Abstract
Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones. Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.