摘要
SNF1基因编码的Snf1p对起始受葡萄糖阻遏基因的转录是必需的。通过重叠延伸PCR和两步PCR法合成了两端各带有352bp和345bp与SNF1序列上下游同源的基因敲除组件。将该敲除组件转化至酿酒酵母YS2,获得被loxP-kan-loxP序列组件替换而产生kanr的阳性克隆子。然后将质粒pSH65转入阳性克隆子并诱导表达Cre酶切除kan基因,进而通过传代丢失质粒pSH65后得到SNF1单倍体缺陷型菌株。重复转化敲除组件实现另一条等位基因的敲除,得到SNF1双倍体缺陷型菌株YS2-△snf1::loxp-kan-loxp。厌氧发酵试验表明该突变株的乙醇产量较出发菌株YS2提高了8.74%。残糖量试验表明在培养基中葡萄糖消耗殆尽后,突变株相对于出发菌株而言并没有利用乙醇作为碳源,乙醇产量保持稳定未有下降。敲除SNF1基因是提高酿酒酵母生物合成乙醇的一条有效途径。
In Saccharomyces cerevisiae, Snflp encoded by SNF1 is essential for expression of glucose-re- pressed genes. To improve ethanol production, the gene knockout cassette with 352 bp and 345 bp ho- mologous sequences of the upstream and downstream of SNF1 was constructed by overlap extension PCR and two-step PCR.Then one SNF1 of diploid yeast cell was replaced by the loxP-kan-loxP cassette and yield kanr clones after transformation with the gene knockout cassette. Positive clone was further transformed with plasmid pSH65 followed by induction in galactose-containing medium to produce the cre enzyme to cut the kan marker. Another SNF1 was knockout by repeating the same procedure. Finally a SNF1 knockout homozygous strain YS2-△snf1：：loxp-kan-loxp was obtained. Anaerobic fermentation showed the mutant strain resulted in a 8. 74% higher ethanol production compared to YS2, while sugar consumption test showed that the ethanol yield of the mutant strain kept stability compared to YS2, Thinking comprehensively, the knockout of SNF1 was a successful pathway to raise the ethanol production.
出处
《药物生物技术》
CAS
CSCD
2009年第2期103-107,共5页
Pharmaceutical Biotechnology
基金
国家"948"(No.2006-G37)
福建省发改委重大项目(No.[2004477])
福建省科技厅平台建设项目(No.2005Q007)
关键词
酿酒酵母
SNF1基因
基因敲除
乙醇
Saccharomyces cerevisiae, SNF1 gene, Gene knockout, Ethanol
