Efficient production of a self-amplifying RNA vaccine sequence with the single subunit RNA polymerase from <i>Klebsiella</i> phage 32 utilizing the native AU initiating dinucleotide

RNA polymerase (RNAP) is the enzyme that produces RNA for an increasingly large variety of uses. The need for efficient industrial production of high quantities of mRNA was exemplified during the SARS-CoV-2 pandemic. While the RNAP from the T7 bacteriophage has been the gold standard for some time, it has certain limitations impeding efficient manufacturing of all RNA types, including production of long transcript self-amplifying RNA (saRNA). Here we present an alternative to T7 RNAP, identified from the Klebsiella bacteriophage 32 (KP32). We show KP32 RNAP to efficiently produce RNA of varying lengths, with a wide temperature, pH and salt tolerance range. We show KP32 RNAP to be capable of efficient production of an 11,551 nt self-amplifying RNA with its critical native initiating dinucleotide, AU. This represented a 9.3-fold improvement in production levels, relative to T7 RNAP.