Elucidation of the product synthesis of the sesquiterpene synthase Cop6 isolated from Coprinus cinereus

Mushrooms are believed to have a great potential for production of bioactive metabolites e. g. terpenes, a group of interesting compounds with diverse chemical properties such as antitumour and antibacterial activity. Cop6 is a terpene cyclase isolated from the mushroom Coprinus cinereus that catalyzes the cyclization of farnesyl diphosphate (FPP) to mainly αcuprenene. In this study gas chromatography combined with mass spectroscopy (GC-MS) is used to analyze the product profile of Cop6 mutants created by PCR based site directed mutagenesis. The goal is to produce trichodiene, the parent hydrocarbon in the biosynthesis of trichothecene antibiotics and mycotoxins. Valine instead of tyrosine in amino acid position 195 resulted in cyclisation of (E)-β-Farnesene and (3Z,6E)-α-Farnesene besides the products of the wild type enzyme. Another mutant with aspartic acid instead of asparagine in position 224 resulted in the synthesis of β-Bisabolene except for α-cuprenene and methionine in position 74 instead of isoleucine killed the activity of the cyclase. Furthermore, an attempt to saturation of position 98 was made, resulting in four mutants. Two of them essentially killed the activity of the cyclase whereas two had minor effect of the product profile compared to the wild type. Table of contents