ORCID Profile
0000-0002-7107-2824
Current Organisations
University of Western Australia
,
University of Cambridge
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Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1OB01766G
Abstract: Burnettiene A is a novel cytotoxic tridecaketide decalin polyketide from Aspergillus burnettii . Its biosynthesis was elucidated by heterologous expression in fungi.
Publisher: American Chemical Society (ACS)
Date: 11-06-2020
Publisher: Elsevier BV
Date: 07-2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D2NP00055E
Abstract: This review provides an overview of CRISPR/Cas-based strategies for biosynthetic gene cluster engineering in filamentous fungi.
Publisher: American Chemical Society (ACS)
Date: 16-02-2022
DOI: 10.1021/ACSSYNBIO.1C00458
Abstract: 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
Publisher: Cambridge University Press (CUP)
Date: 07-09-2021
DOI: 10.33774/CHEMRXIV-2021-P016T
Abstract: Chemical exploration of the recently described Australian fungus, Aspergillus burnettii, uncovered a new metabolite, burnettiene A. Here, we characterise the structure of burnettiene A as a polyene-decalin polyketide. Bioinformatic analysis of the genome of A. burnettii identified a putative biosynthetic gene cluster for burnettiene A (bue), consisting of eight genes and sharing similarity to the fusarielin gene cluster. Introduction of the reassembled bue gene cluster into Aspergillus nidulans for heterologous expression resulted in the production of burnettiene A under native promoters. Omission of bueE encoding a cytochrome P450 led to the production of preburnettiene A, confirming that BueE is responsible for catalysing the regiospecific multi-oxidation of terminal methyl groups to carboxylic acids. Similarly, bueF was shown to encode an ester-forming methyltransferase, with its omission resulting in the production of the tricarboxylic acid, preburnettiene B. Introduction of an additional copy of the transcription factor bueR under the regulation of the gpdA promoter significantly improved the heterologous production of the burnettienes. Burnettiene A displayed strong in vitro cytotoxicity against mouse myeloma NS-1 cells (MIC 0.8 µg/mL).
Publisher: Springer US
Date: 2022
DOI: 10.1007/978-1-0716-2273-5_5
Abstract: Filamentous fungi produce a wide ersity of secondary metabolites, whose biosynthesis is encoded in biosynthetic gene clusters (BGCs). As novel BGCs are often found in fungal species that are genetically intractable or difficult to cultivate, heterologous expression is increasingly being used for compound discovery. In addition, heterologous expression is a useful strategy to elucidate the function of the genes within a BGC and shed light on their enzymatic mechanisms. Here, we describe a method for BGC elucidation using multi-marker AMA1-based pYFAC vectors for episomal expression in the fungal host Aspergillus nidulans. The pYFAC vectors have the advantage of high transformation efficiency and support high compound production. In addition, different pathway intermediates can be easily evaluated by testing different vector combinations. This protocol encompasses different AMA1-based strategies for BGC expression such as cloning of a BGC native sequence, promoter exchange or transcription factor overexpression. We also describe procedures for A. nidulans protoplasting, transformation, and small-scale culture analysis of strains containing AMA1 vectors.
Publisher: Cold Spring Harbor Laboratory
Date: 14-01-2020
DOI: 10.1101/2020.01.12.903286
Abstract: 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 dLbCas12a D156R -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.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.YMPEV.2016.03.026
Abstract: It has become clear that sRNAs play relevant regulatory functions in bacteria. However, a comprehensive understanding of their biological roles considering evolutionary aspects has not been achieved for most of them. Thus, we have characterized the evolutionary and phylogenetic aspects of the Sinorhizobium meliloti mmgR gene encoding the small RNA MmgR, which has been recently reported to be involved in the regulation of polyhydroxybutyrate accumulation in this bacterium. We constructed a covariance model from a multiple sequence and structure alignment of mmgR close homologs that allowed us to extend the search and to detect further remote homologs of the sRNA gene. From our results, mmgR seemed to evolve from a common ancestor of the α-proteobacteria that erged from the order of Rickettsiales. We have found mmgR homologs in most current species of α-proteobacteria, with a few exceptions in which genomic reduction events or gene rearrangements seem to explain its absence. Furthermore, a strong microsyntenic relationship was found between a large set of mmgR homologs and homologs of a gene encoding a putative N-formyl glutamate amidohydrolase (NFGAH) that allowed us to trace back the evolutionary path of this group of mmgR orthologs. Among them, structure and sequence traits have been completely conserved throughout evolution, namely a Rho-independent terminator and a 10-mer (5'-UUUCCUCCCU-3') that is predicted to remain in a single-stranded region of the sRNA. We thus propose the definition of the new family of α-proteobacterial sRNAs αr8, as well as the subfamily αr8s1 which encompass S. meliloti mmgR orthologs physically linked with the downstream open reading frame encoding a putative NFGAH. So far, mmgR is the trans-encoded small RNA with the widest phylogenetic distribution of well recognized orthologs among α-proteobacteria. Expression of the expected MmgR transcript in rhizobiales other than S. meliloti (Sinorhizobium fredii, Rhizobium leguminosarum and Rhizobium etli) was confirmed by Northern blot. These findings will contribute to the understanding of the biological role(s) of mmgR in the α-proteobacteria.
Publisher: Cold Spring Harbor Laboratory
Date: 20-08-2021
DOI: 10.1101/2021.08.20.457072
Abstract: 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.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Indra Roux.