A temperature dependent pilin promoter for production of thermostable enzymes in Thermus thermophilus

Abstract

Background Enzymes from thermophiles are of great interest for research and bioengineering due to their stability and efficiency. Thermophilic expression hosts such as Thermus thermophilus [T. thermophilus] can overcome specific challenges experienced with protein production in mesophilic expression hosts, such as leading to better folding, increased protein stability, solubility, and enzymatic activity. However, available inducible promoters for efficient protein production in T. thermophilus HB27 are limited. Results In this study, we characterized the pilA4 promoter region and evaluated its potential as a tool for production of thermostable enzymes in T. thermophilus HB27. Reporter gene analysis using a promoterless β-glucosidase gene revealed that the pilA4 promoter is highly active under optimal growth conditions at 68 °C and downregulated during growth at 80 °C. Furthermore, growth in minimal medium led to significantly increased promoter activity in comparison to growth in complex medium. Finally, we proved the suitability of the pilA4 promoter for heterologous production of thermostable enzymes in T. thermophilus by producing a fully active soluble mannitol-1-phosphate dehydrogenase from Thermoanaerobacter kivui [T. kivui], which is used in degradation of brown algae that are rich in mannitol. Conclusions Our results show that the pilA4 promoter is an efficient tool for gene expression in T. thermophilus with a high potential for use in biotechnology and synthetic biology applications.

Extremophiles in a changing world

“…Thermus thermophilus is a particularly suitable candidate as a production platform for thermostable proteins, given its very high natural transformation frequencies (Cava et al 2009 ; Averhoff et al 2021 ). Six inducible promoters, the P arg , P dnaK , P scs-mdh P nar , P sip , and P pilA4 promoters, have been characterized and two systems for efficient protein over-production have been reported (Moreno et al 2003 , 2005 ; Fujino et al 2020 ; Kirchner and Averhof 2023 ). However, it has to be noted that the P nar promoter is only induced under anaerobic conditions in the presence of nitrate and the P sip is only induced by addition of 10 mM silica, which leads to growth inhibition (Moreno et al 2005 ; Fujino et al 2020 ).…”