Large scale microfluidic CRISPR screening for increased amylase secretion in yeast

• Published in: Lab on a chip 2023-08, Vol.23 (16), p.374-3715
• Main Authors: Johansson, S. Andreas, Dulermo, Thierry, Jann, Cosimo, Smith, Justin D, Pryszlak, Anna, Pignede, Georges, Schraivogel, Daniel, Colavizza, Didier, Desfougères, Thomas, Rave, Christophe, Farwick, Alexander, Merten, Christoph A, Roy, Kevin R, Wei, Wu, Steinmetz, Lars M
• Format: Article
• Language: English
• Subjects: alpha-Amylases - genetics; alpha-Amylases - metabolism; Amylases; Amylases - metabolism; Cell cycle; CRISPR; Endosomes; Gene expression; Genes; Microfluidics; Perturbation; Proteins; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Screening
• ISSN: 1473-0197; 1473-0189
• DOI: 10.1039/d3lc00111c

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae , we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion. Large scale perturbation of gene expression in yeast using CRISPR libraries, coupled with high-throughput screening using fluorescence-based sorting of microfluidic droplets, to identify genes important for increased α-amylase secretion.