Establishing and engineering heterologous production systems for argyrin and α-pyrone antibiotics

Myxobacterial natural products have proven to be a valuable source of antibacterial and anticancer compounds in the last decades. This thesis covers the biosynthesis elucidation, heterologous production as well as structure and yield optimization of two potent myxobacterial compounds. The work on coralloypronin resulted in the design of an efficient heterologous production platform in the myxobacterial model organism Myxococcus xanthus. Research on the established heterologous producer led to identification of specific biochemical processes and resulted in isolation and structure elucidation of two novel coralloypronin derivatives. Furthermore, gene deletion experiments led to discovery of hypothetical self-resistance mechanism, which seems to be conserved in most myxobacterial RNA polymerase (RNAP) inhibitor biosynthetic gene clusters (BGC). The BGC of argyrin, a cyclic octapeptide produced by nonribosomal peptide synthetase (NRPS), was identified in Cystobacter sp. SBCb004. The heterologous expression of synthetic argyrin BGC was achieved in M. xanthus, which, after medium optimization, led to a significant yield improvement over the wild type producer. The production of argyrin could be even further improved by utilization of different promoter systems with a specifically optimized leader sequence. Furthermore, precursor directed biosynthesis was applied to produce a library of novel argyrin derivatives.Myxobakerielle Naturstoffe haben sich in den letzten Jahrzehnten als wertvolle Ressource für Wirkstoffe gegen Bakterien und Krebs bewährt. Diese Dissertation umfasst die Biosyntheseaufklärung, die heterologe Produktion sowie Struktur und Ausbeuteoptimierung zweier wirksamer myxobakterieller Verbindungen. Die Arbeit über Corallopyronin führte zum Design einer effizienten myxobakteriellen Produktionsplattform im myxobakteriellen Modellorganismus Myxococcus xanthus. Die Erforschung dieses etablierten heterologen Produzenten führte zur Identifizierung spezifischer biochemischer Prozesse, die in der Isolation und Strukturaufklärung zweier neuer Corallopyroninderivate resultierte. Zusätzliche Gendeletionsexperimente führten zur Entdeckung eines hypothetischen Selbstresistenzmechanismus, der in myxobakteriellen RNA Polymerase (RNAP) Inhibitor Biosynthese-Genclustern (BGC) konserviert zu sein scheint. Das BGC des zyklischen Oktapeptids Argyrin wurde in einem Nichtribosomalen Peptid Synthetase (NRPS) Gencluster in Cystobacter sp. SBCb004 entdeckt. Die heterologe Expression eines synthetischen Argyrin BGC in M. xanthus führte nach Medienoptimierung zu einer signifikanten Steigerung der Ausbeute im Vergleich zum Wildtyp. Zusätzliche Steigerungen der Argyrinproduktion wurden durch Integration verschiedener Promotoren mit einer optimierten Leader Sequenz erreicht. Zudem konnte durch die Fütterung spezieller Vorläufermoleküle eine Bibliothek neuer Argyrinderivate produziert werden

Association analysis between genetic variability of selected non-coding rnas with growth and carcass traits in chicken

Mikro RNA so kratke nekodirajoče RNA, ki uravnavajo izražanje genov. Preko vplivov na razne biološke poti v organizmu lahko prispevajo tudi k spremembi fenotipa in posledično k uspešnosti prireje rejnih živali. S tem postanejo miRNA uporabne tudi kot biooznačevalci v selekciji živali. Za analizo smo izbrali miRNA gen gga-mir-1658 pri kokoši. Namen te naloge je bil: 1. pridobiti znane genomske podatke o gga-mir-1658 pri kokoši in 2. z molekularno-genetskimi metodami analizirati povezavo med SNP-ji (rs316300182 in rs1059378319) v gga-mir-1658 ter desetimi pitovnimi in klavnimi lastnostmi kokoši. V prvem delu smo informacije o gga-mir-1658 iz osmih podatkovnih zbirk in bioinformacijskih orodij združili v atlas regulatornih elementov. Z asociacijsko analizo smo nato potrdili povezavo med SNP-jem rs1059378319 in maso jeter (p = 0,05). Živali z genotipom CC so imele za 5,91 g težja jetra (p = 0,02) kot živali z genotipom CT. Povezava med rs316300182 in maso jeter je bila le nakazana (p = 0,07). Živali z genotipom CC so imele za 5,66 g težja jetra (p = 0,06) kot živali z genotipom CG. Povezave ostalih izmerjenih lastnosti s prej omenjenima SNP-jema niso bile značilne. Rezultati nakazujejo na to, da je gga-mir-1658 zanimiva za nadaljnje raziskave. S testiranjem na večji in pasemsko bolj raznoliki populaciji bo možno bolje raziskati njen funkcionalni vpliv in njen potencial biooznačevalca v selekciji kokoši.MicroRNAs are small noncoding RNAs that regulate gene expression. Through effects on various biological pathways in organism, miRNAs contribute to phenotype changes and consequently on efficiency of animal production. That is why miRNAs became useful as biomarkers in animal selection. For the analysis we selected miRNA gene gga-miR-1658. The aim of this study was: 1.) to retrieve known genomic information about gga-mir-1658 in chicken and 2.) to analyze the association between two SNPs (rs316300182 and rs1059378319) in gga-mir-1658 gene and ten growth and carcass traits using molecular-genetic methods. Firstly, we designed an atlas of regulatory elements for gga-mir-1658 from eight databases and bioinformatics tools. In the second part, we confirmed an association between rs1059378319 and liver mass (p = 0.05). Animals with CC genotype had 5.91g heavier liver (p = 0.02) than animals with CT genotype. Association between SNP rs316300182 and liver mass (p = 0.07) did not meet the level of significance, but was close. Animals with CC genotype had for 5.66g heavier liver (p = 0.06) as animals with a genotype CG. Other growth and carcass traits did not reach significant associations with these two SNPs. Results suggest that gga-mir-1658 is interesting for further studies. Testing on a larger population and including additional breeds is required to functionally characterize it and potentially apply it as a biomarker in chicken selection programs

