High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics

Abstract

Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 104 clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes.

Recent advances of microbial breeding via heavy-ion mutagenesis at IMP

“…) and droplet‐based microfluidics technology (Beneyton et al . ). To our knowledge, microtitre plate wells screening method is usually established when positive microbial strains had obvious phenotype diversity or a bacteriostatic effect (Liu et al .…”

“…Hence, establishment of a high-throughput screening method will gain in importance for microbial trait improvements. Currently, several kinds of prevailing high-throughput screening methods have been developed for obtaining positive strains from mutant libraries, such as microtitre plate wells , Fourier transform infrared (FT-IR) and Raman spectroscopy techniques (Liu and Huang 2016), Fluorescence-activated cell sorting (FACS) ) and droplet-based microfluidics technology (Beneyton et al 2016). To our knowledge, microtitre plate wells screening method is usually established when positive microbial strains had obvious phenotype diversity or a bacteriostatic effect .…”

The Promises and the Challenges of Biotransformations in Microflow

“…A 196‐fold enrichment of an α‐amylase producing Aspergillus niger ( A. niger ) after a single round was demonstrated using ≈10 nL droplets that allowed germination of filamentous fungal spores. Screening of the 10 4 clone UV‐mutated library using microfluidic droplets was performed in 90 min requiring only $14, while a robotic microtiter plate‐based platform would need 16 days and $8770 for the screening of the same amount of mutants . The UHTS has also been applied in the engineering of an artificial computationally designed aldolase .…”

Droplet microfluidics for synthetic biology

“…64 This allows for accurate microbial performance assessments and subsequent droplet sorting to collect microbes with desirable phenotypes typically at ∼1 kHz, and potentially up to 30 kHz. 65 These characteristics give FADS an access to various phenotypic screenings such as screening for cell culture 66 and single cell secretion level of proteins and metabolites, 6,67,68 that are not accessible to FACS as FACS assay is inherently limited to fluorescence within the cell or on the cell membrane. 69 Huang et al used droplet microfluidics to screen 10 5 to 10 6 of UV-irradiated S. cerevisiae variants with desired α-amylase secretion rates (Fig.…”