乙酰乳酸合成酶基因(budB)整合表达载体的构建

为了进一步了解2,3-丁二醇合成路径中关键酶的表达量与产量之间的关系,本研究根据Gen Bank中乙酰乳酸合成酶基因(bud B)的基因序列设计引物,以产酸克雷伯氏菌(Klebsiella oxytoca)HD79基因组DNA为模板,通过PCR扩增技术得到目标酶基因,目的片段全长1680 bp。以表达载体p RSFDuet-1为骨架,利用基因工程手段构建整合表达载体p R1SW-bud B。电转化法将其转化至菌株HD79感受态细胞中,通过高浓度Kan筛选和PCR双重鉴定验证后测定ALS酶活力。结果表明,乙酰乳酸合成酶基因整合表达载体构建成功,酶活可达1.0627 U/mg,比原始菌株提高了4.35倍。在一定程度上为实现2,3-丁二醇的工业化生产奠定基础。

The Construction of Acetolactate Synthase Gene Integration Expression Vector

In order to learn more about the connections between the expression levels and the yield of the key enzymes in 2,3-BD synthesis route. In this study the Acetolactate Synthase gene (budB) from Klebisella oxytoca HD79 was amplified by PCR with primers designed according to the sequence of budB in Gene Bank, which is 1680bp. The multicopying integrated expression vector pR1SW-budB was constructed using expression vector pRSFDuet-1 as a framework by meas of genetic engineering. The plasmid pR1SW-budB was transformed into Klebisella oxytoca HD79 by electric conversion, then screened through high concentration Kan and PCR for double characterization and transformants were carried out on the determination of enzyme activity. The result demonstrated that integration of expression vector of acetolactate synthase gene was constructed successful. ALS was mostly expressed with the activity of 1.0627 U/mg, which was improved for 4.35 times. That the yield of 2,3-BD as well as lay the foundation of the industrialization of 2,3-BD production in some degree.