Characterization of a Two-Sided Orthogonal Translation System and Its Application to Violacein Biosynthesis

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Microbial Biotechnology

Background:

• Microbial production of valuable compounds faces a growth-biosynthesis trade-off.
• Orthogonal translation systems (OTS) offer potential to reallocate resources from cell growth to biosynthesis.
• The in vivo efficacy and orthogonality of OTS require thorough evaluation.

Purpose of the Study:

• To systematically evaluate orthogonal rRNA-mRNA pairs in Escherichia coli.
• To identify an OTS with robust two-sided orthogonality and high translation efficiency.
• To apply the optimized OTS to enhance violacein biosynthesis.

Main Methods:

• Systematic testing of orthogonal rRNA-mRNA pairs in E. coli using superfolder green fluorescent protein (sfGFP) as a reporter.
• Application of the identified high-performance OTS to the violacein biosynthetic pathway.
• Quantification of violacein production under the endogenous and orthogonal translation systems.

Main Results:

• A specific orthogonal rRNA-mRNA pair demonstrated robust two-sided orthogonality and high translation efficiency in E. coli.
• Application of this OTS to violacein biosynthesis increased production by 6.30- to 7.77-fold.
• The OTS sustained violacein production into the stationary phase, overcoming growth-biosynthesis limitations.

Conclusions:

• A well-characterized two-sided OTS can effectively improve microbial production by reallocating translational capacity in vivo.
• This approach offers a promising strategy for enhancing the biosynthesis of valuable compounds.
• Optimized OTS represent a powerful tool for synthetic biology and metabolic engineering applications.