Genetic Engineering of Long Chain Dicarboxylic Acid Production in Yeast

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The research plan constituted of following three parts. (1) Construction of new dicarboxylic acid-producing yeast by using genetic engineering system which was developed for n-alkane-assimilating yeast Candida maltosa in our laboratory. (2) Improvement of production efficiency and purity of the dicarboxylic acid by overproducing specific cytochrome P-450. (3) Improvement of dicarboxylic acid production by disruption of acyl-CoA oxidase gene which works in degradation of the product.In 1994, we analyzed various genes encoding cytochromes P-450 in C.maltosa.and attained overproduction of specific cytochrome P-450 by GAL gene promoters. We also analyzed the substrate specificity and catalyzed products of the cytochromes. In 1995, disruption of C.maltosa POX2 gene encoding acyl-CoA oxidase was successfully accomplished. Production of dicarboxylic acid from n-tetradecane by the disrupted strain was subsequently confirmed by the Japan Energy group who had fermentation and detection equipments. We constructed a vector that was stably maintained in C.maltosa and isolated cytochrome P-450 reductase gene, of which overexpression will further improve alkane oxidation by C.maltosa.