Development of an Efficient Recombinant Protein Expression System in Clostridium saccharoperbutylacetonicum Based on the Bacteriophage T7 System

Recombinant proteins have broad applications. However, there is a lack of a recombinant protein expression system specifically for large-scale production in anaerobic hosts. Here, we developed a powerful and stringently inducible protein expression system based on the bacteriophage T7 system in the strictly anaerobic solvent-producing Clostridium saccharoperbutylacetonicum. With the integration of a codon optimized T7 RNA polymerase into the chromosome, a single plasmid carrying a T7 promoter could efficiently drive high-level expression of the target gene in an orthogonal manner, which was tightly regulated by a lactose-inducible system. Furthermore, by deleting beta-galactosidase genes involved in lactose metabolism, the transcriptional strength was further improved. In the ultimately optimized strain TM-07, the transcriptional strength of the T7 promoter showed 9.5-fold increase compared to the endogenous strong promoter P-thl . The heterologous NADP(+)-dependent 3-hydroxybutyryl-CoA dehydrogenase (Hbd1) from C. kluyveri was expressed in TM-07, and the yield of the recombinant protein reached 30.4-42.4% of the total cellular protein, surpassing the strong protein expression systems in other Gram-positive bacteria. The relative activity of Hbd1 in the crude enzyme was 198.0 U/mg, which was 8.3-fold higher than the natural activity in C. kluyveri. The relative activity of the purified enzyme reached 467.4 U/mg. To the best of our knowledge, this study represents the first application of the T7 expression system in Clostridium species, and this optimized expression system holds great potential for large-scale endotoxin-free recombinant protein production under strictly anaerobic conditions. This development paves the way for significant advancements in biotechnology and opens up new avenues for industrial applications.

Automated summary

This study developed a recombinant protein expression system specifically for large-scale production in anaerobic hosts, and successfully applied it in Clostridium species. By integrating a codon optimized T7 RNA polymerase, introducing a T7 promoter, and deleting beta-galactosidase genes involved in lactose metabolism, high-level expression of the target gene was achieved. The system was used to express NADP(+)-dependent 3-hydroxybutyryl-CoA dehydrogenase in an anaerobic strain, resulting in a significant increase in recombinant protein yield compared to other expression systems. This optimized system has great potential for large-scale recombinant protein production under strictly anaerobic conditions.