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Trichoderma reesei Contains a Biosynthetic Gene Cluster That Encodes the Antifungal Agent Ilicicolin H.

ABSTRACT: The trili biosynthetic gene cluster (BGC) from the well-studied organism Trichoderma reesei was studied by heterologous expression in the fungal host Aspergillus oryzae. Coexpression of triliA and triliB produces two new acyl tetramic acids. Addition of the ring-expanding cytochrome P450 encoded by triliC then yields a known pyridone intermediate to ilicicolin H and a new chain-truncated shunt metabolite. Finally, addition of the intramolecular Diels-Alderase encoded by triliD affords a mixture of 8-epi ilicicolin H and ilicicolin H itself, showing that the T. reesei trili BGC encodes biosynthesis of this potent antifungal agent. Unexpected A. oryzae shunt pathways are responsible for the production of the new compounds, emphasising the role of fungal hosts in catalysing diversification reactions.

SUBMITTER: Shenouda ML

PROVIDER: S-EPMC8705728 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Trichoderma reesei Contains a Biosynthetic Gene Cluster That Encodes the Antifungal Agent Ilicicolin H.

Shenouda Mary L ML Ambilika Maria M Cox Russell J RJ

Journal of fungi (Basel, Switzerland) 20211201 12

The trili biosynthetic gene cluster (BGC) from the well-studied organism Trichoderma reesei was studied by heterologous expression in the fungal host Aspergillus oryzae. Coexpression of triliA and triliB produces two new acyl tetramic acids. Addition of the ring-expanding cytochrome P450 encoded by triliC then yields a known pyridone intermediate to ilicicolin H and a new chain-truncated shunt metabolite. Finally, addition of the intramolecular Diels-Ald ...[more]

PMID: 34947016

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Project description:BackgroundGiven the global rise in antimicrobial resistance, the discovery of novel antimicrobial agents and production processes thereof are of utmost importance. To this end we have activated the gene cluster encoding for the biosynthesis of the potent antifungal compound ilicicolin H in the fungus Trichoderma reesei. While the biosynthetic gene cluster (BGC) is silent under standard cultivation conditions, we achieved BGC activation by genetically overexpressing the transcription factor TriliR.ResultsSuccessful activation was confirmed by RT-qPCR, proteomic and metabolomic analyses. Metabolomic profiling upon BGC expression revealed high-yield production of ilicicolin H. To elucidate the enzymatically highly diverse functionality of this BGC, we employed a combination of overexpression and deletions of individual genes in the BGC. While we hardly observed any of the previously reported side- or shunt products associated with heterologous ilicicolin H expression, we discovered that Trichoderma reesei produces a novel member of the ilicicolin family using a metabolomic molecular networking approach. This new compound, ilicicolin K, is expressed in substantial amounts in the genetically engineered Trichoderma reesei. Ilicicolin K differs from ilicicolin H in its structure by a second hydroxylation of the tyrosine derived phenol and an additional ring formed by an intramolecular ether bridge of the hydroxyl group at the pyridone towards the tyrosine moiety of the molecule. Bioactivity tests of ilicicolin K revealed a strong antifungal activity against Saccharomyces cerevisiae and a moderate activity against the human pathogen Candida auris, an emerging multi-drug resistant fungus.ConclusionsBy activating a silent BGC in T. reesei, we obtained a high-yielding strain for the production of the antifungal compounds ilicicolin H and the novel ilicicolin K. These two compounds share some structural properties and are thus highly likely to act on the fungal cytochrome bc1 complex, a component of the mitochondrial repository chain. However, they possess different bioactive properties, which might suggest that ilicicolin K may overcome certain limitations of ilicicolin H.

Project description:Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2',3'-dihydrosorbicillin to sorbicillinol and 2',3'-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2',3'-dihydrosorbicillin and 2',3'-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.

Project description:BackgroundThe ascomycete Trichoderma reesei is one of the most efficient industrial producers of cellulase. Gene targeting by homologous recombination is a key technique for improving strains and constructing mutants. In T. reesei, tku70 (homologous to human KU70) was deleted to block non-homologous end-joining, which led to 95% of transformants exhibiting homologous recombination.ResultsTwo genes located in close proximity to tku70 were identified: the ferrochelatase gene hem8 (tre78582, homologous to Aspergillus niger hemH and Cryptococcus neoformans HEM15) and a putative DNA methylation modulator-2 gene dmm2 (tre108087, homologous to Neurospora crassa dmm-2). Genome-wide surveys of 324 sequenced fungal genomes revealed that the homologues of the three genes of interest are encoded in tandem in most Sordariomycetes. The expression of this three-gene cluster is regulated by blue light. The roles of these three genes were analyzed via deletion and complementation tests. The gene hem8 was originally described as a novel and highly distinct auxotrophic marker in T. reesei and we found that the product protein, HEM8, catalyzes the final step in heme biosynthesis from highly photoreactive porphyrins. The lethal phenotype of the hem8 deletion could be overcome by hematin supplementation. We also studied the functions of tku70 and dmm2 in DNA repair using mutagen sensitivity experiments. We found that the Δtku70 strain showed increased sensitivity to bleomycin, which induces DNA double-strand breaks, and that the Δdmm2 strain was sensitive to bleomycin, camptothecin (an inhibitor of type I topoisomerases), and hydroxyurea (a deoxynucleotide synthesis inhibitor). The double-mutant Δtku70&dmm2 showed higher sensitivity to hydroxyurea, camptothecin, and bleomycin than either of the single mutants. Knockout of dmm2 significantly increased cellulase production.ConclusionsOur data show, for the first time, that ferrochelatase encoded by hem8 catalyzes the final step in heme biosynthesis from highly photoreactive porphyrins and that dmm2 encodes a putative DNA methylation modulator-2 protein related to DNA repair and cellulase expression in T. reesei. Our data provide important insights into the roles of this three-gene cluster in T. reesei and other Sordariomycetes and show that the DNA methylation modulator DMM2 affects cellulase gene expression in T. reesei.

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Trichoderma reesei Contains a Biosynthetic Gene Cluster That Encodes the Antifungal Agent Ilicicolin H.

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<i>Trichoderma reesei</i> Contains a Biosynthetic Gene Cluster That Encodes the Antifungal Agent Ilicicolin H.

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