Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency

Frontiers in Bioengineering and Biotechnology, vol. 7, pp. 458

Abstract

High gene expression of enzymes partaking in recombinant production pathways is a desirable trait among cell factories belonging to all different kingdoms of life. High enzyme abundance is generally aimed for by utilizing strong promoters, which ramp up gene transcription and mRNA levels. Increased protein abundance can alternatively be achieved by optimizing the expression on the post-transcriptional level. Here, we evaluated protein synthesis with a previously proposed optimized gene expression architecture, in which mRNA stability and translation initiation are modulated by genetic parts such as self-cleaving ribozymes and a bicistronic design, which have initially been described to support the standardization of gene expression. The optimized gene expression architecture was tested in Pseudomonas taiwanensis VLB120, a promising, novel microbial cell factory. The expression cassette was employed on a plasmid basis and after single genomic integration. We used three constitutive and two inducible promoters to drive the expression of two fluorescent reporter proteins and a short acetoin biosynthesis pathway. The performance was confronted with that of a traditional expression cassette harboring the same promoter and gene of interest but lacking the genetic parts for increased expression efficiency. The optimized expression cassette granted higher protein abundance independently of the expression basis or promoter used proving its value for applications requiring high protein abundance.

Authors 3

1. Dário Neves Aachen Institute of Applied Microbiology

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany

2. Lars M. Blank Aachen Institute of Applied Microbiology

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany

3. Birgitta E. Ebert corresponding Aachen Institute of Applied Microbiology

   RWTH Aachen University · The University of Queensland · Commonwealth Scientific and Industrial Research Organisation

   Affiliation as printed

   Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia

   CSIRO Synthetic Biology Future Science Platform, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Brisbane, QLD, Australia

   Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany