A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis .

Anaerobic fungi are powerful platforms for biotechnology that remain unexploited due to a lack of genetic tools. These gut fungi encode the largest number of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom, making them attractive for applications in renewable energy and sustainability. However, efforts to genetically modify anaerobic fungi have remained limited due to inefficient methods for DNA uptake and a lack of characterized genetic parts. We demonstrate that anaerobic fungi are naturally competent for DNA and leverage this to develop a nascent genetic toolbox informed by recently acquired genomes for transient transformation of anaerobic fungi. We validate multiple selectable markers (HygR and Neo), an anaerobic reporter protein (iRFP702), enolase and TEF1A promoters, TEF1A terminator, and a nuclear localization tag for protein compartmentalization. This work establishes novel methods to reliably transform the anaerobic fungus Neocallimastix frontalis , thereby paving the way for strain development and various synthetic biology applications.