ONO Hisayo Osaka Univ.Eng. Assis.Prof., 遺伝情報実験施設, 助手 (40224274)
YAMASHITA Mitsuo Osaka Univ.Eng. Assoc.Prof, 大学院・工学研究科, 助教授 (40220347)
KANEKO Yoshinobu Osaka Univ.Eng. Assoc.Prof., 大学院・工学研究科, 助教授 (90161182)
ASHIKARI Toshihiko Suntory Senior Res., 基礎研究所, 主席研究員
|Budget Amount *help
¥12,500,000 (Direct Cost : ¥12,500,000)
Fiscal Year 2000 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1999 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1998 : ¥8,000,000 (Direct Cost : ¥8,000,000)
Probiotic bacteria, such as lactic acid and propionic acid bacteria and bifidobacteria, are believed to contribute to promoting human health in different ways. Consequently, genetic engineering to improve dairy lactobacilli has been actively pursued, promising numerous benefits in food production and health promotion.
In this project, host-vector systems for lactic acid and propionic acid bacteria have been developed. The shuttle vectors pRN14 and pPK705 were constructed from indigenous plasmids derived from Lactobacillus plantarum L137 and Propionibacter acidipropionici, respectively. Successful development of transformation systems in various species of Lactobacillus and Propionibacterium using shuttle vectors pRN14 and pPK705 prompted to develop as expression system for heterologous genes in lactic acid and propionic acid bacteria. Our choA gene from Streptomyces has been widely used as a diagnostic tool or for degradation of cholesterol in several species of lactic acid bacteria. In
these studies, promoters from E.coli and Streptomyces were used for the expression of choA.In contrast, we constructed a plasmid, pTACO-1, that consisted of the acetyl coenzyme A carboxylase (acc) promoter and the aminoterminal region of the first open reading frame of the acc operon, which is essential for fatty acid biosynthesis, plus choA in the promoter probe vector pCVE1. Furthermore, we constructed an expression vector for lactic acid bacteria using the acc promoter-choA cassette and a shuttle vector, pRN14. For propionibacteria, native promoters (P138, P8, P116, and P4) were isolated from P.freudenrechii subsp. freudenreichii IFO 12424 using the promoter probe vector pCVE1. The Propionibacterium promoters allow as strong expression of cholesterol oxidase in E.coli were used to construct an expression vector for propionibacteria with the shuttle vector pPK705.
Thus, the extension of the functional diversity in probiotic bacteria will increase their potential utility as a "health care microbe" for bringing about desirable compositional changes in food, such as reduction of cholesterol content in dairy products. Less