一种新型的基因转化系统允许将所需基因多次整合到酿酒酵母染色体中。

A novel system of genetic transformation allows multiple integrations of a desired gene in Saccharomyces cerevisiae chromosomes.

作者信息

Guerra Odanir Garcia, Rubio Ileana G S, da Silva Filho Claudionor Gomes, Bertoni Regiane Aparecida, Dos Santos Govea Rute Cardoso, Vicente Elisabete José

机构信息

Department of Microbiology, Biomedical Sciences Institute, University of São Paulo-USP, Av. Prof. Lineu Prestes 1374, Cidade Universitária, São Paulo, Brazil.

出版信息

J Microbiol Methods. 2006 Dec;67(3):437-45. doi: 10.1016/j.mimet.2006.04.014. Epub 2006 Jul 10.

DOI:10.1016/j.mimet.2006.04.014

PMID:16831478

Abstract

Increasing industrial competitiveness and productivity demand that recombinant yeast strains, used in many different processes, be constantly adapted and/or genetically improved to suit changing requirements. Among yeasts, Saccharomyces cerevisiae is the best-studied organism, and the most frequently employed yeast in industrial processes. In the present study, laboratory strains and industrial S. cerevisiae strains were stably transformed with a novel vector containing the glucoamylase cDNA of Aspergillus awamori flanked by delta-sequences (deltaGlucodelta), and lacking a positive selection marker. Co-transformation with known plasmids allowed selection by auxotrophic complementation of the leu2 mutation and/or geneticin resistance (G418). In all cases, several copies of the deltaGlucodelta vector were inserted into the genome of the yeast cell without selective pressure, showing 100% stability after 80 generations. Transformation frequency of the new vector was similar for S. cerevisiae laboratory strains and industrial wild-type S. cerevisiae strains. This novel genetic transformation system is versatile and suitable to introduce several stable copies of a desired expression cassette into the genome of different S. cerevisiae yeast strains.

摘要

日益增长的工业竞争力和生产力要求，用于许多不同工艺的重组酵母菌株需不断适应和/或进行基因改良，以满足不断变化的需求。在酵母中，酿酒酵母是研究最深入的生物体，也是工业生产中最常用的酵母。在本研究中，实验室菌株和工业用酿酒酵母菌株用一种新型载体进行稳定转化，该载体包含泡盛曲霉葡糖淀粉酶cDNA，两侧为δ序列（δGlucodelta），且无阳性选择标记。与已知质粒共转化可通过亮氨酸2突变的营养缺陷型互补和/或遗传霉素抗性（G418）进行选择。在所有情况下，δGlucodelta载体的多个拷贝在无选择压力的情况下插入酵母细胞基因组，经过80代后显示出100%的稳定性。新载体对酿酒酵母实验室菌株和工业野生型酿酒酵母菌株的转化频率相似。这种新型基因转化系统用途广泛，适合将所需表达盒的多个稳定拷贝引入不同酿酒酵母菌株的基因组。