Identification of a Novel Biosynthetic Gene Cluster in Aspergillus niger Using Comparative Genomics

Abstract

Previously, DNA microarrays analysis showed that, in co-culture with Bacillus subtilis, a biosynthetic gene cluster anchored with a nonribosomal peptides synthetase of Aspergillus niger is downregulated. Based on phylogenetic and synteny analyses, we show here that this gene cluster, NRRL3_00036-NRRL3_00042, comprises genes predicted to encode a nonribosomal peptides synthetase, a FAD-binding domain-containing protein, an uncharacterized protein, a transporter, a cytochrome P450 protein, a NAD(P)-binding domain-containing protein and a transcription factor. We overexpressed the in-cluster transcription factor gene NRRL3_00042. The overexpression strain, NRRL3_00042OE, displays reduced growth rate and production of a yellow pigment, which by mass spectrometric analysis corresponds to two compounds with masses of 409.1384 and 425.1331. We deleted the gene encoding the NRRL3_00036 nonribosomal peptides synthetase in the NRRL3_00042OE strain. The resulting strain reverted to the wild-type phenotype. These results suggest that the biosynthetic gene cluster anchored by the NRRL3_00036 nonribosomal peptides synthetase gene is regulated by the in-cluster transcriptional regulator gene NRRL3_00042, and that it is involved in the production of two previously uncharacterized compounds.

Improving candidate Biosynthetic Gene Clusters in fungi through reinforcement learning

“…Testing: The decisions taken by the reinforcement learning agent are evaluated on candidate BGCs obtained for the Aspergillus niger NRRL3 genomic sequence (publicly available at https://gb.fungalgenomics.ca/portal ) by three tools: TOUCAN ( Almeida et al , 2020 ), fungiSMASH ( Blin et al , 2021 ) and DeepBGC ( Hannigan et al , 2019 ). Aspergillus niger is an organism of interest given its ubiquitous presence, and its importance for industrial processes and biotechnology, which makes it a relevant species in the study of BGC discovery ( Aguilar-Pontes et al , 2018 ; de Vries et al , 2017 ; Evdokias et al , 2021 ). To obtain test candidate BGCs from A.niger amino acid sequence, we extracted sequentially sliding windows of fixed 10 000 amino acid length with a 30% window overlap [see Almeida et al (2020) for details].…”

“…They may also present other components, such as cluster-specific transcription factors, transporters and hypothetical proteins ( Keller, 2015 ). Fungal BGCs are known to vary considerably in composition (similar clusters with different components), and location (cluster regions overlapping or spanning multiple chromosomes) even among closely related species ( Evdokias et al , 2021 ; Keller, 2019 ; Kjærbølling et al , 2020 ).…”

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

“…112 CRISPR/ Cas9 integration was also used to overexpress a cluster-specic transcription factor by incorporating an additional copy of the coding sequence downstream of a native strong promoter located in the genome. 113 CRISPR/Cas gene editing can also be used to replace the sequence within a gene, enabling the study of mutations in the native context of a gene (Fig. 4C).…”

“…112 CRISPR/Cas9 integration was also used to overexpress a cluster-specific transcription factor by incorporating an additional copy of the coding sequence downstream of a native strong promoter located in the genome. 113…”

Systems Biology in Fungal Research

“…The modeling of metabolism and metabolic networks is a powerful strategy to build upon our fundamental knowledge of fungal pathogens and to explore the role of metabolite production within the biological system. For Aspergillus niger , altered metabolite production was observed upon regulation of a novel biosynthetic gene cluster associated with transcriptional regulation, which was identified following comparative genomics analyses [ 12 ]. The anchoring of the biosynthetic gene cluster by a nonribosomal peptide synthetase is notable due to bioactive roles (e.g., toxins, siderophores, pigments, antibiotic, cytostatics, immunosuppressants, or anticancer agents).…”