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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Gene Expression Regulation

Background:

• T7 RNA polymerase (RNAP) is a powerful tool for gene expression and protein production in prokaryotes.
• A key limitation of T7 RNAP in eukaryotes is the lack of 5' methyl guanosine caps on transcripts, hindering its utility.
• Existing T7 RNAP systems lack broad applicability and efficiency in eukaryotic systems.

Purpose of the Study:

• To develop an orthogonal gene regulatory system for eukaryotic hosts.
• To enhance T7 RNAP efficiency by incorporating a capping mechanism.
• To improve recombinant protein production in eukaryotic systems.

Main Methods:

• Evolved a fusion enzyme combining T7 RNAP with an African swine fever virus capping enzyme in Saccharomyces cerevisiae.
• Isolated and characterized highly active variants of the engineered fusion enzyme.
• Demonstrated programmable gene expression control using T7 RNAP-based genetic circuits in yeast and mammalian cells.

Main Results:

• Engineered fusion enzyme variants showed approximately two orders of magnitude higher protein expression compared to wild-type T7 RNAP.
• Successfully demonstrated programmable gene expression control in yeast.
• Validated enhanced performance of the engineered variants in mammalian cells.

Conclusions:

• Developed a robust and orthogonal gene regulatory system for diverse eukaryotic hosts.
• The engineered T7 RNAP fusion enzyme significantly enhances protein expression and gene regulation efficiency.
• This system broadens the versatility and applicability of synthetic biology in eukaryotic systems.