Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase

Abstract

Vibrio natriegens is the fastest-growing bacteria, and its doubling time is less than 10 min. At present, the T7 expression system has been introduced into V. natriegens for heterologous protein expression, including the commercial strain V_max¹ and the variant VnDX,² which is a backup expression chassis of Escherichia coli BL21(DE3). However, the strength of the existing T7 expression system is not optimal for every recombinant protein. The different expression strengths of T7 RNA polymerase (T7 RNAP) can be obtained by changing the promoter and ribosome binding site (RBS) sequences of T7 RNAP at different transcription and translation levels. In this work, we obtained a robust VnDX variant library with the fine-tuning T7 RNAP using the industrially used enzyme glucose dehydrogenase (GDH) as the reporter protein. Among this library, the variant VnDX-tet, whose promoter of T7 RNAP was changed from P_lacUV5 to P_tet, showed that the reporter enzyme GDH activity was increased by 109% by the T7 expression system. Similarly, variants with different T7 RNAP translation levels were obtained by changing RBS sequences upstream of T7 RNAP, and the results showed that the variant VnDX-RBS12/pGDH had the highest GDH activity, which increased by 12.6%. The VnDX variant library constructed in this study with different T7 expression strengths provides a choice for expressing various recombinant proteins, greatly expanding the application of V. natriegens.

Keywords: T7 RNA polymerase; Vibrio natriegens; fine-tuning; glucose dehydrogenase; recombinant protein expression.