How to maximize protein production in plants? Novel guidance for codon optimization in diverse species

Abstract Description: Gene engineering to maximize protein production is critical for biotechnology and agricultural applications. The conventional recommendation to maximize protein expression is codon optimization, altering the gene sequence to match the codon use of the host species’ genes, but does this approach truly confer optimal expression? To assess how codon preference affects protein synthesis, we analyzed the translation efficiency (TE) of genes from Arabidopsis, tomato, maize, and rice by combining deep sequencing of ribosome footprints (Ribo-seq) and RNA-seq data. When comparing high TE and low TE genes in maize and rice, the high TE genes show a strong bias favoring codons with G or C in the third position (GC3). Interestingly, Arabidopsis also exhibits a small but significant preference for higher GC3 in high TE genes. To test the effect of GC3 content directly, we generated firefly luciferase (FLUC) reporters with the same peptide sequence but differing GC3 content. In maize, Arabidopsis, and tobacco BY2 protoplasts, the highest GC3 FLUC had 107-, 5-, and 2.5-times higher activity, respectively, compared to the low GC3 FLUC. This supports that increasing GC3 content improves protein expression in these species, especially in maize, but also in species like Arabidopsis and tobacco that have low GC3 content in their native genes. To further assess if higher GC3 may confer improved protein expression in diverse species, we analyzed the coding sequences of 74 photosynthetic species. We observed notable variations in GC3 content distributions, even between relatively recently diverged species. Importantly, we found that higher GC3 content is associated with higher TE in every species, excluding several algae. Our findings contradict the conventional recommendation to match the codon use of the host. Rather, maximizing GC3 appears to improve protein expression in diverse species, providing new guidance to increase GC3 content when conducting codon optimization in plants.

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