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

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

THE IMPORTANCE AND EFFECTS OF PACKAGING IN THE NEW PRODUCT DEVELOPMENT PROCESS

V zadnjih letih je tehnologija napredovala in tako se rojeva vsak dan veliko novih izdelkov. Na trg pride veliko novih izdelkov, ampak le malo izdelkov zaslovi in se njihov življenjski cikel podaljša. Za podjetja pa je ustvarjanje izdelkov nuja. Potrebno je poiskati tiste kompetitivne lastnosti izdelkov in jih ponuditi na trg. Uskladiti pa moramo tudi časovni razvoj izdelka. Zaradi velike konkurence se velikokrat zgodi, da te kakšno drugo podjetje prehiti z izdelkom. V okviru izdelka se moramo zavedati, da noben izdelek ne pritegne potencialnega kupca samo z obstojem. Potrebno je pritegniti kupce s propagando, oglaševanjem in vse to vsebuje tudi embalaža. Razviti moramo embalažo, ki opravlja svoje prvotne funkcije in izstopa. Namen diplomskega seminarja je bil predvsem ugotoviti kako vpliva embalaža na ljudi in s katerimi sestavinami najbolj pritegne kupce. Iz analiziranih podatkov smo prišli do zaključka, da embalaža vpliva na ljudi vendar se mnogi sploh ne zavedajo tega. Pomembna je kreativnost pri ustvarjanju embalaže in ustvariti embalažo izdelka, ki pritegne ljudi in se jim tudi vtisne v spomin.In the recent years technology has advanced and so new products are created every day. A lot of new product enter the market but only a few become famous and therefore extend their cycle of existence lengthens. For companies creating new products is crucial. It is important to find those competitive characteristics of products and offer them to the market. It is also very important to adjust the developing time of a product. Because the competition is high it often happens that another company comes out with the product first. As far as the product goes we have to be aware that no product attracts the customer just by existing. It is vital to attract consumers with propaganda, advertising and packing. Packing has to enhance the functions as well as stand out. The purpose of the diploma seminar is to find out how the packing influences people and which characteristics attract the most consumers. From the data analysis I came to the conclusion that packing influences people but a lot of them are not aware of this. Creativity is vital when creating a package that is why we must create one that attracts and stays in the consumers memory

The impact of reading on language development in the preschool children

The importance of the role played by children's literature in the child's mental, social and linguistic development and in the development of his or her basic academic skills, such as reading andwriting, has been confirmed by numerous studies. A central issue in developmental psychology is what activities related to children's books exert an influence on the child's development and in what ways. Thisinterest in children's books and in child language development places our research into two scientific disciplines, viz. psychology and linguistics. The study explores the impact of systematic and regular readingof selected children's books in preschool institutions on the development of language competences in children aged four to six years, boys and girls. Other contributing factors whose relevance for languagedevelopment has been either postulated by theories or highlighted by empirical studies, are also observed - e.g. parents' education, number of books in the family, quality of education in the family (frequencyof conversations, visits to cultural events, reading books together, etc.). The children included in the study all attend a preschool institution with an educational program which is based on the national curriculumand which targets also the language area. The children in the experimental group are submitted to additional reading of selected children's literature. The development of children's linguistic competences isfollowed using two methods: analysis of answers on The Vane evaluation of language scale (The Vane-L) and analysis of transcripts of story retelling after the child has been read H. Ch. Andersen's fairy taleThe Princess and the Pea. The results show, that the children who were systematically read selected children's books in their preschool groups, achieved significantly higher scores on the standardized Vanelanguage development scale and on the unstandardized test of retelling a story. Correlations between some of the children's results achieved on both tests and the quality of language education in the family(mother's education, family environment and family activities) were low but statistically significant. The results of the study may also serve as an expert platform underlying introduction of various curricularcomponents and methods specifically targeting language development

Elementi koji reguliraju biosintezu tetraciklinskih antibiotika u genskim nakupinama: otcG gen pozitivno regulira proizvodnju oksitetraciklina u vrste Streptomyces rimosus

