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Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency.
Front Bioeng Biotechnol. 2019; 7:458.FB

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+Show Affiliations

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

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32039175

Citation

Neves, Dário, et al. "Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency." Frontiers in Bioengineering and Biotechnology, vol. 7, 2019, p. 458.
Neves D, Vos S, Blank LM, et al. Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency. Front Bioeng Biotechnol. 2019;7:458.
Neves, D., Vos, S., Blank, L. M., & Ebert, B. E. (2019). Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency. Frontiers in Bioengineering and Biotechnology, 7, 458. https://doi.org/10.3389/fbioe.2019.00458
Neves D, et al. Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency. Front Bioeng Biotechnol. 2019;7:458. PubMed PMID: 32039175.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency. AU - Neves,Dário, AU - Vos,Stefan, AU - Blank,Lars M, AU - Ebert,Birgitta E, Y1 - 2020/01/24/ PY - 2019/09/29/received PY - 2019/12/19/accepted PY - 2020/2/11/entrez PY - 2020/2/11/pubmed PY - 2020/2/11/medline KW - Pseudomonas taiwanensis VLB120 KW - bicistronic design KW - high gene expression KW - mRNA stability KW - ribozymes KW - synthetic biology SP - 458 EP - 458 JF - Frontiers in bioengineering and biotechnology JO - Front Bioeng Biotechnol VL - 7 N2 - 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. SN - 2296-4185 UR - https://www.unboundmedicine.com/medline/citation/32039175/Pseudomonas_mRNA_2.0:_Boosting_Gene_Expression_Through_Enhanced_mRNA_Stability_and_Translational_Efficiency L2 - https://doi.org/10.3389/fbioe.2019.00458 DB - PRIME DP - Unbound Medicine ER -
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