MULTIPLE-COPY INTEGRATION OF THE ALPHA-GALACTOSIDASE GENE FROM CYAMOPSIS-TETRAGONOLOBA INTO THE RIBOSOMAL DNA OF KLUYVEROMYCES-LACTIS
MULTIPLE-COPY INTEGRATION OF THE ALPHA-GALACTOSIDASE GENE FROM CYAMOPSIS-TETRAGONOLOBA INTO THE RIBOSOMAL DNA OF KLUYVEROMYCES-LACTIS
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DOI：
10.1007/bf00351696
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发表时间：
1992-04-01
期刊：
影响因子：
2.5
通讯作者：
PLANTA, RJ
中科院分区：
文献类型：
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作者：
BERGKAMP, RJM;KOOL, IM;PLANTA, RJ
我们开发了一种载体系统，用于高拷贝数整合到酵母乳酸克鲁维酵母的核糖体 DNA 中。该系统类似于为酿酒酵母开发的 pMIRY 系统。构建含有用于靶向同源重组的乳酸克鲁维酵母rDNA的一部分以及具有各种启动子缺失的酿酒酵母TRP1基因的质粒，并在转化至乳酸克鲁维酵母后分析拷贝数和稳定性。发现这些质粒在每个细胞中存在约 60 个拷贝，并且在非选择性条件下的生长过程中稳定维持。使用该载体系统，我们表达了包含酿酒酵母 GAL7 启动子、SUC2（转化酶）信号序列和来自植物 Cyamopsis tetragonoloba 的 α-半乳糖苷酶编码基因的融合构建体。尽管这些整合质粒的最大拷贝数仅为15左右，但我们仍然获得了高水平的α-半乳糖苷酶产量（250 mg/l），分泌效率约为95%。与含有相同融合构建体的染色体外乳酸克鲁维酵母载体相比，多拷贝整合体显示出高得多的α-半乳糖苷酶生产水平以及在非选择性条件下相当高的稳定性。
We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for alpha-galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of alpha-galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher alpha-galactosidase production level and a considerably higher stability under non-selective conditions.