基于rDNA序列的酵母整合载体的构建及应用

Construction and application of Saccharomyces cerevisiae integration vector based on rDNA sequence

目的构建一个基于rDNA序列的酵母整合载体。方法通过PCR扩增得到酿酒酵母rDNA序列;通过常规分子生物学技术构建以rDNA序列为整合位点的酵母整合载体。为了确证该载体的效能,将青蒿紫穗槐-4,11-二烯合酶基因克隆到该载体上,构建了含紫穗槐-4,11-二烯合酶基因的整合型酿酒酵母工程菌。提取该工程酵母的基因组,通过紫穗槐-4,11-二烯合酶基因的特异引物进行扩增,确认紫穗槐-4,11-二烯合酶基因是否已整合到酵母基因组上。发酵酵母工程菌,对发酵产物进行GC-MS检测,确认是否产生紫穗槐-4,11-二烯。结果构建了1个酵母整合载体,具有如下特点:①能将外源基因表达盒序列整合到酿酒酵母rDNA序列上,增加目的基因的拷贝数;②选择标记能反复应用,方便获得多拷贝的整合形式;③整合片段不需要酶切线性化,节省了内切酶,而且简化了操作程序。酵母基因组PCR结果表明,紫穗槐-4,11-二烯合酶基因已经整合到了酵母基因组上。以朱栾倍半萜为标准品,对该酿酒酵母工程菌的代谢产物进行GC-MS检测,发现其能产生紫穗槐-4,11-二烯,产量达到(91.33±7.57)mg/L朱栾倍半萜当量。结论初步成功构建了一个基于rDNA序列的酵母整合载体。

Objective To construct a stable integration vector with high efficiency and multi-copy characteristics. Methods rDNA sequence of Saccharomyces cerevisiae was amplified by PCR. A stable integration vector based on rDNA sequence was constructed by conventional molecular biological methods and named as pMECAURAR. To characterize the plasmid efficiency, the gene encoding amorpha-4,11-diene synthase （ADS） from Artemisia annua was subcloned into the vector pMECAURAR resulting in the yeast expression vector pADSRDNA. The recombinant vector pADSRDNA was used to transform the Saccharomyces cerevisiae strain W303A to obtain engineered yeast producing amorpha-4,11-diene. The yeast genome was isolated. To determine the ADS gene integration into yeast genome, PCR reaction was performed using the yeast genome as template and ADS specific primers. Then the production was analyzed and quantified by GC-MS based on the standard curve of valencene. Results An integration vector, named pMECAURAR, was constructed to stably express in Saccharomyces cerevisiae. This plasmid has the following characteristics. First, the rDNA genes, which consist of approximately 140 copies of a tandemly repeated segment in the yeast genome, were used as a homologous recombination sequence to increase multiple copies of the plasmid. Another characteristic of the plasmid was that it contains the 3.8 kb hisG-URA3-hisC fragment, which allows for repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Moreover, the integration vector does not have to be linearized, which means no enzymatic treatment and simplified processing. PCR result of engineered yeast genome showed the ADS gene had been introduced into the yeast genome by homologous recombination. The fermentations were analysed by GC-MS and the results showed the engineered yeast could produce amorpha-4,11-diene and the production of amorpha-4,11-diene reached （91.33 ± 7.57）mg/L （valencene equivalent）. Conclusion A stable integration vector with high efficiency and multi-copy characteristics was successfully constructed.