A novel gene expression system for Ralstonia eutropha based on the T7 promoter

Muzi Hu,Zhongkang Li,Chunzhi Zhang,Siwei Li,Bin Xiong,Li Liu,Xueli Zhang,Changhao Bi

doi:10.1186/s12866-020-01812-9

Abstract

BackgroundRalstonia 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.ResultsIn 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.ConclusionsThe R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

Highlights

Construction of a T7 gene expression system for R. eutropha The T7 RNA polymerase gene was cloned from E. coli BL21(DE3), modified to be driven by the L-arabinose induced promoter PBAD, and the rfp gene was cloned under the control of a T7 promoter
We found that rfp was expressed in E. coli S17–1, but was not expressed in R. eutropha C5 with or without L-arabinose induction
We considered that the failure of expression in R. eutropha might due to the multiple copies of the T7 RNA polymerase genes expressed from the multicopy plasmid

Summary

Introduction

Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Known as Cupriavidus necator, is a facultative chemolithoautotrophic bacterium which is able to fix CO2 via the Calvin-Benson-Bassham (CBB) cycle [1]. It is a model microorganism for studying the metabolism of polyhydroxyalkanoates (PHAs) [1, 2]. Hu et al BMC Microbiology (2020) 20:121 was a more than 100-fold increase compared to OPH expression in E. coli [12] This indicated that R. eutropha is a potential industrial protein production host

Methods

Results

Discussion

Conclusion