Rational Design of Polyketide Natural Products

Compounds of polyketide origin possess a wealth of pharmacological effects, including antibacterial, antifungal, antiparasitic, anticancer and immunosuppressive activities. Many of these compounds and their semi-synthetic derivatives are used today in the clinic. The first complete gene cluster encoding the polyketide antibiotic actinorhodin was cloned twenty years ago. The erythromycin gene cluster followed in 1990, and since then most of the gene clusters encoding commercially important drugs have also been cloned, sequenced and their biosynthetic mechanisms studied in great detail. Recent advances in the area of biosynthetic engineering of the enzymes involved in polyketide biosynthesis are presented in this review. The biosynthesis of a typical polyketide can be divided into three separate steps, including (i) choice of starter unit, (ii) the choice of extender units and the degree of beta-keto group reduction, and (iii) post-PKS tailoring of the basic polyketide backbone. Each of these steps represents a potential opportunity for the introduction of structural modification. The technologies to achieve this have now been highly developed and transferred into the industrial arena. The power of biosynthetic engineering will be exemplified by manipulations carried out on the erythromycin and the rapamycin biosynthetic gene clusters

Aplicación de la herramienta de edición genética CRISPR-Cas9 para la generación de grandes deleciones en el genoma Streptomyces rimosus

Sección: Siguiendo la pista[ES] Mediante la aplicación de la técnica de CRISPR-Cas9 se ha conseguido la deleción de fragmentos de ADN de 245 kpb a 450 kpb del cromosoma de la cepa productora de oxitetraciclina, Streptomyces rimosus ATCC 10970. Estas deleciones afectaron a la misma región de un extremo del cromosoma que, según el análisis bioinformático, contiene varios agrupamientos genéticos que codifican para enzimas implicadas en la biosíntesis de metabolitos secundarios. La generación de grandes deleciones en esta parte del cromosoma de S. rimosus probablemente hará que las cepas modificadas sean más robustas, reduciendo de esta forma la inestabilidad morfológica y genética. Para ello, mediante diferentes procedimientos de clonación se ensamblaron 3 construcciones en Escherichia coli de plásmidos que contenían la maquinaria CRISPR-Cas9, para posteriormente ser introducidas independientemente en S. rimosus usando técnicas de conjugación, para conseguir las deleciones correspondientes. A pesar de las grandes deleciones generadas, los clones modificados de S. rimosus seguían manteniendo la morfología de la cepa original de referencia, pudiendo representar atractivas factorías de células microbianas para la producción de diferentes metabolitos

Characterization of S-layer proteins of potential probiotic starter culture Lactobacillus brevis SF9B isolated from sauerkraut

Abstract S-layers represent the simplest biological membranes developed during the evolution and are one of the most abundant biopolymers on Earth. Current fundamental and applied research aim to reveal the chemical structure, morphogenesis and function of S-layer proteins (Slps). This is the first paper that describes the Slps of certain Lactobacillus brevis strain isolated from sauerkraut. The whole genome sequence (WGS) analysis of the L. brevis SF9B strain uncovered three genes encoding the putative Slps, but merely one, identified as similar to the SlpB of L. brevis ATCC 14869, was expressed. Slp-expressing SF9B cells exhibited increased survival in simulated gastrointestinal (GI) conditions and during freeze-drying. Their survival in stress conditions was additionally enhanced by microencapsulation, especially when using alginate with gelatine as a matrix. Thus prepared cells were subjected to simulated GI conditions and their mortality was only 0.28 ± 0.45 log CFU/mL. Furthermore, a correlation between the high surface hydrophobicity and the remarkable aggregative capacity of SF9B strain was established. The results indicate a prominent role of Slps in adhesion to mucin, extracellular matrix (ECM) proteins, and particularly to Caco-2 cells, where the removal of Slps utterly abolished the adhesiveness of SF9B cells for 7.78 ± 0.25 log CFU/mL.Peer reviewe

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering

Lower ototoxicity and absence of hidden hearing loss point to gentamicin C1a and apramycin as promising antibiotics for clinical use

