Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSMolecular biological basis for statin resistance in naturally statin-producing organisms. Ana Rems, Rasmus Frandsen. DTU Systems Biology, TechnicalUniversity of Denmark, Kongens Lyngby, Denmark.Secondary metabolites can be toxic to the organism producing them; therefore gene clusters for biosynthesis of secondary metabolites often includegenes responsible for the organism’s self-resistance to the toxic compounds. One such gene cluster is the compactin (ML-236B) cluster in Penicilliumsolitum. Compactin is an inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, and is used as a precursor for production of the cholesterolloweringdrug pravastatin. The compactin gene cluster includes two genes encoding proteins that may confer the self-resistance to compactin and itssecretion [1]. The mlcD gene encodes a putative 'HMG-CoA reductase-like protein’, and mlcE encodes a putative efflux pump. However, the function ofthese two putative proteins has not yet been confirmed. We aim to elucidate the biological basis for compactin resistance in the compactin-producingorganism. A codon-optimized version of the mlcD gene was inserted into the Saccharomyces cerevisiae genome. The constructed yeast strain was testedfor sensitivity to lovastatin, a statin structurally similar to compactin, by growing the strain on media containing lovastatin. The strain showed an increasedresistance to lovastatin compared to the wild-type strain. Furthermore, we investigated if MlcD confers the resistance by functional complementation ofthe endogenous HMG-CoA reductases in S. cerevisiae. There are two isozymes of HMG-CoA reductase in yeast, HMG1 and HMG2, both involved in thesterol biosynthetic pathway, which leads to the synthesis of ergosterol. Following deletion of HMG1 and HMG2 genes in S. cerevisiae, we inserted the mlcDgene into the knockout mutants, and tested the resulted strains for sensitivity to lovastatin. The HMG1 and HMG2 knockout mutants were unable to growon minimal media and had an increased sensitivity to lovastatin on rich media. However, insertion of the mlcD gene restored the growth of the yeastmutants and increased their resistance to lovastatin. These results show that MlcD complements the activity of the deleted HMG-CoA reductases, enablingsynthesis of ergosterol in yeast. In addition MlcD confers statin resistance by being insensitive to the inhibiting effects of statins. Reference: [1] Abe Y.,Suzuki T., Ono C., Iwamoto K., Hosobuchi M., Yoshikawa H. Mol Genet Genomics 2002, 267, 5:636-46.Engineering Cyclic Peptide Biosynthesis in Poisonous Mushrooms. Hong Luo, John S. Scott Craig, Robert M. Sgambelluri, Sung-Yong Hong, Jonathan D.Walton. Department of Energy Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824, United States.Ninety percent of fatal mushroom poisonings are caused by alpha-amanitin and related bicyclic peptides found in some species of Amanita, Galerina,Lepiota, and Conocybe. We showed that the amatoxins (mainly amanitins) and related phallotoxins are synthesized on ribosomes in A. bisporigera and theunrelated mushroom G. marginata. The primary gene products are short (34-35 amino acid) proproteins that are initially processed by a dedicated prolyloligopeptidase. A genome survey sequence of A. bisporigera suggested that it has a repertoire of over 40 cyclic peptides, all produced on a singlebiosynthetic scaffold. Members of this extended gene family are characterized by conserved upstream and downstream amino acid sequences, includingtwo invariant proline residues, flanking a six to ten-amino acid “hypervariable” region that encodes the amino acids found in the mature toxins (orpredicted toxins). The evidence indicates that A. bisporigera has evolved a combinatorial strategy that could in principle biosynthesize billions of smallcyclic peptides. In order to study the other steps in amanitin biosynthesis, and to engineer novel cyclic peptides, we have developed a transformationstrategy for the amanitin-producing mushroom G. marginata. This first transformation method uses Agrobacterium-mediated transformation followed byhygromycin selection. Taking advantage of this platform, we are introducing artificial toxin genes that are deliberately designed to provide insights into thepathway. The synthetic genes include those that encode the cyclic octapeptide beta-amanitin, the heptapeptides phalloidin and phallacidin, examples ofthe toxin gene family known from A. bisporigera but not G. marginata, and randomly generated artificial sequences. Currently, thousands of transformantshave been generated through an efficient pipeline and the transformants are being analyzed for production of the expected products. If successful, thenovel peptides will be screened in a number of assays including RNA polymerase (the site of action of alpha-amanitin), membrane ion channels,pathogenic bacteria, and cancer cell lines.80