Tang Longpan, Yu Jincong, Dai Danfeng, Fang Baishan
Key Laboratory for Industrial Biotechnology of Fujian Higher Education (Huaqiao University), Xiamen 361021, China.
Wei Sheng Wu Xue Bao. 2011 Apr;51(4):504-9.
To improve expression level of glycerol dehydrogenase gene gldA in Escherichia coli by means of codon optimization.
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-gldA4, 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.
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.
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.
通过密码子优化提高大肠杆菌中甘油脱氢酶基因gldA的表达水平。
针对gldA起始密码子紧邻的下游区域，通过选择富含AT的同义密码子设计优化序列，使该区域的AT含量增加而不改变相应氨基酸。然后根据大引物PCR对野生型基因gldA-WT进行定点诱变，从而获得物理优化的基因gldA-4。将gldA-4克隆到pET-32a(+)中构建表达质粒pET-gldA4，将其转化到大肠杆菌BL21(DE3)中获得工程菌大肠杆菌-4，与之对比的涉及gldA-WT的工程菌命名为大肠杆菌-WT。将大肠杆菌-4和大肠杆菌-WT在摇瓶中发酵后，以甘油为底物测定表达产物的酶活性。
gldA-4的第二、第五和第六密码子中的四个碱基与gldA-WT不同，优化基因的AT含量高达80.0%，高于野生型基因的53.3%。此外，大肠杆菌-4粗提物中的酶活性为191.3 U/mL，是大肠杆菌-WT的48.3 U/mL的三倍多。
该优化方案快速简便，但确实提高了脱氢酶的活性。它有可能成为提高目标基因异源表达水平的通用方法。