Trabajo presentado en el 42nd Annual MidWinter Meeting of the Association of Otorhinolaryngology, celebrado en Baltimore (Estados Unidos) del 9 al 13 de febrero de 2019.[Background]: Spread of antimicrobial resistance and shortage of novel antibiotics have led to an urgent need for new antibacterials (Maura et al. 2016, Curr Opin Microbiol 33: 41-46; Tacconelli et al. 2018, Lancet Infect Dis 18: 318-327). Although aminoglycoside antibiotics (AGs) exhibit potent antimicrobial activity, their use has been largely restricted due to serious sideeffects, mainly nephrotoxicity and ototoxicity (Forge and Schacht 2000, Audiol Neurootol 5: 3-22; Huth et al. 2011, Int J Otolaryngol 2011: 937861). It is therefore of great importance to identify AGs of strong antibacterial activity that lack their most harmful side effects.[Methods]: A large number of AGs were tested against a series of multidrug-resistant clinical isolates of the ESKAPE panel; of these, five AGs showing strong antibacterial activity were selected to evaluate their ototoxicity. A stepwise approach was followed, aiming at setting up a protocol that could be used in future high-throughput screenings. In vitro tests were initially conducted by assessing the viability of two established otic cell lines following AG treatment, and subsequently on murine cochlear organotypic cultures, by analysing hair cell survival. In vivo work was then carried out on a guinea pig model, following local round window application of the AGs.[Results]: Commercial gentamicin mixture (GM), the GM congener gentamicin C1a (GM C1a), apramycin (Apra), paromomycin (Paro) and neomycin (Neo) were selected for ototoxicity testing. In vitro analyses confirmed GM and Neo as the most toxic of the tested AGs, and Apra and Paro as those with the lowest toxicity; interestingly, GM C1a appeared to be less toxic than GM. Regarding the in vivo work, a dose-dependent effect of AGs on outer hair cell (OHC) survival and compound action potentials (CAPs) showed that GM C1a and Apra were the least toxic. Strikingly, although no changes were observed in CAP thresholds and OHC survival following treatment with low concentrations of Neo, GM and Paro, the number of inner hair cell (IHC) synaptic ribbons and the CAP amplitudes were reduced. This indication of hidden hearing loss was not observed with GM C1a or Apra at such concentrations.[Conclusion]: These findings have: (a) validated our screening approach, approach that will now be used for high-throughput testing of newly isolated AG congeners, (b) revealed the IHCs as the inner ear’;s most vulnerable element to AG treatment, and (c) identified GM C1a and Apra as promising bases for the development of clinically useful antibiotics

