Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design

Different codon optimization algorithms are available that aim at improving protein production by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5′‐UTR structure and related ribosome binding site availability. This is a serious drawback, because the influence of codon usage on mRNA secondary structures, especially near the start of a gene, may strongly influence translation initiation. In this study, we aim to reduce the effect of codon usage on translation initiation by applying a bicistronic design (BCD) element. Protein production of several codon‐optimized gene variants is tested in parallel for a BCD and a standard monocistronic design (MCD). We demonstrate that these distinct architectures can drastically change the relative performance of different codon optimization algorithms. We conclude that a BCD is indispensable in future studies that aim to reveal the impact of codon optimization and codon usage correlations. Furthermore, irrespective of the algorithm used, using a BCD does improve protein production compared with an MCD. The overall highest expression from BCDs for both GFP and RFP is at least twofold higher than the highest levels found for the MCDs, while for codon variants having very low expression from the MCD, even 10‐fold to 100‐fold increases in expression were achieved by the BCD. This shows the great potential of the BCD element for recombinant protein production.