Raynal A, Guerineau M
Mol Gen Genet. 1984;195(1-2):108-15. doi: 10.1007/BF00332732.
Cellobiose, the last product in cellulose degradation, is converted into two molecules of glucose by a beta-glucosidase. S. cerevisiae does posses the structural gene for a beta-glucosidase, but it is very poorly expressed; we thus decided to isolate and characterize that of Kluyveromyces fragilis. We constructed in E. coli HB101 strain a genomic library of the Kluyveromyces fragilis Y610 strain (ATCC 12424), a yeast able to grow on cellobiose and which constitutively produces the beta-glucosidase. The structural gene for beta-glucosidase was identified by its expression in E. coli. The initial isolated cosmid KF1 contained an insert of 35 Kb and by successive subcloning the insert size was reduced to 3.5 Kb (KF4). This cloned beta-glucosidase gene introduced in S. cerevisiae by transformation is expressed at a level of about 500 times that of K. fragilis. We checked by Southern hybridization that the high expression level was not due to a rearrangement of K. fragilis DNA during the cloning experiments. Nevertheless to obtain yeast transformants able to grow on cellobiose a yeast strain whose permeability to sugar is increased must be used and this last point is discussed.
纤维二糖是纤维素降解的最终产物，它由β - 葡萄糖苷酶转化为两分子葡萄糖。酿酒酵母确实拥有β - 葡萄糖苷酶的结构基因，但该基因表达水平很低；因此我们决定分离并鉴定脆壁克鲁维酵母的β - 葡萄糖苷酶。我们在大肠杆菌HB101菌株中构建了脆壁克鲁维酵母Y610菌株（ATCC 12424）的基因组文库，该酵母能够利用纤维二糖生长并组成型产生β - 葡萄糖苷酶。通过其在大肠杆菌中的表达鉴定出β - 葡萄糖苷酶的结构基因。最初分离的粘粒KF1含有一个35 Kb的插入片段，通过连续亚克隆，插入片段大小减小到3.5 Kb（KF4）。通过转化导入酿酒酵母的这个克隆的β - 葡萄糖苷酶基因的表达水平约为脆壁克鲁维酵母的500倍。我们通过Southern杂交检查发现，高表达水平并非克隆实验过程中脆壁克鲁维酵母DNA重排所致。然而，为了获得能够在纤维二糖上生长的酵母转化体，必须使用对糖通透性增加的酵母菌株，最后一点将进行讨论。
