罗伊乳酸杆菌甘油脱水酶基因的克隆及其在大肠杆菌中的表达

Cloning and expression of Lactobacillus reuteri glycerol dehydratase gene in Escherichia coli

摘要:

迅速升温的生物柴油投资热导致了其副产物甘油的大量积累, 这一现状使得开发和利用甘油生产各种精细化工产品备受关注。本实验通过构建基因工程菌来生物转化甘油生产3-羟基丙醛, 为甘油下游产品的开发开辟了一条新途径。3-羟基丙醛是一种重要的化学中间体, 同时也是一种有效地抗菌剂和生物组织的固定剂, 在化学工业中具有广泛的应用前景。实验主要利用甘油脱水酶N末端序列, 并根据NCBI中公布的甘油脱水酶的氨基酸序列设计了一对克隆引物, 并以菌株罗伊乳酸杆菌(Lactobacillus reuteri)的基因组DNA为模板进行PCR扩增, 获得约为1.6 kb的片段, 将其克隆到T载体上进行测序, 对测序结果进行分析, 重新设计两端含有EcoR I和Hind III酶切位点的表达引物, 利用PCR扩增得到了甘油脱水酶基因, 该基因片段长度为1674 bp, 编码558个氨基酸。将所得片段定向克隆到pET28b载体中, 转化至大肠杆菌BL21感受态细胞中, 筛选阳性单克隆。经IPTG诱导后, 进行SDS-PAGE电泳, 在约65 kD处检测出一蛋白表达条带, 另外还对该重组菌进行比活力测定, 最高比活力可达1.14 U/mg, 比野生型菌株提高了86.88%。

Abstract:

There is growing interest in biodiesel and this results in the accumulation of glycerol. The exploitation and application of glycerol has attracted more and more attention. In the current study, glycerol was biotransformed to produce 3-hydroxypropionaldehyde by genetic engineering bacteria. It is known that 3-hydroxypopionaldehyde has been widely used as an important intermediate for chemicals, effective antimicrobial agent, and fix agent for tissues. A pair of primers was designed on the basis of the sequence of both NH2-terminus and the amino acid sequence of glycerol dehydratase reported by NCBI, and a fragment about 1.6 kb was obtained by PCR amplification using the total genome DNA of Lactobacillus reuteri as template, then the fragment was cloned to the pMD18-T vector and sequenced. Two specific primers were designed according to the obtained sequence, and a fragment with length of 1674 bp was amplified using PCR with these two specific primers. Consequently, the resulting products were digested with EcoR I and Hind III and ligated using T4 DNA ligase to the pET28b vector digested with the same enzymes. The recombinant plasmid, named pET28b-dhaB, was transformed into E. coli BL21. The positive clones were induced with IPTG and the expression products were further analyzed by SDS-PAGE, indicating that protein with a molecule weight of around 65 kD was obtained. Furthermore, the glycerol dehydratase activity was evaluated and compared with the wild type strain as well.