Biology subjects

Chooi, Y.-H.

Publications and source records attributed to Chooi, Y.-H..

9 recordsLinked to original sources

bioRxiv2024

Next generation AMA1-based plasmids for enhanced heterologous expression in filamentous fungi

Episomal AMA1-based plasmids are increasingly used for expressing biosynthetic pathways and CRISPR/Cas systems in filamentous fungi cell factories due to their high transformation efficiency and multicopy nature. However, gene expression from AMA1 plasmids has been observed to be highly heterogeneous in growing mycelia. To overcome this limitation, here we developed next-generation AMA1-based plasmids that ensure homogenous and strong expression. We achieved this by evaluating various degradation tags fused to the auxotrophic marker gene on the AMA1 plasmid, which introduces a more stringent selection pressure throughout multicellular fungal growth. With these improved plasmids, we observed in Aspergillus nidulans a 5-fold increase in the expression of a fluorescent reporter, a doubling in the efficiency of a CRISPRa system for genome mining, and a up to a 10-fold increase in the production of heterologous natural product metabolites. This strategy has the potential to be applied to diverse filamentous fungi.

synthetic biology↗

bioRxiv2023

The Velvet transcription factor PnVeA regulates necrotrophic effectors and secondary metabolism in the wheat pathogen Parastagonospora nodorum

The fungus Parastagonospora nodorum causes septoria nodorum blotch on wheat. The role of the fungal Velvet-family transcription factor VeA in P. nodorum development and virulence was investigated here. Deletion of the P. nodorum VeA ortholog, PnVeA, resulted in growth abnormalities including pigmentation, abolished asexual sporulation and highly reduced virulence on wheat. Comparative RNA-Seq and RT-PCR analyses revealed that the deletion of PnVeA also decoupled the expression of major necrotrophic effector genes. In addition, the deletion of PnVeA resulted in an up-regulation of four predicted secondary metabolite (SM) gene clusters. Using liquid-chromatography mass-spectrometry, it was observed that one of the SM gene clusters led to an accumulation of the mycotoxin alternariol. PnVeA is essential for asexual sporulation, full virulence, secondary metabolism and necrotrophic effector regulation.

microbiology↗

bioRxiv2023

CAGECAT: The CompArative GEne Cluster Analysis Toolbox for rapid search and visualisation of homologous gene clusters

BackgroundCo-localized sets of genes that encode specialized functions are common across microbial genomes and occur in genomes of larger eukaryotes as well. Important examples include Biosynthetic Gene Clusters (BGCs) that produce specialized metabolites with medicinal, agricultural, and industrial value (e.g. antimicrobials). Comparative analysis of BGCs can aid in the discovery of novel metabolites by highlighting distribution and identifying variants in public genomes. Unfortunately, gene-cluster-level homology detection remains inaccessible, time-consuming and difficult to interpret. ResultsThe comparative gene cluster analysis toolbox (CAGECAT) is a rapid and user-friendly platform to mitigate difficulties in comparative analysis of whole gene clusters. The software provides homology searches and downstream analyses without the need for command-line or programming expertise. By leveraging remote BLAST databases, which always provide up-to-date results, CAGECAT can yield relevant matches that aid in the comparison, taxonomic distribution, or evolution of an unknown query. The service is extensible and interoperable and implements the cblaster and clinker pipelines to perform homology search, filtering, gene neighbourhood estimation, and dynamic visualisation of resulting variant BGCs. With the visualisation module, publication-quality figures can be customized directly from a web-browser, which greatly accelerates their interpretation via informative overlays to identify conserved genes in a BGC query. ConclusionOverall, CAGECAT is an extensible software that can be interfaced via a standard web-browser for whole region homology searches and comparison on continually updated genomes from NCBI. The public web server and installable docker image are open source and freely available without registration at: https://cagecat.bioinformatics.nl

bioinformatics↗

bioRxiv2021

Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

Building strains of filamentous fungi for stable long-term heterologous expression of large biosynthetic pathways is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase-mediated cassette exchange. We built A. nidulans strains harboring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21 kb DNA fragment in a single step. We further evaluated the integration at two loci by analyzing the expression of a fluorescent reporter and the production of a heterologous polyketide metabolite. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterized aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

synthetic biology↗

bioRxiv2020

CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGC could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans. Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multi-gene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12aD156R-VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2019

The substrate-dependent stereoselectivity of the multicopper oxidase (MCO)-catalyzed oxidative coupling in the biosynthesis of the bisnaphthopyrone viriditoxin

VdtB, the multiple-copper oxidase (MCO) from the bisnaphthopyrone (M)-viriditoxin biosynthetic pathway in Paecilomyces variotii, was shown to catalyze regioselective 6,6'-coupling of semi-viriditoxin (1). The stereoselectivity of the oxidative coupling reaction for the production of the atropisomer (M)-viriditoxin, however, was controlled by VdtD, a non-catalytic dirigent protein from the pathway. In this work, VdtB either alone or together with VdtD were investigated for its stereoselective control upon coupling of other monomeric naphthopyrone derivatives from the pathway with different minor structural variations in terms of presence/absence of O-methylation at C7-position and C3-C4{Delta} 2 double bond on the pyrone ring, and the different side-chain modifications. We showed that VdtB could favour either M- or P-form coupling in a substrate-dependent manner. For some substrates, VdtB could catalyze oxidative coupling in an enantiomerically selective manner. The efficiency of the VdtD in exerting stereoselective control of the oxidative coupling reaction also varies between substrates. The results point to a model whereby VdtB and VdtD form a VdtB-ligand-VdtD complex in which the stereochemical outcome of the coupling reaction depends on how the substrate interacts with both proteins, based on the substrate structure. Our findings contributed to a more comprehensive understanding of dirigent protein-mediated MCO-catalyzed stereoselective oxidative coupling reactions in fungi.

