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

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

Author Correction:CRISPR-based transcriptional activation tool for silent genes in filamentous fungi (Scientific Reports, (2021), 11, 1, (1118), 10.1038/s41598-020-80864-3)

The Supplementary Information published with this Article contained errors. In Note S2, the text formatting including green italics, red bold, yellow underline, purple text and blue underline was omitted. The original Supplementary Information file is provided below. These errors have now been corrected in the Supplementary Information file that accompanies the original Article

A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to ten glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalyzing the final step in patulin biosynthesis. The A. niger strain expressing ten copies of the patE::6xHis expression cassette produced about 70 μg/mL PatE protein in the culture medium with a purity just under 90%.</p

Gyógyszerhatóanyagok szennyezőinek izolálása kromatográfiás műveletekkel

Dolgozatom célja, hogy a Mupirocin D szennyező kinyerésére alkalmas preparatív kromatográfiás módszert fejlesszek ki. Ismertetem a kromatográfia elméleti hátterét és a Mupirocin hatóanyagra vonatkozó fontosabb információkat.BSc/BABiomérnökg

A genetic tool for transcriptional regulation in Penicillium chrysogenum

The filamentous fungus Penicillium chrysogenum is the major producer of the beta-lactam antibiotic penicillin. In addition to the penicillin biosynthesis cluster, the genome of P. chrysogenum contains a large number of uncharacterized secondary metabolite gene clusters. However, many of these clusters are not expressed under laboratory conditions. This thesis summarizes the steps of developing a novel, orthogonal, synthetic transcriptional regulation system in P. chrysogenum. The system utilizes transcriptional regulatory genes to express the gene of interests with a strong but controllable manner. The build system can be used for heterologous expression or silenced genes, even whole gene clusters can be activated with it.MSc/MABiomérnöki MScg