Rana in potovanje – Sofoklejev Filoktet in Homerjeva Odiseja v drami in uprizoritvi Inflammation du verbe vivre (Vnetje glagola živeti) Wajdija Mouawada

Avtorica v članku analizira primer besedila in njegove uprizoritve, ki ju je navdihnila nenadna smrt v ožji ustvarjalni ekipi njunega avtorja. Kanadsko-libanonski dramatik, režiser in igralec Wajdi Mouawad je v solo predstavi Inflammation du verbe vivre (Vnetje glagola živeti), uprizorjeni v pariškem gledališču Théâtre National de la Colline leta 2015, prepletel antično grško literarno in mitološko izročilo z osebno izpovedjo o izgubi prijatelja in sodelavca Roberta Davreuja, vse to pa nadgradil še z družbenokritičnim prikazom razmer v sodobni Grčiji. Predstava je nastala kot predzadnji del cikla uprizoritev sedmih ohranjenih Sofoklejevih tragedij s skupnim naslovom Le dernièr jour de sa vie (Zadnji dan njegovega življenja), ki jih je Mouawad nameraval režirati v Davreujevih novih prevodih. Proces žalovanja ob smrti, ki je zaustavila projekt, je povezal z dokumentiranjem procesa nastajanja besedila, ki ga je v obliki svojevrstne gledališko-cinematične žalostinke tudi režiral in v njej nastopil. Lik Filokteta je spojil z likom Odiseja, toda ne tistega, ki si v Sofoklejevi tragediji prizadeva, da bi mu s spletko izmaknil lok, temveč tistega, ki pri Homerju po zavzetju Troje deset let išče pot domov na Itako in ki v podzemlju obišče senco Tejrezija