biochemistry↗

bioRxiv2019

Volatile molecules secreted by the wheat pathogen Parastagonospora nodorum are involved in development and phytotoxicity

Septoria nodorum blotch is a major disease of wheat caused by the fungus Parastagonospora nodorum. Recent studies have demonstrated that secondary metabolites, including polyketides and non-ribosomal peptides, produced by the pathogen play important roles in disease and development. However, there is currently no knowledge on the composition or biological activity of the volatile organic compounds (VOCs) secreted by P. nodorum. To address this, we undertook a series of growth and phytotoxicity assays and demonstrated that P. nodorum VOCs inhibited bacterial growth, were phytotoxic and suppressed self-growth. Mass spectrometry analysis revealed that 3-methyl-1-butanol, 2-methyl-1-butanol, 2-methyl-1-propanol and 2-phenylethanol were dominant in the VOC mixture and phenotypic assays using these short chain alcohols confirmed that they were phytotoxic. Further analysis of the VOCs also identified the presence of multiple sesquiterpenes of which four were identified via mass spectrometry and nuclear magnetic resonance as {beta}-elemene, -cyperone, eudesma-4,11-diene and acora-4,9-diene. Subsequent reverse genetics studies were able to link these molecules to corresponding sesquiterpene synthases in the P. nodorum genome. However, despite extensive testing, these molecules were not involved in either of the growth inhibition or phytotoxicity phenotypes previously observed. Plant assays using mutants of the pathogen lacking the synthetic genes revealed that the identified sesquiterpenes were not required for disease formation on wheat leaves. Collectively, these data have significantly extended our knowledge of the VOCs in fungi and provided the basis for further dissecting the roles of sesquiterpenes in plant disease.

microbiology↗

bioRxiv2019

The fungal gene cluster for biosynthesis of the antibacterial agent viriditoxin

BackgroundViriditoxin is one of the classical secondary metabolites produced by fungi and that has antibacterial and other activities; however, the mechanism of its biosynthesis has remained unknown.\n\nResultsHere, a gene cluster responsible for its synthesis was identified, using bioinformatic approaches from two species that produce viriditoxin and then through gene disruption and metabolite profiling. All eight genes in the cluster in Paecilomyces variotii were mutated, revealing their roles in the synthesis of this molecule and establishing its biosynthetic pathway which includes an interesting Baeyer-Villiger monooxygenase catalyzed reaction. Additionally, a candidate catalytically-inactive hydrolase was identified as being required for the stereoselective biosynthesis of (M)-viriditoxin. The localization of two proteins were assessed by fusing these proteins to green fluorescent protein, revealing that at least two intracellular structures are involved in the compartmentalization of the synthesis steps of this metabolite.\n\nConclusionsThe full pathway for synthesis of viriditoxin was established by a combination of genomics, bioinformatics, gene disruption and chemical analysis processes. Hence, this work reveals the basis for the synthesis of an understudied class of fungal secondary metabolites and provides a new model species for understanding the synthesis of biaryl compounds with a chiral axis.

microbiology↗

bioRxiv2018

Acquisition and loss of secondary metabolite clusters shaped the evolutionary path of three recently emerged phytopathogens of wheat

O_LIWhite grain disorder is a recently emerged wheat disease in Australia, caused by three Botryosphaeriaceae spp.; Eutiarosporella darliae, E. pseudodarliae, and E. tritici-australis. The disease cycle of these pathogens and the molecular basis of their interaction with wheat are poorly understood. To address this, we undertook a comparative genomics approach to identify potential pathogenicity factors.\nC_LIO_LISubsequent genome analysis revealed that each of the white grain disorder species harbour modular polyketide synthase genes. To our knowledge, this is the first report of fungi harbouring such genes. Further comparative analysis using the modular polyketide synthase genes discovered their presence in the closely related Macrophomina phaseolina. Phylogenetic analysis implicates horizontal acquisition of these genes from a bacterial or a protist species.\nC_LIO_LIBoth E. darliae and E. pseudodarliae possess a secondary metabolite cluster with multiple polyketide/non-ribosomal peptide synthase genes (Hybrid-1, -2, and -3). In contrast, only remnant and partial genes homologous to this cluster were identified at a syntenic locus in E. tritici-australis suggesting loss of this cluster. Homologues of Hybrid-2 in other fungi have been proposed to facilitate disease induction in woody plants. Subsequent assays confirmed that E. darliae and E. pseudodarliae were both pathogenic on woody plant hosts, but E. tritici-australis was not, implicating woody plants as potential host reservoirs for the fungi. We hypothesise that loss of the cluster in E. tritici-australis represents a committed lifestyle jump to grasses.\nC_LIO_LICombined, our observations relating to the secondary metabolite potential of the WGD Eutiarosporella spp. have contributed novel data to the field by expanding the range of known fungal secondary metabolite genes, and helped develop our understanding of the lifestyle and potential host-range of a recently emerged pathogen of wheat.\nC_LI

plant biology↗