The Errant Labor of the Humanities: Festschrift Presented to Stipe Grgas

In preparing this Festschrift, we had in mind a specific inflection of the concept of errancy, one that comes from the rich and layered work of the American scholar and philosopher William V. Spanos, who conceived it as a way of rendering the logos and telos of the American project subject to thorough rethinking and redefinition, both in history and at present. By calling attention to the complementarity of the work of the two scholars, Grgas and Spanos, who both hone their critical skills on the theme of the logic of the American project, we do not so much intend to claim a direct influence but rather wish to highlight the confluence, commingling, and inspiration that working in the humanities may engender. This commonality is featured in the work of Spanos and in the work of Grgas as a dedicated and passionate engagement with the practices and possibilities inscribed in the discipline, which also requires the scholar to move beyond the given and inhabit what Spanos calls a meta-level of thinking. Grgas’s work, located at the intersection of several disciplines within the humanities and social sciences (which is reflected in the principal themes of this Festschrift), reveals precisely such a commitment that has in the course of his long, fruitful and versatile career charted out a scholarly position always in the process of becoming, and never quite stabilized and domesticated. Grgas’s academic career has been as diverse as the humanistic disciplinary habitus allows: a provocative and popular lecturer, a researcher of tireless intellectual curiosity, a scholar testing the boundaries of disciplines, an enthusiastic and motivating mentor, a thoughtful and sensitive translator, or, as one of the contribution shows, an unobtrusive poet, Grgas has always displayed a remarkable intellectual energy in every aspect of his engagement with the varied and nowadays often embattled debates in the humanities. However, Grgas’s work as an Americanist, cultural theorist, translator, writer, mentor, and teacher doesn’t merely reflect the exciting, if uncontainable, shifts marking the discipline in the last couple of decades; rather, his intellectual labor has been committed to offering a new way of comprehending this change, its scope, direction, and consequences, so as to create an intense web of connections and interrelations where different disciplines talk to one another, without hastening to provide answers so much as to provoke the right kind of questions. The questioning and questing nature of Grgas’s work has marked his writing from the start, but it has intensified in his later writing as the humanities find themselves facing a whole new set of questions for the new millennium. His sustained effort to bring a new awareness of economic issues to discussions of culture and literature in the recent period has been both timely and critically engaged in its reflection on why this issue is particularly significant at this point in history. The layout of the Festschrift may be said to loosely reflect and acknowledge Grgas’s scholarly interests that have charted out his career in the field of the humanities. (from the Editors’ Preface)In preparing this Festschrift, we had in mind a specific inflection of the concept of errancy, one that comes from the rich and layered work of the American scholar and philosopher William V. Spanos, who conceived it as a way of rendering the logos and telos of the American project subject to thorough rethinking and redefinition, both in history and at present. By calling attention to the complementarity of the work of the two scholars, Grgas and Spanos, who both hone their critical skills on the theme of the logic of the American project, we do not so much intend to claim a direct influence but rather wish to highlight the confluence, commingling, and inspiration that working in the humanities may engender. This commonality is featured in the work of Spanos and in the work of Grgas as a dedicated and passionate engagement with the practices and possibilities inscribed in the discipline, which also requires the scholar to move beyond the given and inhabit what Spanos calls a meta-level of thinking. Grgas’s work, located at the intersection of several disciplines within the humanities and social sciences (which is reflected in the principal themes of this Festschrift), reveals precisely such a commitment that has in the course of his long, fruitful and versatile career charted out a scholarly position always in the process of becoming, and never quite stabilized and domesticated. Grgas’s academic career has been as diverse as the humanistic disciplinary habitus allows: a provocative and popular lecturer, a researcher of tireless intellectual curiosity, a scholar testing the boundaries of disciplines, an enthusiastic and motivating mentor, a thoughtful and sensitive translator, or, as one of the contribution shows, an unobtrusive poet, Grgas has always displayed a remarkable intellectual energy in every aspect of his engagement with the varied and nowadays often embattled debates in the humanities. However, Grgas’s work as an Americanist, cultural theorist, translator, writer, mentor, and teacher doesn’t merely reflect the exciting, if uncontainable, shifts marking the discipline in the last couple of decades; rather, his intellectual labor has been committed to offering a new way of comprehending this change, its scope, direction, and consequences, so as to create an intense web of connections and interrelations where different disciplines talk to one another, without hastening to provide answers so much as to provoke the right kind of questions. The questioning and questing nature of Grgas’s work has marked his writing from the start, but it has intensified in his later writing as the humanities find themselves facing a whole new set of questions for the new millennium. His sustained effort to bring a new awareness of economic issues to discussions of culture and literature in the recent period has been both timely and critically engaged in its reflection on why this issue is particularly significant at this point in history. The layout of the Festschrift may be said to loosely reflect and acknowledge Grgas’s scholarly interests that have charted out his career in the field of the humanities. (from the Editors’ Preface

