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，基因工程改良乳制品乳酸菌受到了积极的关注，有望在食品生产和健康促进方面带来许多好处。在这个项目中，我们开发了乳酸菌和丙酸菌的宿主载体系统。以植物乳杆菌L137和丙酸丙酸杆菌L137为出发菌株，构建了穿梭载体pRN14和pPK705。利用穿梭载体pRN14和pPK705在不同种类的乳酸菌和丙酸菌中成功地建立了转化体系，为异源基因在乳酸菌和丙酸菌中的表达奠定了基础。我们来自链霉菌的ChoA基因已被广泛用作诊断工具或在几种乳酸菌中降解胆固醇。在…中在更多的研究中，我们利用来自大肠杆菌和链霉菌的启动子来表达choA。相反，我们构建了一个含有乙酰辅酶A羧基酶(Acc)启动子和acc操纵子第一个开放阅读框氨基末端区域的载体pTACO-1，该操纵子是脂肪酸生物合成所必需的，外加启动子探针载体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。对于丙酸菌，从弗劳登雷奇亚种分离到天然启动子(P138、P8、P116和P4)。Freudenreichii IFO 12424，使用启动子探测载体pCVE1。由于丙酸杆菌启动子在大肠杆菌中有很强的胆固醇氧化酶表达，因此利用穿梭载体pPK705构建了丙酸菌的表达载体。因此，益生菌功能多样性的扩大将增加它们作为“保健微生物”的潜在用途，从而在食品中产生理想的组成变化，如降低乳制品中的胆固醇含量。较少

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