MOLECULAR ASPECTS OF CONSTRUCTION OF BIOCATALYSTS BASED ON THE CELLULAR FUNCTION OF METHYLOTROPHIC YEASTS

点击翻译按钮获取中文摘要

In this project, we developed biocatalysts for useful compounds production based on the cell functions of methylotrophic yeasts.1) Construction of heterologous gene expression system : Genes for formate dehydrogenase and dihydroxyacetone synthase of Candida boidinii were cloned. Each enzyme was confirmed to be indispensable for methanol metabolism of the yeast using each gene disrupted strain. The ability to gene expression systems with these promoters were found to be comparable with that with the alcohol oxidase gene (AODI) promoter, and the strong gene expression systems were newly constructed by the use of these promoters. These three expression systems can be used on media containing different carbon sources according to the inducers of the promoters. The AODI-disrupted strain was very useful for the production of the peroxisomal enzymes.2) Construction of catalase producing transformant : The catalase gene was cloned from C.boidinii, and determined the peroxisomal targeting signal(PTS1) When the PTSl was truncated and transformed into C.boidinii, a large part of catalase activity was found in the cytosol fraction. The transformant was found to useful to the treatment of wastwater containing a high concentration of H_2O_2.3) Methyl formate synthesis : A new enzyme, methyl formate synthase was found, the genes for the activity were cloned, and the enzymatic properties were determined. We developed the biocatalysis for methyl formate production using some methylotrophic yeasts.4) Utilization of D-amino acid oxidase (DAO) for the synthesis of useful compounds : The gene for DAO was cloned from C.boidinii. The enzyme was confirmed to be located in the peroxisome. The gene for DAO from Trigonopsis variabilis which is used for production of cephalosporin C was cloned and the expression in the C.boidinii system was investigated.