The expanding CRISPR toolbox for natural product discovery and engineering in filamentous fungi.

Covering: up to May 2022Fungal genetics has transformed natural product research by enabling the elucidation of cryptic metabolites and biosynthetic steps. The enhanced capability to add, subtract, modulate, and rewrite genes via CRISPR/Cas technologies has opened up avenues for the manipulation of biosynthetic gene clusters across diverse filamentous fungi. This review discusses the innovative and diverse strategies for fungal natural product discovery and engineering made possible by CRISPR/Cas-based tools. We also provide a guide into multiple angles of CRISPR/Cas experiment design, and discuss current gaps in genetic tool development for filamentous fungi and the promising opportunities for natural product research.

Heterologous Expression of Fungal Biosynthetic Pathways in Aspergillus nidulans Using Episomal Vectors.

Filamentous fungi produce a wide diversity 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.

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.

Characterisation and heterologous biosynthesis of burnettiene A, a new polyene-decalin polyketide from Aspergillus burnettii.

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-1).

Polyketides produced by the entomopathogenic fungus Metarhizium anisopliae induce Candida albicans growth.

Metarhizium anisopliae is an important entomopathogenic species and model for arthropod-fungus interaction studies. This fungus harbors a diverse arsenal of unexplored secondary metabolite biosynthetic gene clusters, which are suggested to perform diverse roles during host interaction and soil subsistence as a saprophytic species. Here we explored an unusual carnitine acyltransferase domain-containing highly reducing polyketide synthase found in the genome of M. anisopliae. Employing heterologous expression in Aspergillus nidulans, two new polyketides were obtained, named BAA and BAB, as well as one known polyketide [(2Z,4E,6E)-octa-2,4,6-trienedioic acid]. Intra-hemocoel injection of the most abundant compound (BAA) in the model-arthropod Galleria mellonella larvae did not induce mortality or noticeable alterations, suggesting that this compound may not harbor insecticidal activity. Also, the potential role of such molecules in polymicrobial interactions was evaluated. Determination of minimum inhibitory concentration assays using distinct fungal species revealed that BAA and BAB did not alter Cryptococcus neoformans growth, while BAA exhibited weak antifungal activity against Saccharomyces cerevisiae. Unexpectedly, these compounds increased Candida albicans growth compared to control conditions. Furthermore, BAA can mitigate the fungicidal effects of fluconazole over C. albicans. Although the exact role of these compounds on the M. anisopliae life cycle is elusive, the described results add up to the complexity of secondary metabolites produced by Metarhizium spp. Moreover, up to our knowledge, these are the first polyketides isolated from filamentous fungi that can boost the growth of another fungal species.

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 BGCs 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, multigene 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.

Phylogenetic distribution and evolutionary pattern of an α-proteobacterial small RNA gene that controls polyhydroxybutyrate accumulation in Sinorhizobium meliloti.

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 diverged 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.