Site-directed Mutagenesis And Expression Of Cyp51 From Ustilago Maydis

Corn smut,caused by the most major pathogens Ustilago maydis,is one of the most serious diseases for cultivated corn and causes serious recurrent epidemics throughout corn growing regions in the world.Every year,the economic loss in agriculture caused by corn smut is significant.Sterol 14α-demethylase(CYP51) is the only member of the cytochrome P450 monooxygenase family present in higher plants,fungi,bacteria and mammals,In all cases it catalyzes a three-step reaction of sterol 14α-demethylation.In fungi,the complex sterol 14α-demethylation reaction presents one of the key steps in sterol biosynthesis,an essential metabolic pathway producing ergosterol.Ergosterol depletion will result in the accumulation of methylated sterol precursors,and affect both membrane integrity and function of membrane-bound proteins,eventually cause inhibition of fungal growth.Inhibitors of fungal sterol 14α-demethylase,termed demethylase inhibitors(DMIs),are the main group of fungicides used in agriculture.In this study,we have investigated the target enzyme of the recombinant CYP51 from U. maydis by combining the molecular cloning technique with site-directed mutagenesis technique.The results obtained list below.1.High expression of recombinant CYP51 from U.maydis.It is found that the truncation of N-terminal amino acid of YHCYP51 were critical for its heterologous expression in E.coli.Full-length YHCYP51 was not expressed with various combinations of expression vectors and strains.Based on U.maydis CYP51 gene structure analysis, two pairs of primers were designed for cloning and expressing the mutants which truncated different transmembrane region(20aa and 35aa).The recombinant plasmids pET28-YH-20 and pET28-YH-35 were constructed by amplification the mutant gene fragments from the original pET28-YH,at the same time another four truncated plasmids were constructed.All of the plasmids were induced in different strains and optimized conditions.A series of experiments leads to the finding that only pET32-YH-35 could be highly expressed at the optimal condition of 30℃induced with 0.5mmol/L IPTG and the P450 activity was maintained.The spectra analysis of recombinant YHCYP51 binding to 4 standard fungicides and to 25 fungicide compounds showed that a XF-synthetic fungicide compound(XF-113) and another ZST-synthetic(ZST-4) were similar to standard fungicides in binding constant.The two compounds are promising to be a new effective antifungal drug.The overexpressed YHCYP51 was purified by Ni-NTA affinity chromatography under denatured conditions.The recombinant shuttle plasmid pAUR123-YH was constructed based on plasmids pMD-YH and pAUR123 and then transformed into Saccharomyces cerevisiae.The positive clone was induced and SDS-PAGE analysis showed a specific band(about 66kD).2.Structure and function analysis of the active site in recombinant YHCYP51.The target enzyme active site amino acid residues can affect the interaction with drugs.We could choice some residues which may be involved in inhibitor binding for mutation. This study chose three sites for site-directed mutagenesis by analysing the homology and conserved of a wide range of fungi and Mycobacterium tuberculosis CYP51 sequence. The two sites Tyr95 and His293 were highly conserved in all living organisms,and Phe206 was conserved in fungi.The results indicated that all of them could affect the interaction with drugs.Y95 and F206 played a role in recombinant YHCYP51 binding to tebuconazole,and F206 might be a target site of high specificity YHCYP51 inhibitors. H293 might be involved in the maintaining of spatial structure.The study of gene expression and site-directed mutagenesis in active site amino acid residues from CYP51 are facilitate to further clarify the mechanism of fungal resistance, and will provide a theoretical basis and new idea for efficient design and development of new anti-fungal drugs.