The Errant Labor of the Humanities: Festschrift Presented to Stipe Grgas

In preparing this Festschrift, we had in mind a specific inflection of the concept of errancy, one that comes from the rich and layered work of the American scholar and philosopher William V. Spanos, who conceived it as a way of rendering the logos and telos of the American project subject to thorough rethinking and redefinition, both in history and at present. By calling attention to the complementarity of the work of the two scholars, Grgas and Spanos, who both hone their critical skills on the theme of the logic of the American project, we do not so much intend to claim a direct influence but rather wish to highlight the confluence, commingling, and inspiration that working in the humanities may engender. This commonality is featured in the work of Spanos and in the work of Grgas as a dedicated and passionate engagement with the practices and possibilities inscribed in the discipline, which also requires the scholar to move beyond the given and inhabit what Spanos calls a meta-level of thinking. Grgas’s work, located at the intersection of several disciplines within the humanities and social sciences (which is reflected in the principal themes of this Festschrift), reveals precisely such a commitment that has in the course of his long, fruitful and versatile career charted out a scholarly position always in the process of becoming, and never quite stabilized and domesticated. Grgas’s academic career has been as diverse as the humanistic disciplinary habitus allows: a provocative and popular lecturer, a researcher of tireless intellectual curiosity, a scholar testing the boundaries of disciplines, an enthusiastic and motivating mentor, a thoughtful and sensitive translator, or, as one of the contribution shows, an unobtrusive poet, Grgas has always displayed a remarkable intellectual energy in every aspect of his engagement with the varied and nowadays often embattled debates in the humanities. However, Grgas’s work as an Americanist, cultural theorist, translator, writer, mentor, and teacher doesn’t merely reflect the exciting, if uncontainable, shifts marking the discipline in the last couple of decades; rather, his intellectual labor has been committed to offering a new way of comprehending this change, its scope, direction, and consequences, so as to create an intense web of connections and interrelations where different disciplines talk to one another, without hastening to provide answers so much as to provoke the right kind of questions. The questioning and questing nature of Grgas’s work has marked his writing from the start, but it has intensified in his later writing as the humanities find themselves facing a whole new set of questions for the new millennium. His sustained effort to bring a new awareness of economic issues to discussions of culture and literature in the recent period has been both timely and critically engaged in its reflection on why this issue is particularly significant at this point in history. The layout of the Festschrift may be said to loosely reflect and acknowledge Grgas’s scholarly interests that have charted out his career in the field of the humanities. (from the Editors’ Preface)In preparing this Festschrift, we had in mind a specific inflection of the concept of errancy, one that comes from the rich and layered work of the American scholar and philosopher William V. Spanos, who conceived it as a way of rendering the logos and telos of the American project subject to thorough rethinking and redefinition, both in history and at present. By calling attention to the complementarity of the work of the two scholars, Grgas and Spanos, who both hone their critical skills on the theme of the logic of the American project, we do not so much intend to claim a direct influence but rather wish to highlight the confluence, commingling, and inspiration that working in the humanities may engender. This commonality is featured in the work of Spanos and in the work of Grgas as a dedicated and passionate engagement with the practices and possibilities inscribed in the discipline, which also requires the scholar to move beyond the given and inhabit what Spanos calls a meta-level of thinking. Grgas’s work, located at the intersection of several disciplines within the humanities and social sciences (which is reflected in the principal themes of this Festschrift), reveals precisely such a commitment that has in the course of his long, fruitful and versatile career charted out a scholarly position always in the process of becoming, and never quite stabilized and domesticated. Grgas’s academic career has been as diverse as the humanistic disciplinary habitus allows: a provocative and popular lecturer, a researcher of tireless intellectual curiosity, a scholar testing the boundaries of disciplines, an enthusiastic and motivating mentor, a thoughtful and sensitive translator, or, as one of the contribution shows, an unobtrusive poet, Grgas has always displayed a remarkable intellectual energy in every aspect of his engagement with the varied and nowadays often embattled debates in the humanities. However, Grgas’s work as an Americanist, cultural theorist, translator, writer, mentor, and teacher doesn’t merely reflect the exciting, if uncontainable, shifts marking the discipline in the last couple of decades; rather, his intellectual labor has been committed to offering a new way of comprehending this change, its scope, direction, and consequences, so as to create an intense web of connections and interrelations where different disciplines talk to one another, without hastening to provide answers so much as to provoke the right kind of questions. The questioning and questing nature of Grgas’s work has marked his writing from the start, but it has intensified in his later writing as the humanities find themselves facing a whole new set of questions for the new millennium. His sustained effort to bring a new awareness of economic issues to discussions of culture and literature in the recent period has been both timely and critically engaged in its reflection on why this issue is particularly significant at this point in history. The layout of the Festschrift may be said to loosely reflect and acknowledge Grgas’s scholarly interests that have charted out his career in the field of the humanities. (from the Editors’ Preface

Kliničke upute za dijagnostiku, liječenje i praćenje bolesnika oboljelih od raka mokraćnog mjehura Hrvatskoga onkološkog društva i Hrvatskoga urološkog društva Hrvatskoga liječničkog zbora [Clinical guidelines for diagnosing, treatment and monitoring patients with bladder cancer - Croatian Oncology Society and Croatian Urology Society, Croatian Medical Association]

Urothelial cancer is the most common bladder cancer. Hematuria is the most common presenting symptom in patients with bladder cancer. The most common diagnostics of bladder cancer is performed by transurethral resection of bladder after which pathohistological diagnosis is set. It is necessary to determine whether the cancer penetrated in muscle layer (muscle-invasive cancer) or not (muscle-noninvasive cancer). Decision on therapeutic modality depends on the clinical stage of disease and on prognostic and risk factors. For muscle non-invasive bladder cancer transurethral resection is preferred with or without intravesical instillation of Bacillus Calmette-Guérin (BCG). For invasive cancer the method of choice is radical cystectomy. Radiotherapy is used in radical and palliative purposes. Metastatic disease is most frequently treated by chemotherapy metotrexate/vinblastine/doxorubicine/cisplatin (MVAC) or gemcitabine/cisplatin (GC). The purpose of this article is to present clinical recommendations to set standards of procedures and criteria in diagnostics, treatment and follow up of patients with bladder cancer in the Republic of Croatia

