(316d) A Genetic Toolkit for Anaerobic Gut Fungi

Anaerobic fungi, found in the rumen of herbivores, are powerful degraders of lignocellulosic biomass and are equipped with a broad suite of enzymes to accomplish this goal. These organisms are found in diverse multi-kingdom communities and have been shown to produce a range of biotechnologically valuable products, including short- and medium-chain fatty acids and bioactive small molecules. However, a major bottleneck remains in the translation of anaerobic fungi to the industrial setting as there is a lack of robust genetic engineering strategies to direct metabolic flux(es) towards desired products, generate protein diversity, and introduce heterologous genes.

We have built and characterized a diverse series of vectors with promoters driving the expression of anaerobe-compatible flavin-based fluorescent proteins in a transient expression system and are advancing the most promising candidates for genomic integration using CRISPR and transposon-based engineering. By mining the transcriptomes across many genera of anaerobic fungi, we identified putatively active mobile genetic elements, consisting of long terminal repeats (LTRs) that flank protein machinery for reverse transcription and genomic integration. We have co-opted this system for heterologous gene insertion by constructing a library of promoter-optimized antibiotic selection markers flanked by LTRs and found evidence of long-term persistence of resistance to hygromycin and expression of a suite of flavin-based fluorescent reporters.

Careful consideration of the life cycle of anaerobic fungi is important to the genetic engineering of these complex organisms. Anaerobic fungal sporangia produce motile, unicellular zoospores that due to their thinner cell wall, are more amenable to genetic manipulation. We have found that fungal zoospores can be sorted by FACS while retaining viability, enabling high throughput sorting for downstream applications. We also show zoospores are capable of the uptake of fluorescently-tagged oligonucleotides and have optimized transformation conditions for high levels of DNA entry and cellular viability. Additionally, we also show that these zoospores can be analyzed by fluorescence-activated cell sorting (FACS) to gain insights into the production of cell-surface proteins involved in lignocellulose degradation.