Development of probiotic bacteria which have an ability of degrading cholesterol and starch

益生菌，如乳酸菌、丙酸菌和双歧杆菌，被认为以不同的方式促进人类健康。因此，改良乳酸菌的基因工程一直在积极进行，在食品生产和健康促进方面有许多好处。本课题开发了乳酸菌和丙酸菌的宿主-载体系统。穿梭载体pRN14和pPK705分别以植物乳杆菌L137和酸性丙酸杆菌的原生质粒构建。利用穿梭载体pRN14和pPK705在多种乳酸菌和丙酸杆菌中成功建立了转化体系，促进了乳酸菌和丙酸菌异种基因表达体系的发展。来自链霉菌的choA基因已被广泛用作几种乳酸菌中胆固醇降解的诊断工具。在更多的研究中，大肠杆菌和链霉菌的启动子被用于choA的表达。相比之下，我们构建了一个质粒pTACO-1，由乙酰辅酶a羧化酶（acc）启动子和acc操纵子第一开放阅读框的氨基末端区域组成，这是脂肪酸生物合成所必需的，加上启动子探针载体pCVE1中的choA。此外，我们利用acc启动子- choa盒和穿梭载体pRN14构建了乳酸菌的表达载体。丙酸菌的原生启动子分别为P138、P8、P116和P4。IFO 12424使用启动子探针载体pCVE1。利用丙酸杆菌启动子在大肠杆菌中具有较强的胆固醇氧化酶表达能力，以穿梭载体pPK705为载体构建丙酸杆菌表达载体。因此，益生菌功能多样性的扩展将增加其作为“保健微生物”的潜在效用，为食品带来理想的成分变化，如降低乳制品中的胆固醇含量。少

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 … More 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