Comparative Analysis of Codon Optimization Tools: Advancing toward a Multi-Criteria Framework for Synthetic Gene Design.

Codon optimization is an essential technique in synthetic biology and biopharmaceutical production, enhancing recombinant protein expression by fine-tuning genetic sequences to match the translational machinery and codon usage preferences of specific host organisms. This study presents a comprehensive comparative analysis of widely used codon optimization tools, focusing on their capacity to reflect host-specific codon biases, design principles, and parameters. Industrially relevant target proteins were evaluated in , , and CHO cells, uncovering significant variability in sequence design and clustering patterns across tools.