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Title: CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932
Authors: Gakuubi, Martin Muthee
Ching, Kuan Chieh
Munusamy, Madhaiyan
Wibowo, Mario
Lim, Chun Teck
Ma, Guang-Lei
Liang, Zhao-Xun
Kanagasundaram, Yoganathan
Ng, Siew Bee
Keywords: Science::Biological sciences
Issue Date: 2022
Source: Gakuubi, M. M., Ching, K. C., Munusamy, M., Wibowo, M., Lim, C. T., Ma, G., Liang, Z., Kanagasundaram, Y. & Ng, S. B. (2022). CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932. Frontiers in Microbiology, 13, 1012115-.
Project: #21719
Journal: Frontiers in Microbiology
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.
ISSN: 1664-302X
DOI: 10.3389/fmicb.2022.1012115
Schools: School of Biological Sciences 
Organisations: Singapore Institute of Food and Biotechnology Innovation (SIFBI), A*STAR
Rights: © 2022 Gakuubi, Ching, Munusamy, Wibowo, Lim, Ma, Liang, Kanagasundaram and Ng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Fulltext Permission: open
Fulltext Availability: With Fulltext
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