Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase

Publication date: Available online 30 June 2018Source: Research in MicrobiologyAuthor(s): Catherine Rouzeau, Adilya Dagkesamanskaya, Krzysztof Langer, Jérôme Bibette, Jean Baudry, Denis Pompon, Véronique Anton-LeberreAbstractAdjustment 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 acc...

Source: Research in Microbiology - July 5, 2018 Category: Microbiology Source Type: research