Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9

The full potential of fungal secondary metabolism has until recently been impeded by the lack of universal genetic tools for most species. However, the emergence of several CRISPR-Cas9-based genome editing systems adapted for several genera of filamentous fungi have now opened the doors for future efforts in discovery of novel natural products and elucidation and engineering of their biosynthetic pathways in fungi where no genetic tools are in place. So far, most studies have focused on demonstrating the performance of CRISPR-Cas9 in various fungal model species, and recently we presented a versatile CRISPR-Cas9 system that can be successfully applied in several diverse Aspergillus species. Here we take it one step further and show that our system can be used also in a phylogenetically distinct and largely unexplored species from the genus of Talaromyces. Specifically, we exploit CRISPR-Cas9-based genome editing to identify a new gene in T. atroroseus responsible for production of polyketide-nonribosomal peptide hybrid products, hence, linking fungal secondary metabolites to their genetic origin in a species where no genetic engineering has previously been performed

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry

Diversity in Secondary Metabolites Including Mycotoxins from Strains of <i>Aspergillus </i>Section <i>Nigri </i>Isolated from Raw Cashew Nuts from Benin, West Africa

<p>In a previous study, raw cashew kernels were assayed for the fungal contamination focusin on strains belonging to the genus Aspergillus and on aflatoxins producers. These sample showed high contamination with Aspergillus section Nigri species and absence o aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, th species of A. section Nigri may produce and thus threaten to contaminate the raw cashe kernels, 150 strains were isolated from cashew samples and assayed for their productio of secondary metabolites using liquid chromatography high resolution mass spectrometr (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological an chemical identification: A.Tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonariu (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). Fro these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalen and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxin groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), an secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts Thirty strains of black Aspergilli were randomly sampled for verification of species identit based on sequences of β-Tubulin and calmodulin genes. Among them, 27 isolates wer positive to the primers used and 11 were identified as A. niger, 7 as A.Tubingensis, 6 as A carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names a based on morphology and chemical features. These strains clustered in 5 clades in A. sectio Nigri. Chemical profile clustering also showed also 5 groups confirming the speciespecific metabolites production.</p