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

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• Codon optimization algorithms enhance protein production by altering coding sequences.
• Existing algorithms often neglect the impact of codon usage on mRNA secondary structures, particularly the 5'-UTR and ribosome binding site accessibility.
• This oversight can significantly affect translation initiation and overall protein yield.

Purpose of the Study:

• To investigate the impact of a bicistronic design (BCD) element on mitigating codon usage effects on translation initiation.
• To compare protein production levels between BCD and standard monocistronic design (MCD) for various codon-optimized gene variants.
• To evaluate the influence of BCD architecture on the performance of different codon optimization algorithms.

Main Methods:

• Implementation of a bicistronic design (BCD) element alongside a standard monocistronic design (MCD).
• Testing protein production of multiple codon-optimized gene variants in parallel using both BCD and MCD.
• Comparative analysis of protein expression levels (GFP and RFP) between the two designs and across different codon optimization strategies.

Main Results:

• The bicistronic design (BCD) architecture significantly alters the relative performance of codon optimization algorithms.
• Protein production using BCD consistently improved compared to MCD, irrespective of the codon optimization algorithm employed.
• BCD achieved at least twofold higher expression for GFP and RFP compared to the highest MCD levels, with 10-fold to 100-fold increases for low-expression MCD variants.

Conclusions:

• A bicistronic design (BCD) is crucial for accurately studying codon optimization effects on translation initiation and codon usage.
• BCD elements demonstrate substantial potential for enhancing recombinant protein production.
• The BCD approach offers a significant improvement over standard monocistronic designs for protein expression levels.