Hayakawa Tohru, Howlader Mohammad Tofazzal Hossain, Yamagiwa Masashi, Sakai Hiroshi
Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan.
Appl Microbiol Biotechnol. 2008 Oct;80(6):1033-7. doi: 10.1007/s00253-008-1560-9. Epub 2008 Aug 27.
Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes. However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches.
苏云金芽孢杆菌产生的Cry4Aa是一种双翅目特异性毒素，因此，对于开发用于控制蚊子的生物杀虫剂具有重要意义。然而，Cry4Aa在大肠杆菌中的表达相对较低，这是其作为生物杀虫剂开发过程中的一个主要缺点。在本研究中，为了建立一个有效的生产系统，根据大肠杆菌基因的G + C含量和密码子偏好，设计并合成了一个1914 bp的编码Cry4Aa的修饰基因（cry4Aa-S1），且不改变编码的氨基酸序列。与原始的cry4Aa基因相比，cry4Aa-S1基因的表达水平显著提高了五倍多。cry4Aa-S1基因的产物对致倦库蚊幼虫的杀虫活性与cry4Aa的产物相同。这表明密码子使用不利是cry4Aa在大肠杆菌中表达不佳的原因之一，因此，将cry4Aa密码子改为符合大肠杆菌中的密码子使用情况可导致Cry4Aa的高效生产。在大肠杆菌中高效生产Cry4Aa可以作为一种有力的措施，为基础分析和应用研究制备足够量的Cry4Aa蛋白。
