甘油脱氢酶基因在大肠杆菌中的密码子优化表达

Expression of glycerol dehydrogenase gene in Escherichia coli by Codon Optimization

摘要:

摘要【目的】利用密码子优化技术，提高甘油脱氢酶基因gldA在大肠杆菌中的表达水平。【方法】针对gldA起始密码子下游区域，优先选择AT含量最高的同义密码子，从而在不改变氨基酸序列的前提下，提高该区域的AT含量。利用大引物PCR的方法对野生型gldA-WT进行定点突变，获得优化型基因gldA-4，与pET-32a(+)连接后，构建表达质粒pET-gldA-4，转入E. coli BL21(DE3)，得到工程菌E. coli-4。同时，设定包含gldA-WT的工程菌E. coli-WT作为对照，摇瓶发酵后，以

Abstract:

Abstract: [Objective] To improve expression level of glycerol dehydrogenase gene gldA in Escherichia coli by means of codon optimization. [Methods] For immediately downstream region of initiation codon in gldA, we designed optimized sequence by choosing higher AT-content synonymous, in order that this region’s AT-content was increased without changing the corresponding amino acids. Then we had wild gene gldA-WT site-directed mutagenesis depending on mega-primers PCR, so that physically optimized gene gldA-4 was acquired. We cloned gldA-4 into pET-32a(+) to construct expression plasmid pET-gldA-4, which was transformed into Escherichia coli BL21(DE3) for gaining engineering bacteria E. coli-4, by contrast engineering bacteria involved gldA-WT named E. coli-WT. After E. coli-4 and E. coli-WT were fermented in shake flasks, we measured enzyme activities of expression products with glycerol as substrate. [Results] Four gldA-4’s bases in the second, fifth and sixth codon were different with gldA-WT, so AT-content of the optimized gene was up to 80.0% higher than the wild gene’s 53.3%. Furthermore, enzyme activity of E. coli-4’s crude extract was 191.3 U/mL more three times than E. coli-WT’s 48.3 U/mL. [Conclusion] This optimization scheme was quick and easy, but indeed increased dehydrogenase’s activity. It possible becomes a universal method to improve heterogenous expression level of target genes.