The expression of bacterial polyketide synthase gene clusters is often controlled by a number of different families of regulatory proteins that can have either a pathway-specific or a pleiotropic mode of action, e.g. the SARP family (Streptomyces antibiotic regulatory proteins), ribosome-associated ppGpp synthetase, γ-butyrolactone-binding regulatory proteins, and two-component regulatory proteins. The molecular genetics of such regulatory mechanisms that govern the biosynthesis of tetracyclines is poorly understood. In this work, a comparative bioinformatic analysis of regulatory genes present in three tetracycline antibiotic gene clusters, namely oxytetracycline (OTC), chlortetracycline and recently cloned chelocardin gene clusters of S. rimosus, S. aureofaciens and Amycolatopsis sulphurea has been performed. A SARP family regulatory protein is located in the chlortetracycline gene cluster, but is not detected in the gene cluster encoding OTC biosynthesis. Interestingly, the only regulatory element identified in chelocardin gene cluster was chdA, an otrR and ctcR homologue from the TetR family of regulators that regulates the expression of the otrB and ctc05 exporter genes in the oxytetracycline and chlortetracycline gene clusters. In the oxytetracycline gene cluster, a new LAL (LuxR) family regulatory gene homologue, otcG, was identified. This homologue is also present in the ctc gene cluster. By gene disruption and overexpression experiments, a \u27conditionally positive\u27 role of otcG in OTC biosynthesis has been demonstrated. The observation, the bioinformatics data and the previous work on phosphate regulation suggest the presence of a more complex, fine tuning role of the otcG gene product in overall expression of genes for OTC biosynthesis.Ekspresiju bakterijskih genskih nakupina poliketidnih sintaza obično reguliraju različiti vrlo specifični proteini koji su uglavnom usmjereni samo na ciljane gene u nakupini gena ili je riječ o regulatornim genima što djeluju šire na stanične procese, takozvani pleiotropni regulatori. Prema načinu djelovanja ti se regulatorni proteini mogu podijeliti u više skupina, kao što su SARP (engl. Streptomyces Antibiotic Regulatory Proteins), skupina ribosomski povezanih regulatornih proteina nazvanih ppGpp sintaze, γ-butirolaktonskih veznih i dvokomponentnih regulatornih proteina. Molekularna genetika takvih regulatornih mehanizama, koji utječu na biosintezu tetraciklina, još je i danas slabo istražena. U ovom je radu prikazana komparativna bioinformatička studija regulatornih proteina triju genskih nakupina koje kodiraju biosintezu oksitetraciklina (OTC) u vrste Streptomyces rimosus, klortetraciklina (u vrste Streptomyces aureofaciens), a i nedavno klonirane genske nakupine za biosintezu kelokardina (CHD) u vrste Amycolatopsis sulphurea. U genskoj nakupini koja kodira biosintezu klortetraciklina nalazi se takozvani SARP regulatorni gen. Međutim, homolog toga gena ne postoji u nakupini gena za biosintezu oksitetraciklina. U nakupini gena za biosintezu kelokardina pronađen je samo jedan regulatorni gen nazvan chdA, homolog gena otrR i ctcR iz skupine TetR regulatornih proteina, koji regulira ekspresiju otrB i ctc05 gena u nakupinama gena za biosintezu oksitetraciklina i klortetraciklina. U genskoj nakupini za biosintezu oksitetraciklina identificiran je novi regulatorni protein iz skupine LAL (LuxR), nazvan OtcG. Taj se genski homolog nalazi i u genskoj nakupini za biosintezu klortetraciklina. Inaktivacijom i povećanom ekspresijom gena otcG eksperimentalno je utvrđeno njegovo djelovanje kao uvjetno pozitivnog regulatora u biosintezi oksitetraciklina. Takvi eksperimentalni rezultati bioinformatičke studije i rezultati koji se odnose na prijašnje istraživanje utjecaja fosfata na reguliranje biosinteze oksitetraciklina upućuju na vrlo kompleksnu organizaciju regulatornih elemenata i njihova djelovanja u procesu biosinteze oksitetraciklina, u kojima sudjeluje i novi regulatorni element, produkt gena otcG

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea : a platform for producing novel tetracycline antibiotics

Tetracyclines (TCs) are medically important antibiotics from the polyketide family of natural products. Chelocardin (CHD), produced by Amycolatopsis sulphurea, is a broad-spectrum tetracyclic antibiotic with potent bacteriolytic activity against a number of Gram-positive and Gram-negative multi-resistant pathogens. CHD has an unknown mode of action that is different from TCs. It has some structural features that define it as 'atypical' and, notably, is active against tetracycline-resistant pathogens. Identification and characterization of the chelocardin biosynthetic gene cluster from A. sulphurea revealed 18 putative open reading frames including a type II polyketide synthase. Compared to typical TCs, the chd cluster contains a number of features that relate to its classification as 'atypical': an additional gene for a putative two-component cyclase/aromatase that may be responsible for the different aromatization pattern, a gene for a putative aminotransferase for C-4 with the opposite stereochemistry to TCs and a gene for a putative C-9 methylase that is a unique feature of this biosynthetic cluster within the TCs. Collectively, these enzymes deliver a molecule with different aromatization of ring C that results in an unusual planar structure of the TC backbone. This is a likely contributor to its different mode of action. In addition CHD biosynthesis is primed with acetate, unlike the TCs, which are primed with malonamate, and offers a biosynthetic engineering platform that represents a unique opportunity for efficient generation of novel tetracyclic backbones using combinatorial biosynthesis