生物合成γ-氨基丁酸酿酒酵母谷氨酸脱羧酶基因的克隆与表达

Molecular Cloning and Expression of Glutamic Acid Decarboxylase Gene from Saccharomyces cerevisiae for Biosynthesis of γ-Aminobutyric Acid

为了提高酵母菌的γ-氨基丁酸产量,本研究以十六烷基三甲基溴化铵法(hexadecyl trimethy ammonium bromide,CTAB)提取的酿酒酵母28基因组DNA为模板,扩增得到序列长度为1 758 bp的GAD1基因,经比对与S.cerevisiae S288c的一致性达到98.58%,并设计引物扩增包括GAD1基因上游启动子及调控序列,下游终止子在内的全长基因序列,长度为3 490 bp。将全长基因序列克隆至高拷贝质粒p UG6,构建重组质粒p28。以菌株28作为出发菌株进行转化,经G418抗性筛选得到转化子,进一步经过聚合酶链式反应(polymerase chain reaction,PCR)实验验证,质粒提取及酶切验证,确证得到重组子DL28。经重组质粒p28的特性研究得知,重组质粒p28具有良好的遗传稳定性,无抗性传代培养10次重组质粒不丢失。转化后进行酶活力测定,重组子DL28的谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)酶活力比菌株28提高73.8%。通过以上实验结果得出结论,GAD基因高表达菌株DL28具有更高的G418抗性和GAD酶活性。

Glutamic acid decarboxylase（GAD） is a rate-limiting enzyme for the biocatalysis of γ-aminobutyric acid（GABA）.Therefore,GAD gene is the key gene to regulate the production of GABA.In order to improve the productive capacity of GABA in yeasts,in the present study,we used the CTAB method to purify S.cerevisiae 28 genomic DNA as the template.A 1 758 bp gene fragment was amplified and sequenced as the GAD1 gene that had 98.58% homology with the S.cerevisiae S288 c gene.The primers were designed for amplifying the 3 490 bp sequence of GAD1 full-length gene,including GAD1 gene upstream promoter,regulatory sequence and downstream terminator.The full-length gene sequence was cloned into plasmid p UG6,to construct the recombinant plasmid p28.The strain 28 was used as the starting strain for the conversion,and the transformant was gained by G418 resistance screening,and further verified by PCR,plasmid extraction and double digest identification as the recombinant DL28.By exploring the characteristics of the recombinant plasmid p28,we found that it had good genetic stability,and the recombinant plasmid was still stable after subculture for 10 generations without resistance training. The enzyme activity was determined after transformation, and the GAD activity of recombinant DL28 was improved by 73.8% when compared with the strain 28.Therefore,GAD gene is highly expressed in recombinant DL28,which also has higher G418 resistance.