Development of genetic transformation methods in anaerobic gut fungi

Development of genetic transformation methods in anaerobic gut fungi at  Fungal Genetics Conference, Pacific Grove, CA, Asilomar Conference Grounds

Abstract: 

Microorganisms hold great promise for applications in biotechnology, including the renewable production of fuels and chemicals, the biosynthesis of therapeutics, and even the creation of novel materials. However, it is well known that only a relatively small percentage of microbial species are isolated and characterized sufficiently to be amenable for engineering purposes, thus limiting our repertoire of available biological tools. This issue is particularly true for hard-to-culture organisms. One such group of organisms, the anaerobic gut fungi (phylum Neocallimastigomycota), hold significant, untapped biotechnological potential. These fungi, which are found predominantly in the digestive tracts of herbivores, possess an expansive array of uncharacterized carbohydrate-active enzymes, indicating a high-degree of potential for applications involving the processing and conversion of lignocellulosic material. Furthermore, as these fungi natively exist in a competitive microbial environment, they are believed to possess diverse secondary metabolites with potential for therapeutic applications. However, studies of anaerobic gut fungi are severely limited by an almost complete lack of genetic tools. The genetic intractability of these organisms also hinders efforts at strain engineering for biotechnological applications. To overcome these restrictions, we are in the process of developing tools and techniques for the genetic manipulation of these fungi. A critical first step in this regard is to develop methods for introducing exogenous DNA into the fungal cells for stable or transient expression. We are investigating multiple mechanisms to carry out this genetic transformation, including natural competency-based transformation, PEG-mediated transformation, electroporation, and bacterially-mediated transformation. Yet, establishing these transformation techniques is made difficult by the extreme nature of the fungi, both in terms of their obligately anaerobic growth as well as the severe ATbias of their genomes. As such, we are adapting existing transformation protocols as well as developing novel approaches to accommodate the unique features of these organisms. The development of the aforementioned systems into functional transformation tools in anaerobic gut fungi will significantly #Fungal26|99 expand the scope of studies that can be performed on these organisms and will usher in the development of engineered fungal strains to address pressing biotechnological applications.