Chaperone‐Tag System for Activity Improvement of RNA Polymerase in Thermophiles

Thermophilic microorganisms hold advantages including resistance to contamination, reduced cooling costs, and enhanced enzyme activity, positioning them as promising candidates for next-generation industrial biotechnology. However, the inactivity of tools developed for mesophiles hampered the development of thermophiles. By investigating the expression of the T7 RNA polymerase (T7RNAP) in Parageobacillus thermoglucosidasius, we found that the low expression levels of heat-shock proteins in thermophiles contribute to the inactivity. Specifically, we identified HSP33, DnaK/J, and GroS/L as key chaperones that synergistically enhance the folding of T7RNAP. Through understanding the potential recruitment effect of HSP33 on DnaK/J, we de novo designed an HSP33-based tag to improve the activity of T7RNAP to a greater extent. To further enhance this recruitment effect, we conducted a systematic collection of the core element HSP33 through evolutionary analysis across various thermophilic microorganisms, and screened a superior tag that significantly boosted the activity of T7RNAP. Ultimately, we demonstrated that the developed Chaperone-Tag system also improved the activity of T3 RNA polymerase in this strain, highlighting the broad applicability of our strategy.