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Breeding of efficient strains of saccharomyces cerevisiae to produce heterologous gene product.

培育高效酿酒酵母菌株以生产异源基因产物。

基本信息

批准号：
63560114
负责人：
SAKAI Takuo
金额：
$ 1.15万
依托单位：
Osaka Prefecture University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

项目摘要

The research on breeding of efficient strains to produce heterologous gene product have been done. A mutant responsible for an overproduction phenotype on heterologous gene product in S. cerevisiae has been isolated, using the gene coding for polygalacturonase (PG) from KZLwvem"s marxianus as a marker.The results obtained are as follows :(1) A mutant responsible for an oversecretion of PG was obtained. The mutant (SSM5-2) produced PG by 10- to 30-fold in parent strain.(2) Contents of lipids in the cell membrane of the mutant was different from those of parent strain, and the conditions for preparing competent cell were also different from parent strain. A simple and efficient method for transformation with plasmids was established.(3) SSM5-2 was temperature-sensitive lethal mutant and it growth was arrested with the production of PG, and it was confirmed to be G2 arrest. The G2 arrest was found to be recovered by Ca^<2+>.(4) A gene which promote the PG production (PGPPF) was found on upstream region of PG gene of K. marxianus. The base sequence of PGPPF was determined.

进行了异源基因产物高效生产菌株的选育研究。一个突变体负责过量生产表型的异源基因产物在S。以KZLwvem marxianus中的多聚半乳糖醛酸酶（PG）基因为标记，从酿酒酵母中分离到一株PG分泌突变株，获得了如下结果：（1）获得了一株PG分泌突变株。突变株SSM 5 -2的PG产量比出发菌株提高了10 ~ 30倍。(2)突变株细胞膜脂质含量与亲本不同，感受态细胞的制备条件也与亲本不同。建立了一种简单、高效的质粒转化方法。(3)SSM 5 -2是一株温度敏感的致死突变株，其生长被抑制，并伴随着PG的产生，被证实为G2期阻滞。Ca^<2+>可使G2期阻滞恢复。(4)在K.马克思主义。测定了PGPPF的碱基序列。