Abstract Detail

Secondary Metabolites

Luo, Hong [1], Angelos, Evan [1], Li, Xuan [2], Sgambelluri, R. Michael [1], Smith, Miranda O. [1], Hong, Sung-Yong [1], Scott Craig, John S. [1], Walton, Jonathan D. [1].

Biosynthesis of Cyclic Peptide Amanita Toxins.

A subset of mushrooms of the genera Amanita, Galerina, Lepiota, and Conocybe contains cyclic peptides, of which alpha-amanitin and related bicyclic peptides are responsible for ninety percent of all fatal mushroom poisonings. We earlier showed that the amatoxins (mainly amanitins) and related phallotoxins are synthesized on ribosomes in A. bisporigera and G. marginata. The primary gene products are short (34-35 amino acids) proproteins that are initially processed by a dedicated prolyl oligopeptidase. The genome of A. bisporigera contains more than 40 sequences related to alpha-amanitin. Members of this gene family are characterized by conserved upstream and downstream amino acid sequences, including two invariant proline residues, flanking a six to ten-amino acid “hypervariable” region that encodes the amino acids found in the mature toxins (or predicted toxins). The evidence indicates that A. bisporigera has evolved a combinatorial strategy that could in principle biosynthesize billions of small cyclic peptides. In order to study the other steps in amanitin biosynthesis, and to engineer novel cyclic peptides, we have developed an Agrobacterium-mediated transformation strategy for the amanitin-producing mushroom G. marginata, enabling us to perform reverse genetic manipulation, i.e., RNAi, gene-targeting (knockout and replacement), and gene introduction. Here we show direct genetic evidence of GmAMA1 and GmPOPB being crucial biosynthetic genes, one P450 being responsible for hydroxylation on the Isoleucine residue, and one flavin-containing monooxygenase (FMO) likely catalyzing sulfoxidation on the cysteine residue. Furthermore we found several regulatory genes, a putative transcription factor, and homologs of G protein and RNA helicase, which significantly altered the toxin production. These recent advances have allowed us to start the process of generating novel cyclic peptides. Currently, many transformants with the GmAMA1 gene being replaced with novel synthetic genes are being analyzed for production of the expected products. If successful, the novel peptides will be screened against a number of targets, including RNA polymerase (the site of action of alpha-amanitin), membrane ion channels, pathogenic bacteria, and cancer cell lines.

1 - Michigan State University, Plant Research Lab, 612 Wilson Road Room 210, E. Lansing, MI, 48824, USA
2 - Kunming University of Science and Technology, Faculty of Environmental Science and Engineering, Kunming, Yunnan, 650093, China

Keywords:
Amanita
cyclic peptide
amatoxin
phallotoxin
secondary metabolite.

Presentation Type: Symposium or Colloquium Presentation
Session: SY3
Location: Room 104 AB/Kellogg Hotel and Conference Center
Date: Monday, June 9th, 2014
Time: 3:30 PM
Number: SY3002
Abstract ID:56
Candidate for Awards:None