Functional analysis of PaxP and PaxQ, two cytochrome P450 monooxygenases required for paxilline biosynthesis in Penicillium paxilli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand

Abstract

The indole-diterpene paxilline is a potent mammalian tremorgenic mycotoxin and a known inhibitor of maxi-K ion channels. The gene cluster encoding the enzymes for the synthesis of this compound was recently cloned from Penicillium paxilli (Young et al. 2001). The cluster comprises a set of core genes required for indole-diterpene biosynthesis, including two cytochrome P450 monooxygenases, paxP and paxQ. Targeted deletion of paxP and paxQ resulted in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively, confirming that both genes are involved in paxilline biosynthesis. The aim of the current work is to establish in vitro that PaxP and PaxQ catalyse the monooxygenation of paspaline and 13-desoxypaxilline, respectively. To achieve this, cDNA copies of both genes were cloned into pGEX-6P-3, to generate pRL2 and pRL4, and the corresponding glutathione-S-transferase (GST) fusion proteins over-expressed in E. coli. However, both GST-fusion proteins accumulated as insoluble inclusion bodies when cultures were incubated at 18°C, 25°C and 37°C. Attempts to express a soluble form of the GST-PaxP by co-expressing this fusion with the chaperones, GroES and GroEL. or by expressing in E. coli, Origami B, a strain (trxB, gor, lacY) designed to facilitate expression of active and soluble proteins, were unsuccessful. GST-PaxP was able to be solubilised by the addition of 0.25% N-laurylsarcosine, and retained some glutathione binding activity, however, the yield was too low to carry out further experiments. GST and thioredoxin fusion expression constructs were designed in which the putative N-terminal trans-membrane region of PaxP and PaxQ was removed to aid solubility in E. coli. These N-terminal modified fusion proteins were still expressed as insoluble protein.