Expanding the RNA polymerase biocatalyst solution space for mRNA manufacture.

Area of Science:

• Biotechnology
• Molecular Biology
• Enzyme Engineering

Background:

• Current mRNA manufacturing relies on a limited set of single-subunit RNA polymerases (RNAPs) for in vitro transcription (IVT).
• Existing RNAPs have variable production phenotypes, hindering optimization for diverse mRNA products, especially larger and more complex molecules.

Purpose of the Study:

• To develop a high-throughput screening platform for functional characterization of large RNA polymerase libraries.
• To identify novel RNA polymerases (RNAPs) and their cognate promoters for improved mRNA biosynthesis.

Main Methods:

• Development of a screening platform integrating in silico and in vitro testing modules.
• Functional characterization of large enzyme libraries to identify novel RNAPs.
• Validation of identified RNAPs in recombinant enzyme-based IVT mRNA production.

Main Results:

• Identification of eight novel, sequence-diverse RNAPs with associated active cognate promoters.
• Validation of these novel RNAPs in mRNA production processes.
• Expansion of the characterized functional RNAP pool by over 130%.

Conclusions:

• The developed screening platform significantly expands the biocatalyst solution space for mRNA manufacturing.
• This advancement enables enhanced optimization of mRNA product yield and quality for specific applications and molecules.
• Provides a rapid method for identifying additional useful enzymes for mRNA synthesis.