Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase.

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.

Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening.

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.