Developing DNA Transformation Methods for Non-model Anaerobic Gut Fungi

Herbivores utilize fungal microbes in their rumen or hindgut to degrade complex lignocellulosic biomass, such as grasses, into constitutive sugars the animal can digest. Harnessing the lignocellulose-degrading capability of these anaerobic gut fungi (AGF) would enable sustainable bio-manufacturing of biofuels and chemicals directly from waste biomass. Currently, we lack reliable genetic methods for these non-model microbes that would otherwise allow us to actively engineer robustness and other useful phenotypes. This study sought to adapt established electroporation methods that have been used for both model and non-model fungi. First, a method was developed to isolate and concentrate the zoospore life-stage, lacking a cell wall, through simultaneous release from agar media. Subsequently, several electroporation workflows were assessed using cell viability as the primary requirement. The spores were washed of salts using centrifugation and observed to tolerate centrifugal forces up to 5000 xg. The protocol was systematically shortened by cutting wash steps that minimally reduced salt content. The electroporation waveform type, the electric field strength, and the buffer temperature were varied in an effort to maintain cell viability. To date, viability after this step has not been observed under any assayed conditions. Future work will further test additional electroporation parameters to ensure cell viability and pursue an alternative, chemical transformation methodology.