Preventing T7 RNA Polymerase Read-through TranscriptionA Synthetic Termination Signal Capable of Improving Bioprocess Stability

The phage-derived T7 RNA polymerase
is the most prominent orthogonal
transcriptions system used in the field of synthetic biology. However,
gene expression driven by T7 RNA polymerase is prone to read-through
transcription due to contextuality of the T7 terminator. The native
T7 terminator has a termination efficiency of approximately 80% and
therefore provides insufficient insulation of the expression unit.
By using a combination of a synthetic T7 termination signal with two
well-known transcriptional terminators (rrnBT1 and T7), we have been
able to increase the termination efficiency to 99%. To characterize
putative effects of an enhanced termination signal on product yield
and process stability, industrial-relevant fed batch cultivations
have been performed. Fermentation of a E. coli HMS174­(DE3)
strain carrying a pET30a derivative containing the improved termination
signal showed a significant decrease of plasmid copy number (PCN)
and an increase in total protein yield under standard conditions.