DIESEL AND BIODIESEL MACRO-SPRAY CHARACTERISTICS

Sažetak Karakteristike ubrizgavanja i formiranja mlaza u prostoru izgaranja jedan su od najvažnijih čimbenika modernih dizelovih motora i imaju značajan utjecaj na efektivne parametre i emisije ispuha. U posljednjih nekoliko desetljeća, biodizelu se kao motornom gorivu sve više posvećuje posebna pažnja. Unatoč tome što ima slične fizičke osobine kao dizelsko gorivo, svejedno veća gustoća, viskoznost i površinski napon mogu ponekad prouzročiti anomalije u procesu ubrizgavanja, kao što su npr. dodir mlaza sa stijenkama komore izgaranja, loše miješanje sa zrakom (prevelike kapljice) i drugo. U radu su prikazani rezultati istraživanja fizičkih svojstava dizelskog i biodizelskog goriva glede karakteristika ubrizgavanja i mlaza goriva. Utjecaj fizičkih osobina goriva proučavan je uz pomoć fizičko-matematičkog modela, simuliranjem procesa ubrizgavanja u konvencionalnom sustavu. Model je bio razvijen na IEPOI - Inštitut za energetiko, procesno in okoljsko inženirstvo Fakulteta za strojništvo, Univerza v Mariboru, koda Bkin, provjeren eksperimentalno na uređaju i na motoru. U posebnom smo uređaju izmjerili makro karakteristike mlaza (domet, kut, oblik), dok je vremensko prodiranje mlaza u mirujući zrak praćeno visokobrzinskom kamerom (2500 snimaka/s). Uz pomoć 3D programa (AVL "Fire"), dopunjena za ova istraživanja na IEPOI, mogli smo pratiti procese u motoru i odrediti ostale parametre mlaza (promjer kapljice i njihovu distribuciju u aksijalnom prosjeku) u ovisnosti o vremenu i odrediti njihove integralne prosječne vrijednosti i usporediti ih s vrijednostima dobivenih eksperimentom. U radu su prikazani rezultati utjecaja fizičkih osobina goriva i radnih uvjeta na karakteristike ubrizgavanja i mlaza goriva.Abstract Characteristics of fuel injection and spray formation in the engine combustion chamber are most important factor of up-to-date diesel engines, influencing their effective performance parameters and exhaust emissions. During last few decades alternative automotive fuels, such as biodiesel have received salient attention. Even though it has similar physical properties as diesel fuel, higher density, viscosity and surface tension of biodiesel may have a dominant influence on the spray formation and sometimes cause spray anomalies - such as contact between fuel and combustion chamber, poor mixing with air (bigger fuel droplets) etc. Results of the investigation about the impact of the diesel and biodiesel fuel physical properties on the injection and spray characteristics are presented in the paper. Physical properties of both have been examined and used in the mathematical model of the conventional injection system. The model was derived in IEPO - Institute for Power, Process and Environmental Engineering Faculty of mechanical engineering, University Maribor, simulation code Bkin, and was verified by experiment carried out on the fuel injection system test bench and on the engine. In a special test chamber the macro-characteristics of the spray injected into motionless air at the atmospheric pressure and room temperature (spray penetration distance, angle, and spray shape) have been measured and spray images were recorded by the high-speed camera (2500 frames/s). Using 3D simulation program (AVL "Fire") and including for the purpose of these investigations a few new estimates, the injection process for both fuels have been simulated and compared with the results obtained by experiments. These results are presented and discussed in the paper

Chemical synthesis of tripeptide thioesters for the biotechnological incorporation into the myxobacterial secondary metabolite argyrin via mutasynthesis

The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure–activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product’s biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-DhaSar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide

First Insight Into Genetic Diversity of Alpine ibex (Capra ibex) in Slovenia

In Europe, the Alpine ibex (Capra ibex) was on the brink of extinction in the 19th century. Therefore, different conservation measures were implemented, and several reintroductions were made in the Alpine arc, starting from the only surviving population in Gran Paradiso, Italy. An extreme historical bottleneck and additional reintroductions have strongly shaped the genetic make-up of recent populations, resulting in significant genetic drift and profound inbreeding across the species range. To support science-based conservation actions, molecular methods have been increasingly used. However, such analyses did not include populations in Slovenia. We analysed neutral loci (partial fragment of mitochondrial cytochrome b, mtDNA) and the adaptive major histocompatibility complex (MHC DRB exon 2) of the Alpine ibex from both Slovenian populations (Julian and Kamnik-Savinja Alps) to understand how past reintroductions and recent management have affected the genetic diversity of the species. Results showed that both populations are genetically severely depleted, carrying only one mtDNA haplotype and one functional allele for MHC DRB exon 2, Caib-DRB*01. This calls for further conservation actions, including the reintroduction of individuals with different genetic background. However, the Alpine ibex is currently considered a non-native species in Slovenia, which makes conservation actions extremely difficult and threatens the long-term survival of the species. Therefore, scientists and population managers are urging policy/decision makers to change the status of the species to the native one and consequently to allow reintroductions. These appeals are supported by previous archaeological data on the existence of bones assigned to Alpine ibex in the Julian Alps, and evidence of severe genetic depletion in current ibex populations confirmed in this study. PRVI VPOGLED V GENETSKO RAZNOLIKOST ALPSKEGA KOZOROGA (Capra ibex) V SLOVENIJI Izvleček: V Evropi je bil alpski kozorog (Capra ibex) v 19. stoletju na robu izumrtja. Izvajali so se različni ukrepi za njegovo ohranjanje. V alpskem loku je bilo izvedenih več ponovnih naselitev, najprej z edino ohranjeno populacijo v kraju Gran Paradiso v Italiji. Izredna okrnjenost vrste v preteklosti in dodatne ponovne naselitve so močno vplivale na genetsko sestavo populacije, kar je povzročilo znaten genetski zdrs in parjenje v sorodstvu na celotnem območju vrste. V podporo znanstveno utemeljenim ukrepom ohranjanja se vse pogosteje uporabljajo molekularne metode, vendar takšne analize niso vključevale populacij v Sloveniji. Da bi razumeli, kako je ponovno naseljevanje in nedavno upravljanje vplivalo na genetsko raznolikost vrste, smo analizirali nevtralne lokuse (delni fragment mitohondrijskega citokroma b, mtDNA) in adaptivni poglavitni histokompatibilnostni kompleks (MHC DRB ekson 2) alpskega kozoroga iz obeh slovenskih populacij (Julijske in Kamniško-Savinjske Alpe). Rezultati so pokazali, da sta obe populaciji genetsko zelo osiromašeni, saj nosita le en haplotip mtDNA in en funkcionalni alel za MHC DRB ekson 2, Caib-DRB*01. Zato so potrebni nadaljnji ukrepi za ohranjanje, vključno s ponovno naselitvijo živali z drugačnim genetskim ozadjem. Vendar alpski kozorog v Sloveniji trenutno velja za tujerodno vrsto, kar zelo otežuje ukrepe za njegovo ohranitev in ogroža dolgoročno preživetje vrste. Znanstveniki in upravljavci populacij zato pozivajo politike/odločevalce, naj spremenijo status vrste v avtohtono in posledično omogočijo ponovno naselitev. Ti pozivi so podprti s predhodnimi arheološkimi podatki o obstoju kosti alpskega kozoroga v Julijskih Alpah in z dokazi o izraziti genetski osiromašenosti sedanjih populacij kozoroga, potrjenimi v tej študiji. Ključne besede: Capra ibex; mitohondrijska DNA; MHC DRB exon2; ponovna naselitev; upravljanj

