Filamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl "tail," and a citrate-derived "head." We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data. We overexpressed the transcription regulator gene akcR and obtained from one liter of culture filtrate 8.5 grams of extract, which are represented by seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised 94.1% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed that in A. oryzae and A. flavus an in-cluster gene displays sequence similarity to cis-aconitate decarboxylase, the orthologue of which in A. niger, NRRL3_00504, is located on a different chromosome. Overexpression of the A. niger NRRL3_00504 and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids, suggesting that NRRL3_00504 encodes an enzyme with hexyl aconitate decarboxylase activity. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.

Fungal secondary metabolites (SMs) are an important source of numerous bioactive compounds largely applied in the pharmaceutical industry, as in the production of antibiotics and anticancer medications. The discovery of novel fungal SMs can potentially benefit human health. Identifying biosynthetic gene clusters (BGCs) involved in the biosynthesis of SMs can be a costly and complex task, especially due to the genomic diversity of fungal BGCs. Previous studies on fungal BGC discovery present limited scope and can restrict the discovery of new BGCs. In this work, we introduce TOUCAN, a supervised learning framework for fungal BGC discovery. Unlike previous methods, TOUCAN is capable of predicting BGCs on amino acid sequences, facilitating its use on newly sequenced and not yet curated data. It relies on three main pillars: rigorous selection of datasets by BGC experts; combination of functional, evolutionary and compositional features coupled with outperforming classifiers; and robust post-processing methods. TOUCAN best-performing model yields 0.982 F-measure on BGC regions in the Aspergillus niger genome. Overall results show that TOUCAN outperforms previous approaches. TOUCAN focuses on fungal BGCs but can be easily adapted to expand its scope to process other species or include new features.

Filamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl "tail" and a citratederived "head". We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data.We overexpressed the transcription regulator gene akcR and obtained from a litre of culture filtrate 8.5 grams of extract containing seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised ~ 95% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed an in-cluster cis-aconitate decarboxylase gene (cadA) in A. oryzae and A. flavus, which in A. niger is located on a different chromosome. Overexpression of the A. niger cadA and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.

Species of the genus Populus commonly known as poplars are one of the most widely used groups of forest trees in North America and Europe, and play a significant ecological role as a pioneer species in boreal forests, and as a dominant species in the riparian forests that serve as wildlife habitats and watersheds. Natural and artificial hybrids of poplars are being extensively used in commercial plantations. However, many hybrid poplar trees are susceptible to Sphaerulina musiva, the pathogenic fungus that causes leaf spots and stem cankers and limits the utility of hybrid poplars as a plantation trees. We isolated an endophytic bacterial strain Bacillus velezensis EB14 from a Populus hybrid which showed a strong antifungal activity against S. musiva. Through mass spectrometric analyses of co-cultured B. velezensis EB14 and S. musiva, we identified five cyclic lipopeptides produced by B. velezensis EB14 – Iturin A1, Iturin A2, Iturin A9, Subtulene A and Fengycin. In addition, B. velezensis EB14 produced four major unidentified compounds in co-cultivation with S. musiva. The cyclopeptide production by B. velezensis EB14 was more pronounced (20-1000 fold) in the co-cultured plates due to elicitation by S. musiva. We also discovered that the native endophytic B. velezensis EB14 strain exhibited different levels of interactions against the endophytic fungal microbiomes of Populus sp. Overall, our results indicate B. velezensis EB14 strain as a promising biocontrol agent that could be used against stem canker and leaf spot diseases caused by S. musiva in Poplar plantations.