator-Receptor System in the Pristinamycin Producer Streptomyces pristinaespiralis.

Pristinamycin biosynthesis in Streptomyces pristinaespiralis is governed by a complex hierarchical signaling cascade involving seven different transcriptional regulators (SpbR, PapR1, PapR2, PapR3, PapR4, PapR5, and PapR6). The signaling cascade is triggered by γ-butyrolactone (GBL)-like effector molecules, whereby the chemical structure of the effector, as well as its biosynthetic origin is unknown so far. Three of the pristinamycin transcriptional regulators (SpbR, PapR3, and PapR5) belong to the type of γ-butyrolactone receptor (GBLR). GBLRs are known to either act as "real" GBLRs, which bind GBLs as ligands or as "pseudo" GBLRs binding antibiotics or intermediates thereof as effector molecules. In this study, we performed electromobility shift assays (EMSAs) with SpbR, PapR3, and PapR5, respectively, in the presence of potential ligand samples. Thereby we could show that all three GBLRs bind synthetic 1,4-butyrolactone but not pristinamycin as ligand, suggesting that SpbR, PapR3, and PapR5 act as "real" GBLRs in S. pristinaespiralis. Furthermore, we identified a cytochrome P450 monooxygenase encoding gene snbU as potential biosynthesis gene for the GBLR-interacting ligand. Inactivation of snbU resulted in an increased pristinamycin production, which indicated that SnbU has a regulatory influence on pristinamycin production. EMSAs with culture extract samples from the snbU mutant did not influence the target binding ability of SpbR, PapR3, and PapR5 anymore, in contrast to culture supernatant samples from the S. pristinaespiralis wild-type or the pristinamycin deficient mutant papR2::apra, which demonstrates that SnbU is involved in the synthesis of the GBLR-interacting ligand.

Genome Sequences of Two Putative Streptogramin Producers, Streptomyces sp. Strains TÜ 2975 and TÜ 3180, from the Tübingen Strain Collection.

Streptomyces sp. TÜ 2975 and TÜ 3180 are two strains from the Tübingen Actinomycetes strain collection. Here, we present the draft genome sequences of TÜ 2975 and TÜ 3180, with sizes of 7.62 Mb and 8.63 Mb, respectively.

Genetic engineering approaches for the fermentative production of phenylglycines.

L-phenylglycine (L-Phg) is a rare non-proteinogenic amino acid, which only occurs in some natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Industrially, more interesting than L-Phg is the enantiomeric D-Phg as it plays an important role in the fine chemical industry, where it is used as a precursor for the production of semisynthetic ß-lactam antibiotics. Based on the natural L-Phg operon from Streptomyces pristinaespiralis and the stereo-inverting aminotransferase gene hpgAT from Pseudomonas putida, an artificial D-Phg operon was constructed. The natural L-Phg operon, as well as the artificial D-Phg operon, was heterologously expressed in different actinomycetal host strains, which led to the successful production of Phg. By rational genetic engineering of the optimal producer strains S. pristinaespiralis and Streptomyces lividans, Phg production could be improved significantly. Here, we report on the development of a synthetic biology-derived D-Phg pathway and the optimization of fermentative Phg production in actinomycetes by genetic engineering approaches. Our data illustrate a promising alternative for the production of Phgs.

A Complex Signaling Cascade Governs Pristinamycin Biosynthesis in Streptomyces pristinaespiralis.

Pristinamycin production in Streptomyces pristinaespiralis Pr11 is tightly regulated by an interplay between different repressors and activators. A γ-butyrolactone receptor gene (spbR), two TetR repressor genes (papR3 and papR5), three SARP (Streptomyces antibiotic regulatory protein) genes (papR1, papR2, and papR4), and a response regulator gene (papR6) are carried on the large 210-kb pristinamycin biosynthetic gene region of Streptomyces pristinaespiralis Pr11. A detailed investigation of all pristinamycin regulators revealed insight into a complex signaling cascade, which is responsible for the fine-tuned regulation of pristinamycin production in S. pristinaespiralis.