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Background  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.    Results  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 .    Conclusions  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 .    Supplementary Information  The online version contains supplementary material available at 10.1186/s12934-021-01696-y.

microbial cell factories

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris