CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932

Abstract

Lophiotrema is a genus of ascomycetous fungi within the family Lophiotremataceae. Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. Lophiotrema sp. F6932 was isolated from white mangrove (Avicennia officinalis) in Pulau Ubin Island, Singapore. Crude extracts from the fungus exhibited strong antibacterial activity, and bioassay-guided isolation and structure elucidation of bioactive constituents led to the isolation of palmarumycin C8 and a new analog palmarumycin CP30. Whole-genome sequencing analysis resulted in the identification of a putative type 1 iterative PKS (iPKS) predicated to be involved in the biosynthesis of palmarumycins. To verify the involvement of palmarumycin (PAL) gene cluster in the biosynthesis of these compounds, we employed ribonucleoprotein (RNP)-mediated CRISPR-Cas9 to induce targeted deletion of the ketosynthase (KS) domain in PAL. Double-strand breaks (DSBs) upstream and downstream of the KS domain was followed by homology-directed repair (HDR) with a hygromycin resistance cassette flanked by a 50 bp of homology on both sides of the DSBs. The resultant deletion mutants displayed completely different phenotypes compared to the wild-type strain, as they had different colony morphology and were no longer able to produce palmarumycins or melanin. This study, therefore, confirms the involvement of PAL in the biosynthesis of palmarumycins, and paves the way for implementing a similar approach in the characterization of other gene clusters of interest in this largely understudied fungal strain.

Diversity and Biosynthetic Potential of Fungi Isolated from St. John’s Island, Singapore

“…Fungi are an understudied resource for the discovery of novel natural products [ 4 , 11 , 16 , 18 , 49 , 50 , 51 ]. Marine fungi account for more than 60% of the 456 newly reported marine microbial natural products [ 52 ], an indication of the huge scale of untapped biosynthetic potential of naturally occurring fungi.…”

“…The onset of the genomic era followed by the exponential rise in availability of genomic data has led to the development of computational tools for genome mining capable of identifying biosynthetic gene clusters (BGCs) involved in the fungal secondary metabolism [ 12 , 13 , 14 ]. However, there is a noticeable disparity between the number of BGCs predicted by genome mining and the actual number of known compounds identified for any given microbial strain [ 15 , 16 , 17 , 18 ]. For instance, a study analyzing Penicillium genomes found 798 BGCs, but only 16% of the these could be connected to a pathway with a known product [ 19 ].…”

Natural epoxyquinoids: isolation, biological activity and synthesis. An update

“…F6932. 46 Palmarumycin C 8 showed inhibitory activity against S. aureus and A. fumigatus with the IC 90 values of 18 and 7 μg mL −1 , respectively.…”

Discovery and characterization of bioactive compounds from endophytic fungi

“…Among the secondary barcode regions that have been used in fungal identification include two other nuclear ribosomal genes, i.e. 18S nuclear small subunit ribosomal DNA (nrSSU) and nuclear large subunit (nrLSU), and protein-coding loci such as the largest (RPB1) and second largest (RPB2) subunits of RNA polymerase II, beta-tubulin (tub2/BenA) and translation elongation factor 1-alpha (TEF1-α) (Dos Reis et al, 2022;Gakuubi et al, 2022b).…”

“…In MEA, the orange-red colour spread to the entire colony (Figure 5.9B). the wild-type strain after 21 days of growth in SDB, PDA and MEA media (Gakuubi et al, 2022b).…”