Codon Pair Optimization (CPO): an algorithm and software tool for synthetic gene design based on codon pair bias in Pichia pastoris

Codon optimization of foreign genes is a common method to improve protein expression level in Pichia pastoris and the current strategy usually 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 in Pichia pastoris by influencing both translational elongation rates and accuracy. Until now, it remains untested in Pichia pastoris whether optimized exogenous genes based on codon pair bias results in higher protein expression levels compared to codon usage bias. In this work, we developed 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 effect of CPO optimization on their protein expression levels in Pichia pastoris. The CPO optimized genes displayed significantly higher expression levels compared to those based on codon usage bias, suggesting CPO is an efficient tool for synthetic gene design for higher heterologous protein expression in Pichia pastors.