Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation.

Area of Science:

• Plant biotechnology
• Molecular biology
• Biopharmaceutical production

Background:

• Codon optimization is crucial for enhancing transgene expression in plants.
• Accurate quantification of plant-produced biopharmaceuticals is challenging.
• Chloroplast translation regulation impacts protein synthesis efficiency.

Purpose of the Study:

• To investigate the impact of codon optimization on transgene expression in plants.
• To validate parallel reaction monitoring (PRM) for quantifying plant-produced biopharmaceuticals.
• To explore translational regulation mechanisms affecting biopharmaceutical production in chloroplasts.

Main Methods:

• Codon optimization of human clotting factor VIII heavy chain (FVIII HC) and polio VIRAL CAPSID PROTEIN1 (VP1) genes using psbA gene codon usage hierarchy.
• Targeted proteomic quantification using parallel reaction monitoring (PRM) and Western blot analysis.
• Analysis of transcript abundance via Northern blot and ribosome profiling.

Main Results:

• Codon optimization significantly increased FVIII and VP1 protein levels, with PRM showing 4.9- to 28.1-fold increases.
• Western blot quantification revealed higher fold-changes (6.3- to 125-fold) due to technical limitations.
• PRM was validated for in planta quantification, particularly for challenging proteins.
• Northern blots confirmed translational, not transcriptional, upregulation; ribosome profiling identified rate-limiting steps and codon-specific ribosome pauses.

Conclusions:

• Codon optimization enhances biopharmaceutical accumulation at the translational level.
• PRM is a robust tool for accurate quantification of plant-produced biopharmaceuticals.
• Understanding translational regulation, including ribosome pausing, is key for further optimization towards clinical applications.