24 research outputs found
Functional reconstitution of the Trypacidin Gene Cluster in Aspergillus fumigatus by Advanced Gene Editing
«Золота» пора кооперації в умовах непу
Рецензія на книгу: Голець В.В. Кооперація і неп (20-і рр. XX ст.). - Чернігів: Просвіта, 2006. - 244 с
Synthetic Biology of Fungal Natural Products
Synthetic biology is an ever-expanding field in science, also encompassing the research area of fungal natural product discovery and production. Until now, different aspects of synthetic biology have been covered in fungal natural product studies from the manipulation of different regulatory elements and heterologous expression of biosynthetic pathways to the engineering of different multidomain biosynthetic enzymes such as polyketide synthases or nonribosomal peptide synthetases. The following review will cover some of the exemplary studies of synthetic biology in filamentous fungi showing the capacity of these eukaryotes to be used as model organisms in the field. From the vast array of different natural products produced to the ease for genetic manipulation, filamentous fungi have proven to be an invaluable source for the further development of synthetic biology tools
Synthetic biology tools for bioprospecting of natural products in eukaryotes
Filamentous fungi have the capacity to produce a battery of natural products of often unknown function, synthesized by complex metabolic pathways. Unfortunately, most of these pathways appear silent, many in intractable organisms, and their products consequently unidentified. One basic challenge is the difficulty of expressing a biosynthesis pathway for a complex natural product in a heterologous eukaryotic host. Here, we provide a proof-of concept solution to this challenge and describe how the entire penicillin biosynthesis pathway can be expressed in a heterologous host. The method takes advantage of a combination of improved yeast in vivo cloning technology, generation of polycistronic mRNA for the gene cluster under study, and an amenable and easily manipulated fungal host, i.e., Aspergillus nidulans. We achieve expression from a single promoter of the pathway genes to yield a large polycistronic mRNA by using viral 2A peptide sequences to direct successful cotranslational cleavage of pathway enzymes
Synthetic biology tools for bioprospecting of natural products in eukaryotes
SummaryFilamentous fungi have the capacity to produce a battery of natural products of often unknown function, synthesized by complex metabolic pathways. Unfortunately, most of these pathways appear silent, many in intractable organisms, and their products consequently unidentified. One basic challenge is the difficulty of expressing a biosynthesis pathway for a complex natural product in a heterologous eukaryotic host. Here, we provide a proof-of concept solution to this challenge and describe how the entire penicillin biosynthesis pathway can be expressed in a heterologous host. The method takes advantage of a combination of improved yeast in vivo cloning technology, generation of polycistronic mRNA for the gene cluster under study, and an amenable and easily manipulated fungal host, i.e., Aspergillus nidulans. We achieve expression from a single promoter of the pathway genes to yield a large polycistronic mRNA by using viral 2A peptide sequences to direct successful cotranslational cleavage of pathway enzymes
Microbial communication leading to the activation of silent fungal secondary metabolite gene clusters
Microorganisms form diverse multispecies communities in various ecosystems. The high abundance of fungal and bacterial species in these consortia results in specific communication between the microorganisms. A key role in this communication is played by secondary metabolites (SMs), which are also called natural products. Recently, it was shown that interspecies ‘talk’ between microorganisms represents a physiological trigger to activate silent gene clusters leading to the formation of novel SMs by the involved species. This review focuses on mixed microbial cultivation, mainly between bacteria and fungi, with a special emphasis on the induced formation of fungal SMs in co-cultures. In addition, the role of chromatin remodeling in the induction is examined, and methodical perspectives for the analysis of natural products are presented. As an example for an intermicrobial interaction elucidated at the molecular level, we discuss the specific interaction between the filamentous fungi Aspergillus nidulans and Aspergillus fumigatus with the soil bacterium Streptomyces rapamycinicus, which provides an excellent model system to enlighten molecular concepts behind regulatory mechanisms and will pave the way to a novel avenue of drug discovery through targeted activation of silent SM gene clusters through co-cultivations of microorganisms
Rewiring of the Austinoid Biosynthetic Pathway in Filamentous Fungi
Filamentous
fungi produce numerous high-value natural products
(NPs). The biosynthetic genes for NPs are normally clustered in the
genome. A valuable NP class is represented by the insecticidal austinoids.
We previously determined their biosynthesis in the fungus <i>Aspergillus calidoustus</i>. After further computational analysis
looking into the austinoid gene clusters in two additional distantly
related fungi, <i>Aspergillus nidulans</i> and <i>Penicillium
brasilianum</i>, a rearrangement of the genes was observed that
corresponded to the diverse austinoid derivatives produced by each
strain. By advanced targeted combinatorial engineering using polycistronic
expression of selected genes, we rewired the austinoid pathway in
the fungus <i>A. nidulans</i>, which then produced certain
compounds of interest under industrially favored conditions. This
was possible by exploiting the presence of genes previously thought
to be irrelevant. Our work shows that comparative analysis of genomes
can be used to not only discover new gene clusters but unearth the
hidden potential of known metabolic pathways
Functional Reconstitution of a Fungal Natural Product Gene Cluster by Advanced Genome Editing
Filamentous fungi
produce varieties of natural products even in
a strain dependent manner. However, the genetic basis of chemical
speciation between strains is still widely unknown. One example is
trypacidin, a natural product of the opportunistic human pathogen <i>Aspergillus fumigatus</i>, which is not produced among different
isolates. Combining computational analysis with targeted gene editing,
we could link a single nucleotide insertion in the polyketide synthase
of the trypacidin biosynthetic pathway and reconstitute its production
in a nonproducing strain. Thus, we present a CRISPR/Cas9-based tool
for advanced molecular genetic studies in filamentous fungi, exploiting
selectable markers separated from the edited locus
SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus.
F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus