What is codon optimization and when is it necessary?

Codon optimization modifies DNA sequences to use codons preferred by the expression host without changing the encoded protein. Different organisms have different codon usage biases based on tRNA abundance. Optimization is necessary when expressing heterologous proteins, especially from organisms with different codon preferences (e.g., human genes in E. coli). Besides codon usage, optimization considers: avoiding rare codons that cause ribosome stalling, removing mRNA secondary structures, eliminating internal restriction sites, and adjusting GC content. Algorithms balance multiple factors. Over-optimization can sometimes reduce expression due to disrupted co-translational folding, so empirical testing is important.