Abstract
All mRNA products are currently manufactured in in vitro transcription (IVT) reactions that utilize single-subunit RNA polymerase (RNAP) biocatalysts. Although it is known that discrete polymerases exhibit highly variable bioproduction phenotypes, including different relative processivity rates and impurity generation profiles, only a handful of enzymes are generally available for mRNA biosynthesis. This limited RNAP toolbox restricts strategies to design and troubleshoot new mRNA manufacturing processes, which is particularly undesirable given the continuing diversification of mRNA product lines toward larger and more complex molecules. Herein, we describe development of a high-throughput RNAP screening platform, comprising complementary in silico and in vitro testing modules, that enables functional characterization of large enzyme libraries. Utilizing this system, we identified eight novel sequence-diverse RNAPs, with associated active cognate promoters, and subsequently validated their performance as recombinant enzymes in IVT-based mRNA production processes. By increasing the number of available characterized functional RNAPs by more than 130% and providing a platform to rapidly identify further potentially useful enzymes, this work significantly expands the RNAP biocatalyst solution space for mRNA manufacture, thereby enhancing the capability for application-specific and molecule-specific optimization of both product yield and quality.