Whole‐genome SNP genotyping unveils ancestral and recent introgression in wild and domestic goats

After the domestication of goats around 10,000 years before the present (BP), humans transported goats far beyond the range of their wild ancestor, the bezoar goat. This brought domestic goats into contact with many wild goat species such as ibex and markhor, enabling introgression between domestic and wild goats. To investigate this, while shedding light on the taxonomic status of wild and domestic goats, we analysed genome-wide SNP data of 613 specimens from 14 taxonomic units, including Capra hircus, C. pyrenaica, C. ibex (from Switzerland, Austria, Germany and Slovenia), C. aegagrus aegagrus, C. a. cretica, C. h. dorcas, C. caucasica caucasica, C. c. severtzovi, C. c. cylindricornis, C. falconeri, C. sibirica sibirica, C. s. alaiana and C. nubiana, as well as Oreamnos americanus (mountain goat) as an outgroup. To trace gene flow between domestic and wild goats, we integrated genotype data of local goat breeds from the Alps as well as from countries such as Spain, Greece, Türkiye, Egypt, Sudan, Iran, Russia (Caucasus and Altai) and Pakistan. Our phylogenetic analyses displayed a clear separation between bezoar-type and ibex-type clades with wild goats from the Greek islands of Crete and Youra clustered within domestic goats, confirming their feral origin. Our analyses also revealed gene flow between the lineages of Caucasian tur and domestic goats that most likely occurred before or during early domestication. Within the clade of domestic goats, analyses inferred gene flow between African and Iberian goats. The detected events of introgression were consistent with previous reports and offered interesting insights into the historical relationships among domestic and wild goats

Whole‐genome SNP genotyping unveils ancestral and recent introgression in wild and domestic goats

After the domestication of goats around 10,000 years before the present (BP), humans transported goats far beyond the range of their wild ancestor, the bezoar goat. This brought domestic goats into contact with many wild goat species such as ibex and markhor, enabling introgression between domestic and wild goats. To investigate this, while shedding light on the taxonomic status of wild and domestic goats, we analysed genome-wide SNP data of 613 specimens from 14 taxonomic units, including Capra hircus, C. pyrenaica, C. ibex (from Switzerland, Austria, Germany and Slovenia), C. aegagrus aegagrus, C. a. cretica, C. h. dorcas, C. caucasica caucasica, C. c. severtzovi, C. c. cylindricornis, C. falconeri, C. sibirica sibirica, C. s. alaiana and C. nubiana, as well as Oreamnos americanus (mountain goat) as an outgroup. To trace gene flow between domestic and wild goats, we integrated genotype data of local goat breeds from the Alps as well as from countries such as Spain, Greece, Türkiye, Egypt, Sudan, Iran, Russia (Caucasus and Altai) and Pakistan. Our phylogenetic analyses displayed a clear separation between bezoar-type and ibex-type clades with wild goats from the Greek islands of Crete and Youra clustered within domestic goats, confirming their feral origin. Our analyses also revealed gene flow between the lineages of Caucasian tur and domestic goats that most likely occurred before or during early domestication. Within the clade of domestic goats, analyses inferred gene flow between African and Iberian goats. The detected events of introgression were consistent with previous reports and offered interesting insights into the historical relationships among domestic and wild goats.Deutscher Akademischer Austauschdienst http://dx.doi.org/10.13039/501100001655Javna Agencija za Raziskovalno Dejavnost RS http://dx.doi.org/10.13039/501100004329Ministry of Science and Higher Education of the Russian Federation http://dx.doi.org/10.13039/501100012190Peer Reviewe