Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons.

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

GPCR-FEX: A Fluoride-Based Selection System for Rapid GPCR Screening and Engineering.

The directed evolution of proteins comprises a search of sequence space for variants that improve a target phenotype, yet identification of desirable variants is inherently limited by library size and screening ability. Selections that couple protein phenotype to cell viability accelerate identification of promising variants by depleting libraries of undesirable variants en masse. Here, we introduce GPCR-FEX, a stringent selection platform that couples G-protein coupled receptor (GPCR) signaling to expression of a fluoride ion exporter (FEX)-GFP fusion gene and concomitant cellular fluoride tolerance in yeast. The GPCR-FEX platform works to deplete inactive GPCR variants from the library prior to high-throughput fluorescence-based cell sorting for rapid, inexpensive screening of receptor libraries that sample an expanded sequence space. Using this system, FEX1 was placed under the control of either PFUS1 or PFIG1, promoters activated upon agonist binding by the native yeast GPCRs, Ste2p or Ste3p. Addition of a C-terminal degron to FEX1p enhanced the dynamic range of cell growth between agonist-treated and untreated cells. Using deep sequencing to enumerate population members, we show rapid selection of a previously engineered Ste2p receptor mutant strain over wild-type Ste2p in a model library enrichment experiment. Overall, the GPCR-FEX platform provides a mechanism to rapidly engineer GPCRs, which are important cellular sensors for synthetic biology.

The Anaerobic Fungi: Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production.

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation.