Engineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou, 350007, China.
Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Sciences, Fujian Normal University, Fuzhou, 350007, China.
Microb Cell Fact. 2021 Nov 4;20(1):209. doi: 10.1186/s12934-021-01696-y.
Codon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias.
In this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris.
Codon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.
密码子优化是提高毕赤酵母中蛋白质表达水平的常用方法，目前的策略是用偏好密码子替换稀有密码子，以匹配密码子使用偏性。然而，密码子对上下文通过影响翻译延伸速度和准确性对翻译效率有深远的影响。到目前为止，基于密码子对偏差优化的基因是否比基于密码子使用偏性的基因产生更高的蛋白质表达水平仍未得到验证。
在这项研究中，引入了一种基于动态规划的算法来开发密码子对优化（CPO），这是一种用于毕赤酵母中合成基因设计的简单有效的密码子对优化软件工具。使用两种报告基因（MT1-MMP E2C6 和 ADAM17 A9B8 scFvs）来测试密码子对偏差和 CPO 优化对其蛋白质表达水平的影响。为 MT1-MMP E2C6 和 ADAM17 A9B8 生成了四个变体，一个变体具有最佳的密码子对上下文，一个变体具有最差的密码子对上下文，一个变体具有无偏的密码子对上下文，另一个变体根据密码子使用进行了优化。Western blot 几乎无法检测到具有最差密码子对上下文的变体的表达水平，而具有最佳密码子对上下文的变体表达良好。基于密码子对上下文的优化序列的 MT1-MMP E2C6 和 ADAM17 A9B8 的表达水平分别比基于密码子使用的表达水平高 5 倍和 7 倍以上。结果表明，基于密码子对上下文的密码子优化在提高毕赤酵母中外源基因的表达方面更有效。
密码子对上下文在毕赤酵母中的蛋白质表达中起着重要作用。本研究开发的密码子对优化（CPO）软件有效地提高了毕赤酵母中外源基因的蛋白质表达水平，表明基于密码子对偏差的基因设计是提高毕赤酵母中重组蛋白表达的一种替代策略。
