Laing E, Pretorius I S
Department of Microbiology, University of Stellenbosch, South Africa.
Gene. 1992 Nov 2;121(1):35-45. doi: 10.1016/0378-1119(92)90159-m.
Nine different expression-secretion cassettes, comprising novel combinations of yeast and bacterial gene promoters and secretion signal sequences, were constructed and evaluated. A pectate lyase-encoding gene (pelE) from Erwinia chrysanthemi was inserted between each one of these expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS1 through pAMS9. These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae, and the pectate lyase production was monitored. Transcription initiation signals for pelE expression were derived from the yeast alcohol dehydrogenase (ADC1P), the yeast mating pheromone alpha-factor (MF alpha 1P) and the Bacillus amyloliquefaciens alpha-amylase (AMYP) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of pectate lyase (PLe) was directed by the signal sequences of the yeast mating pheromone alpha-factor (MF alpha 1S), B. amyloliquefaciens alpha-amylase (AMYS) and Er. chrysanthemi pectate lyase (pelES). The ADC1P-MF alpha 1S expression-secretion system proved to be the most efficient control cassette for the expression of pelE and the secretion of PLe in S. cerevisiae.
构建并评估了9种不同的表达-分泌盒，这些表达-分泌盒包含酵母和细菌基因启动子以及分泌信号序列的新组合。将来自菊欧文氏菌的果胶酸裂解酶编码基因（pelE）插入这些表达-分泌盒与酵母基因终止子之间，构建出重组酵母整合穿梭质粒pAMS1至pAMS9。将这些源自YIp5的质粒转化并稳定整合到酿酒酵母实验室菌株的基因组中，并监测果胶酸裂解酶的产生情况。pelE表达的转录起始信号来自酵母乙醇脱氢酶（ADC1P）、酵母交配信息素α因子（MFα1P）和枯草芽孢杆菌α淀粉酶（AMYP）基因启动子。转录终止信号来自酵母色氨酸合成酶基因终止子（TRP5T）。果胶酸裂解酶（PLe）的分泌由酵母交配信息素α因子（MFα1S）、枯草芽孢杆菌α淀粉酶（AMYS）和菊欧文氏菌果胶酸裂解酶（pelES）的信号序列引导。ADC1P-MFα1S表达-分泌系统被证明是酿酒酵母中pelE表达和PLe分泌最有效的调控盒。
