M o SA I C: Codon Harmonization of Monte Carlo-Based Simulated Annealing for Linked Codons in Heterologous Protein Expression

odons), for the harmonization of a set of linked codons, which differs from conventional codon harmonization by focusing on the codon sets rather than individual ones. Our MoSAiC demonstrates robust computational performance on ribosomal proteins (S18, S15, S10, and L11) as model systems. Among them, the harmonized gene of RP S18 was expressed and compared with the expression of the wild-type gene. The harmonized gene clearly yielded a larger quantity of the protein, from which the amount of the soluble protein was also significant. These results underscored the potential of the linked codon harmonization approach to enhance the expression and functionality of sensitive proteins, setting the stage for more efficient production of recombinant proteins in various biotechnological and pharmaceutical applications.