Codon compression and novel codon creation for multiplex non-canonical amino acid incorporation

Genetic code expansion (GCE) provides a robust platform for engineering protein function via the site-specific incorporation of noncanonical amino acids (ncAAs). While traditional methods are primarily limited to single site incorporation, the growing demand for complex functional modifications in protein engineering has established the simultaneous incorporation of distinct ncAAs as a critical frontier in synthetic biology. This review systematically classifies strategies for such multiplex ncAA incorporation into two principal frameworks. The first framework involves natural codon compression, which exploits the degeneracy of the genetic code to liberate nonsense or rare sense codons via genome-wide recoding. The second framework centers on novel codon creation to expand the coding space, encompassing approaches such as quadruplet codons, unnatural base pairs, and modified mRNA codons. We evaluate these methodologies based on host fitness, coding capacity, and incorporation efficiency to delineate the multifaceted constraints restricting complex genetic encoding. Finally, we propose an integrated future pipeline combining partial codon compression, adaptive laboratory evolution, and artificial intelligence-assisted design to overcome current barriers in multiplex genetic encoding.