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

1)异源基因表达系统的构建：克隆了假丝酵母甲酸脱氢酶和二羟丙酮合成酶基因。使用每个基因中断的菌株证实了每种酶对酵母的甲醇代谢是必不可少的。用这些启动子构建基因表达系统的能力与乙醇氧化酶基因(Aodi)启动子的能力相当，并利用这些启动子构建了新的强基因表达系统。根据启动子的诱导子的不同，这三种表达系统可用于含有不同碳源的培养基上。2)过氧化氢酶产生转化子的构建：从波氏梭菌中克隆了过氧化氢酶基因，并测定了过氧化氢酶靶向信号(PTS1)。当PTS1被截短并转化到波氏弧菌中时，在胞浆中发现大部分过氧化氢酶活性。该转化子可用于处理高浓度H_2O_2废水中的甲酸甲酯合成：发现了一种新的酶--甲酸甲酯合成酶，克隆了该酶的活性基因，并对其酶性质进行了测定。4)利用D-氨基酸氧化酶(DAO)合成有用化合物：DAO基因是从波氏梭菌中克隆出来的。该酶被证实位于过氧化酶体中。克隆了用于生产头孢菌素C的变色胡芦巴中的DAO基因，并研究了其在C.boidinii系统中的表达。

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.