title: Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.
abstract: BACKGROUND
  Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.
  
  
  RESULTS
  Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).
  
  
  CONCLUSIONS
  Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.
authors.affiliation: Ecole Supérieure de Physique et de Chimie industrielles de la Ville de Paris (ESPCI Paris), CNRS UMR 8231, 10 rue Vauquelin, 75005, Paris, France.|CNRS, University of Bordeaux, CRPP, UPR 8641, 115 Avenue Albert Schweitzer, 33600, Pessac, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Beneyton
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78352, Jouy-en-Josas, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Thomas
authors.affiliation: Ecole Supérieure de Physique et de Chimie industrielles de la Ville de Paris (ESPCI Paris), CNRS UMR 8231, 10 rue Vauquelin, 75005, Paris, France.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Griffiths
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78352, Jouy-en-Josas, France.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.affiliation: Ets J. Soufflet/CRIS-OSIRIS, Quai Sarrail, BP12, 10400, Nogent-sur-Seine, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Drevelle
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78352, Jouy-en-Josas, France. tristan.rossignol@inra.fr.
authors.forename: Tristan
authors.identifier: http://orcid.org/0000-0003-0718-0684
authors.initials: T
authors.lastname: Rossignol
mesh_terms: D057055:Aspartic Acid Proteases / Q000235:genetics / Q000378:metabolism*; D001234:Aspergillus niger / Q000235:genetics; D055162:Biocatalysis; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057166:High-Throughput Screening Assays / Q000379:methods*; D006868:Hydrolysis; D044085:Microfluidics / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases; D043366:Cellulose 1,4-beta-Cellobiosidase; D057055:Aspartic Acid Proteases
keywords: Droplet-based microfluidics; Heterologous expression; High-throughput enzymatic screening; Recombinant fungal protein; Yarrowia lipolytica