Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• T7 RNA polymerase (RNAP) is widely used for gene expression in prokaryotes.
• A key limitation of T7 RNAP in eukaryotes is the lack of 5' methyl guanosine caps on transcripts, hindering efficiency.
• Orthogonal gene expression systems are crucial for advanced synthetic biology applications.

Purpose of the Study:

• To develop an improved T7 RNAP system for eukaryotic gene expression.
• To overcome the capping deficiency of T7 RNAP in eukaryotic hosts.
• To create a robust and versatile orthogonal gene regulatory system for synthetic biology.

Main Methods:

• Evolved a fusion enzyme combining T7 RNAP with an African swine fever virus capping enzyme.
• Utilized *Saccharomyces cerevisiae* (yeast) for enzyme evolution.
• Tested engineered variants in yeast and mammalian cells for protein expression and gene circuit function.

Main Results:

• Isolated highly active fusion enzyme variants.
• Achieved protein expression levels approximately two orders of magnitude higher than wild-type T7 RNAP.
• Demonstrated programmable gene expression control using T7 RNAP-based genetic circuits in yeast.
• Validated enhanced performance in mammalian cells.

Conclusions:

• The engineered fusion enzyme provides a robust, orthogonal gene regulatory system for eukaryotes.
• This system significantly enhances protein expression and gene regulation efficiency.
• The developed technology expands the utility of T7 RNAP in diverse eukaryotic synthetic biology applications.