Sorting for secreted molecule production using a biosensor-in-microdroplet approach

Abstract

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.

The expanding CRISPR toolbox for natural product discovery and engineering in filamentous fungi

“…This could be circumvented making use of microfluidics to sort large libraries and biosensors or bioactivities to screen for small molecules production. 133 For example, recently ∼200 engineered As. oryzae strains were screened to produce heterologous terpenoids with anti-inflammatory proprieties by automated robotics in a biofoundry set up.…”

Engineered Living Materials For Sustainability

“…In addition, when two mutually immiscible liquids intersect in a microchannel, one of them may form highly uniform droplets due to liquid/liquid interfacial tension and shear forces. Microfluidics approaches are designed to precisely control fluids for complex micromanipulations such as single-cell encapsulation and high-throughput screening for various applications, including DNA sequencing, drug synthesis, and directed evolution. − There are different microfluidic platforms (e.g., microchannel-based microfluidics, microchamber-based microfluidics, and droplet microfluidics). Droplet microfluidics allows the in situ encapsulation, incubation, and manipulation of living cells in semipermeable microcapsules or microbeads with uniform size distributions, and it has gained specific attention in materials engineering (Figure f) .…”

Plant Flavonoid Production in Bacteria and Yeasts

“…Both rational and irrational engineering toolboxes have been described in detail for E. coli and S. cerevisiae, but not for non-conventional microbes such as Y. lipolytica. Recently, Bowman et al (2021) addressed this problem by developing a droplet-based high-throughput screening technique that enables the co-encapsulation of two different microbes in a single droplet: one is a mutant cell that produces naringenin, and the other is a sensor cell that translates the naringenin concentration inside the droplet into an altered level of gene expression. Different flavonoids can be monitored by simply changing the sensor cells using this method.…”