Identification of Lipstatin-Producing Ability in Streptomyces virginiae CBS 314.55 Using Dereplication Approach

Streptomicete su poznate kao bogat izvor bioaktivnih metabolita, poput sekundarnog metabolita lipstatina, koji ima vrlo karakterističnu strukturu β-laktona, a upotrebljava se kao intermedijar u proizvodnji lijeka za mršavljenje, komercijalnog naziva orlistat. Dublje razumijevanje distribucije identičnih ili strukturno sličnih spojeva, izoliranih iz taksonomski sličnih skupina mikroorganizama, bitno je za otkrivanje novih biološki aktivnih spojeva ili kvalitetnijih sojeva što proizvode poznate metabolite od velikog značaja za medicinu. Samo su dva soja, neovisni izolati vrste S. toxytricini, poznati kao proizvođači lipstatina. Prema današnjim taksonomskim spoznajama, vrsta S. toxytricini pripada fenotipskoj skupini S. lavendulae. Taksonomskom dereplikacijom i ispitivanjem in vitro inhibicije lipaze pomoću p-nitrofenolnih derivata C4 i C16 masnih kiselina istraženi su odabrani sojevi fenotipske skupine S. lavendulae iz javno dostupnih kolekcija mikroorganizama. Utvrđeno je da bakterijska kultura soja Streptomyces virginiae CBS 314.55 izrazito inhibira lipazu. Pomoću metoda HPLC i LC-MS/MS po prvi je put otkriveno da ovaj novi soj proizvodi metabolit strukture identične lipstatinu. Dokazano je da se novi soj S. virginiae morfološki i fiziološki bitno razlikuje od sojeva S. toxytricini, ali da im je proizvodni potencijal sličan. Time je potvrđena mogućnost učinkovite primjene metode dereplikacije za brzu identifikaciju novih industrijskih sojeva iz javno dostupnih kolekcija mikroorganizama.Streptomyces species are prolific producers of bioactive metabolites, such as β-lactone-containing lipstatin produced by Streptomyces toxytricini, an intermediate used in semi-synthetic process for production of anti-obesity drug orlistat. Understanding the distribution of identical or structurally similar molecules produced by a taxonomic group is of particular importance when trying to isolate novel biologically active compounds or strains producing known metabolites of medical importance with potentially improved properties. Until now, only two independent isolates of S. toxytricini species have been known to be producers of lipstatin. According to the current taxonomic criteria, S. toxytricini belongs to Streptomyces lavendulae phenotypic cluster. Taxonomy-based dereplication approach coupled with in vitro assay was applied to screen the S. lavendulae phenotypic cluster for production of lipstatin-like lipase inhibitors using synthetic p-nitrophenol derivatives of C4 and C16 lipids. Screening the available strains from public collections belonging to S. lavendulae phenotypic cluster, high lipase inhibitory activity was identified in the Streptomyces virginiae CBS 314.55 culture supernatants. HPLC and LC-MS/MS confirmed lipstatin production by a new Streptomyces species for the first time. We have demonstrated that the new lipstatin-producing strain S. virginiae morphologically and physiologically differs from S. toxytricini substantially; however, the production capacity of the newly identified lipstatin-producing species S. virginiae is comparable to S. toxytricini. We have thus demonstrated the effectiveness of a simple and affordable dereplication approach for identification of potentially novel and useful industrial strains available in public culture collections

Reference-grade genome and large linear plasmid of Streptomyces rimosus: pushing the limits of Nanopore sequencing

[EN] Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtainedSIThis work was supported as part of the European project “Thoroughly Optimised Production Chassis for Advanced Pharmaceutical Ingredients” (grant ID 720793, European Union’s Horizon 2020 Research and Innovation Program) and by the Slovenian Research Agency (P4-0116, P4-0077, and P1-0034). L.S. is supported by a Slovenian Research Agency young researcher grant (35220200570), and M.T. is supported by grant C3330-19-952047 funded by Republic of Slovenia Ministry of Education, Science, and Sport and the European Union European Regional Development Fun