Synthetic biology tools for transcriptional activation and regulation of biosynthetic gene clusters in filamentous fungi

The increasing number of sequenced fungal genomes revealed a vast of natural product biosynthetic gene clusters (BGCs) with unknown produced compounds. However, most of the fungal SM BGCs are transcriptionally silent under laboratory growth conditions, and the signal or stimulus triggering the transcriptional activation of these clusters is often unknown, rendering these BGC “cryptic”. Obviously, considering the enormous diversity of the fungal kingdom and the notion that BGCs are widespread, characterized SMs so far represent only the tip of the ice-berg. With the advent of synthetic biology novel applications and tools have been developed for fungi, aiming at the transcriptional activation of cryptic BGCs. The objective of this thesis was to develop novel synthetic biology tools for filamentous fungi, with the aim of transcriptional activation of transcriptionally silent and “cryptic” BGCs, as well as the controlled production of BGC encoded secondary metabolites in the fungal host

A hepcidin, egy máj által termelt hormon szerepe a vasanyagcsere szabályozásában = The role of hepcidin, a liver-produced hormone in the regulation of iron metabolism

A hepcidin az emlős szervezetekben található a közelmúltban felfedezett hormon, amelynek két fő funkciója van emlősökben: rendelkezik egy antimikrobiális hatással és szabályozza a szervezet vasanyagcseréjét. A hepcidin fő hatása a vér vas szintjének csökkentése, amely a ferroportin molekulához, a hepcidin receptorához való kötődés révén valósul meg. A ferroportin az egyetlen ismert vas exportáló molekula, amely a májsejtek, bélhámsejtek és a makrofágok membránjában található. A hepcidin megkötését követően a ferroportin a sejtbe kerülve degradálódik, következményes vasretenciót előidézve. Kísérleteink egy részében a ferroportin hepcidinkötő jellegzetességeit vizsgáltuk, valamint azt, hogy a hepcidin magasabb rendű szerkezetének mi a hatása a két molekula közti kapcsolatra. Eredményeink szerint a hepcidin szokatlan hajtű szerkezete elengedhetetlen a receptorához való kötődésben. A hepcidin a májban egy 84 aminosav hosszúságú preprohormonként szintetizálódik, amelyet a furin hasít az érett 25 aminosavas formává. Ez a hasítás a sejten belül és a vérben is történhet. A hormon különböző érési alakjaihoz kapcsolódó fehérjéket keresve azonosítottuk az alfa-1 antitripszint, amelyről bebizonyosodott, a prepro- és prohormonhoz kötődik, az érett molekulához viszont nem. Bebizonyítottuk, hogy az alfa-1 antitripszin a hormon korai érési formáihoz a májsejtekben és a vérben is kötődik. Ez a fehérjeinterakció a hepcidin érésében egy fontos poszttranszlációs szabályozási lépésnek látszik. | Hepcidin is a lately discovered hormon with two functions in mammalians: it has an antimicrobial effect and it regulates the iron metabolism of the body. The main effect of hepcidin is the reduction of iron level in the blood. This happens through the binding to ferroportin, the receptor of hepcidin. Ferroportin is the only known iron exporter molecule of humans, which is located in the membrane of hepatocytes, intestinal cells, and macrophages. After binding hepcidin, ferroportin is internalized, and degraded intracellularly, so iron retention will happen. We carried out experiments to analyze the hepcidin binding features of ferroportin, also the effect of the higher structure of hepcidin in this intermolecular association. The unusual hairpin structure of hepcidin seems to be essential in binding to ferroportin. Hepcidin is synthesized in the liver as an 84 amino acid preprohepcidin, and cleaved by furin into the mature 25 amino acid form. This cleavage may occur intracellularly or in the blood. By performing a bacterial two-hybrid screening we found a protein, alpha-1 antitrypsin which interacts with preprohepcidin, and prohepcidin, but not with the mature peptide. We proved that the binding of alpha-1 antitrypsin to the premature hepcidin forms happens intracellularly, as well as in the blood. This interaction seems to be an important posttranslational regulation step of hepcidin maturation

Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi

Filamentous fungi are highly productive cell factories, many of which are industrial producers of enzymes, organic acids, and secondary metabolites. The increasing number of sequenced fungal genomes revealed a vast and unexplored biosynthetic potential in the form of transcriptionally silent secondary metabolite biosynthetic gene clusters (BGCs). Various strategies have been carried out to explore and mine this untapped source of bioactive molecules, and with the advent of synthetic biology, novel applications, and tools have been developed for filamentous fungi. Here we summarize approaches aiming for the expression of endogenous or exogenous natural product BGCs, including synthetic transcription factors, assembly of artificial transcription units, gene cluster refactoring, fungal shuttle vectors, and platform strains

Toll-like receptor 4 and NOD2/CARD15 mutations in Hungarian patients with Crohn's disease: Phenotype-genotype correlations

AIM: To determine common NOD2/CARD15 mutations and TLR4 D299G polymorphism in Hungarian patients with CD. METHODS: A total of 527 unrelated patients with CD (male/female: 265/262, age: 37.1 (SD 7.6) years) and 200 healthy subjects were included. DNA was screened for possible NOD2/CARD15 mutations by denaturing high-performance liquid chromatography (confirmed by direct sequencing). TLR4 D299G was tested by PCR-RFLP. RESULTS: NOD2/CARD15 mutations were found in 185 patients (35.1%) and in 33 controls (16.5%, P< 0.0001). SNP8/R702W (10.8% vs 6%, P = 0.02), SNP13/3020insC (19.4% vs 5%, P< 0.0001) and exon4 R703C (2.1% vs 0%, P = 0.02) mutations were more frequent in CD, while the frequency of SNP12/G908R was not increased. The frequency of TLR4 D299G was not different (CD: 9.9% vs controls: 12.0%). Variant NOD2/CARD15 allele was associated with an increased risk for CD (OR(het) = 1.71, 95% CI = 1.12-2.6, P = 0.0001, OR(two-risk alleles) = 25.2, 95% CI = 4.37-, P< 0.0001), early disease onset (carrier: 26.4 years vs non-carrier: 29.8 years, P = 0.0006), ileal disease (81.9% vs 69.5%, OR = 1.99, 95% CI = 1.29-3.08, P = 0.02, presence of NOD2/CARD15 and TLR4: 86.7% vs 64.8%), stricturing behavior (OR = 1.69, 95% CI = 1.13-2.55, P = 0.026) and increased need for resection (OR= 1.71, 95% CI: 1.13-2.62, P = 0.01), but not with duration, extraintestinal manifestations, familial disease or smoking. TLR4 exhibited a modifier effect: age of onset in wt/TLR4 D299G carriers: 27.4 years vs NOD2mut/TLR D299G: 23 years (P = 0.06), in NOD2mut/wt: 26.7 years. CONCLUSION: These results confirm that variant NOD2/CARD15 (R702W, R703C and 3020insC) alleles are associated with earlier disease onset, ileal disease, stricturing disease behavior in Hungarian CD patients. In contrast, although the frequency of TLR4 D299G polymorphism was not different from controls, NOD2/TLR4 mutation carriers tended to present at earlier age. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved