Abstract
BackgroundCodon 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.ResultsIn 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.ConclusionsCodon-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.
Highlights
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
Developing a software tool based on codon‐pair context for efficient synthetic gene optimization in Pichia pastoris Studies unveiled that codon-pair context is biased across species in the three domains of life [5, 6, 21,22,23,24,25,26,27]
Single codon optimization according to codon usage bias (CUB) is based on the performance of the codons in proteins, which only consider the performance of the codon in the current position, but cannot optimize the adjacent codons according to the relationships of front and back position of the current codon
Summary
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. Codonpair 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. Huang et al Microb Cell Fact (2021) 20:209 organisms in gene evolution [1,2,3,4] This phenomenon is known as codon usage bias (CUB). The codons in genes determine the amino acid sequences, and are one of the important determinants for translational efficiency. Studies showed that the usage of synonymous codons has preference, and the contexts of codons have certain patterns in protein coding sequences. CPB has been relatively ignored compared to CUB when considering for gene synthesis
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