A droplet-based workflow for the genetic transformation of a filamentous fungus.

Rapid screening of Trichoderma reesei expressing green fluorescent protein (GFP).

Advantages of droplet culture of T. reesei protoplasts over the classical method.

High throughput sorting for beneficial fungal transformants based on GFP expression.

Droplet-based microfluidic technology is a powerful tool for single-cell cultivation and rapid isolation of bacteria, yeasts and algae. However, it has been of limited use for studies of filamentous fungi due to the fast growth of their branched hyphae. The long regeneration time for fungal protoplasts and low-throughput screening methods are inherent problems for current genetic transformation techniques. Therefore, we have developed a novel droplet-based method for the filamentous fungus Trichoderma reesei expressing green fluorescent protein (GFP) as a marker. This approach presented several outstanding advantages over the traditional transformation method, including a 7-fold reduction in time for T. reesei protoplast regeneration, an 8-fold increase in regeneration frequency, and a screening speed of up to 8,000 droplets min−1. In this study, we encapsulated and incubated the gfp-transformed T. reesei protoplasts in droplets for 24 h, screened the droplets in a high-throughput assay, and eventually collected a transformant library with over 96 % of the candidates transformed with the marker gene. This versatile approach should make fungi more amenable to genetic manipulation and encourage strain improvements for industrial applications.

green fluorescent protein

water-in-oil droplets

fluorescence-activated cell sorting

fluorescence-activated droplet sorting

Potato Dextrose Agar plus Triton X-100

carboxymethyl cellulose

gene encoding Aequorea coerulescens GFP

gene encoding orotidine-5′-phosphate decarboxylase

Water-in-oil droplet culture

High-throughput screening

Filamentous fungi

Genetic transformation

Green fluorescent protein

© 2022 The Author(s). Published by Elsevier B.V.