High-throughput screening for industrial enzyme production hosts by droplet microfluidics

Abstract

A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α-amylase production, close to the theoretical maximum enrichment. Furthermore, a 10 5 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

“…By alternatively transforming microfluidic emulsions to double emulsions (12) or hydrogel beads equipped with polyelectrolyte shells (13), sorting in standard flow cytometers also becomes possible. However, to date, all ultrahigh-throughput screening campaigns implemented in microcompartmentalized formats (including microcapillary arrays) have so far relied on detection of a fluorescent product (13)(14)(15)(16)(17)(18)(19)(20). When assays that lack this type of readout are to be used (e.g., involving instead the widely used absorbance detection), droplet screening is currently impossible: Colony screening assays (relying on precipitation of insoluble product and thus encumbered by poor dynamic range and assay quality) or microwell-plate screening with sophisticated robots (higher quality but expensive in terms of capital expenditure and running costs) remain the only options.…”

Intra-droplet acoustic particle focusing: simulations and experimental observations

“…The interest in droplet microfluidics has emerged because droplets are perfectly suited as miniaturized reaction chambers for encapsulation and investigation of cells at the singlecell level (Schneider et al 2013;Shembekar et al 2016), and the technology allows for improved genome engineering and screening of cells (Agresti et al 2010;Sjostrom et al 2014). In most of these applications, having the possibility to precisely handle the particles inside the droplets expands the complexity of the assays that can be performed on-chip.…”

Metabolite profiling of microfluidic cell culture conditions for droplet based screening

“…5 Droplet microfluidics has been used for a number of single cell or single molecule applications, for example, functional screening of hybridoma cells, 6 the study of sickling in red blood cells, 7 and the improvement of enzyme production in cells using directed evolution. 8 Droplet incubation can be divided into on-and off-chip approaches. Examples of on-chip methods are delay channels, used to incubate droplets for shorter periods of time 9 and serpentine channels, 10 droplet traps, 11 and dropspots 12 used for longer incubation times.…”

“…Examples of on-chip methods are delay channels, used to incubate droplets for shorter periods of time 9 and serpentine channels, 10 droplet traps, 11 and dropspots 12 used for longer incubation times. Off-chip methods are used for cell culture in droplets, e.g., by collecting the emulsion in syringes 8,13 or in a Pasteur pipette under a layer of media 14 and PDMS sealed vials for thermocycling. 15 None of these put droplets in direct contact with the surrounding atmosphere.…”