Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase.

Autor: Rouzeau C; LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Dagkesamanskaya A; LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Langer K; Laboratoire Colloïdes et Matériaux Divisés, From the Institute of Chemistry, Biology and Innovation (CBI), ESPCI ParisTech, CNRS, UMR 8231, PSL Research University, 10 rue Vauquelin, 75005, Paris, France., Bibette J; Laboratoire Colloïdes et Matériaux Divisés, From the Institute of Chemistry, Biology and Innovation (CBI), ESPCI ParisTech, CNRS, UMR 8231, PSL Research University, 10 rue Vauquelin, 75005, Paris, France., Baudry J; Laboratoire Colloïdes et Matériaux Divisés, From the Institute of Chemistry, Biology and Innovation (CBI), ESPCI ParisTech, CNRS, UMR 8231, PSL Research University, 10 rue Vauquelin, 75005, Paris, France., Pompon D; LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Anton-Leberre V; LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France. Electronic address: veronique.leberre@insa-toulouse.fr.
Jazyk: angličtina
Zdroj: Research in microbiology [Res Microbiol] 2018 Jul - Aug; Vol. 169 (6), pp. 335-342. Date of Electronic Publication: 2018 Jun 30.
DOI: 10.1016/j.resmic.2018.06.002
Abstrakt: Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
(Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)
Databáze: MEDLINE
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