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Title: Identification of a hybrid PKS-NRPS required for the biosynthesis of NG-391 in Metarhizium anisopliae var. anisopliae

Author
item Donzelli, Bruno
item Krasnoff, Stuart
item CHURCHILL, A. C. - Cornell University
item Vandenberg, John
item Gibson, Donna

Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2010
Publication Date: 7/7/2010
Citation: Donzelli, B., Krasnoff, S., Churchill, A.L., Vandenberg, J.D., Gibson, D.M. 2010. Identification of a hybrid PKS-NRPS required for the biosynthesis of NG-391 in Metarhizium anisopliae var. anisopliae. Current Genetics. 50:151-162.

Interpretive Summary: Fungi in the genus Metarhizium can infect many insect pests, making them useful for development as insect biological control agents. We have been using a gene-knockout strategy in order to identify compounds that may be involved in the insect host- fungus interaction. This study describes a hybrid polyketide synthase/peptide synthetase responsible for the production of a family of unique mutagenic metabolites called NG 391 and 393, fusarin-like compounds previously reported by our group. Gene knockout strains of the fungus do not produce the NG compounds, but their growth rate, reaction to oxidative stress, and infectivity to insects as compared to the wild type strain is not affected. We did find that the gene is expressed during the infection cycle within insects, and affected by fungal colony density. This work adds to our understanding of the basic biology of the fungus and its interaction with the insect host; it also contributes to our knowledge of factors for improving its safety and efficacy as a biocontrol agent.

Technical Abstract: A 19,818 kb genomic region harboring six predicted ORFs was identified in M. anisopliae ARSEF 2575. ORF4, putatively encoding a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) was targeted using Agrobacterium-mediated gene knockout. Homologous recombinants failed to produce detectable levels of the fusarin-like compound NG-391, indicating the involvement of this locus in its biosynthesis. ORF4 (NGS1) deletion mutants had no significant changes in virulence levels against beet armyworm, Spodoptera exigua larvae, and in resistance to hydrogen peroxide-generated oxidative stress compared to the wild type strain. The use of an NGS1 promoter-GFP reporter fusion showed that gene expression occurs during early exponential growth and is affected by M. anisopliae ARSEF 2575 cell density. RT-PCR, performed using total RNA extracted from the S. exigua–fungus interaction, revealed that NGS1 is expressed during the infection process.