Presentation Information
[P02-187]Development of a High-Level Expression System for Psychrophile-Based Simple Biocatalysts (PSCat)
○Ziwen Wang1, Akiko HIDA1, Junichi KATO1, Takahisa TAJIMA1 (1. Hiroshima University (Japan))
Keywords:
Psychrophile,High-level expression system,Biocatalysis,Chromosomal integration
Microbial metabolic engineering is widely used for producing valuable chemicals, but host metabolism often diverts substrates into by-products, reducing yields and requiring extensive pathway optimization. Cell-free systems offer higher conversion efficiency by operating outside living cells, yet they require costly enzyme preparation and suffer from limited enzyme stability. To bridge this gap, a psychrophile-based simple biocatalyst (PSCat) system was developed. This approach uses cold-adapted bacteria as hosts to express relatively thermostable enzymes. By applying moderate heat treatment, the native host enzymes are inactivated, allowing only the introduced enzymes to establish biosynthetic pathways that produce target compounds without forming by-products. This combination retains the high efficiency of cell-free approaches at a much lower cost and with tolerance to toxic products. However, the expression capability of the current PSCat platform remains constrained by the host's native transcription machinery.
To address this limitation, a T7 RNA polymerase expression system, inspired by the well-established BL21(DE3)-pET system in E. coli, is being developed in the PSCat host strain S. livingstonensis Ac10 to substantially enhance enzyme expression levels and productivity. A basic polymerase cassette was designed based on the DE3 structure with modifications adapted for Ac10 and integrated into the host chromosome through homologous recombination. To further improve inducibility and reduce leakage before induction, an improved cassette with enhanced repressor expression is being constructed. In parallel, a broad-host-range expression vector has been designed with cognate promoter and terminator elements. The constructed system will be evaluated for inducibility and leakage using GFP as a reporter and compared with the conventional PSCat platform. Upon validation, this system will be applied to a model biotransformation to demonstrate enhanced productivity.
To the best of our knowledge, this represents the first report of such a high-level expression system developed in a well-established psychrophilic host. This system is expected to enhance the biosynthetic capability of PSCat and also benefit the use of Ac10 as a cold-adapted recombinant protein expression host, for which it was originally developed.
To address this limitation, a T7 RNA polymerase expression system, inspired by the well-established BL21(DE3)-pET system in E. coli, is being developed in the PSCat host strain S. livingstonensis Ac10 to substantially enhance enzyme expression levels and productivity. A basic polymerase cassette was designed based on the DE3 structure with modifications adapted for Ac10 and integrated into the host chromosome through homologous recombination. To further improve inducibility and reduce leakage before induction, an improved cassette with enhanced repressor expression is being constructed. In parallel, a broad-host-range expression vector has been designed with cognate promoter and terminator elements. The constructed system will be evaluated for inducibility and leakage using GFP as a reporter and compared with the conventional PSCat platform. Upon validation, this system will be applied to a model biotransformation to demonstrate enhanced productivity.
To the best of our knowledge, this represents the first report of such a high-level expression system developed in a well-established psychrophilic host. This system is expected to enhance the biosynthetic capability of PSCat and also benefit the use of Ac10 as a cold-adapted recombinant protein expression host, for which it was originally developed.
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