Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

Abstract

BackgroundEngineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment.ResultsUsing sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains.ConclusionsWe report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0611-7) contains supplementary material, which is available to authorized users.

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose

“…A. robustus and N. californiae yielded a total accumulated sugar concentration of 4.5 ± 0.4 and 4.0 ± 0.6 g/L, respectively. We expect that cellobiose is primarily hydrolyzed to glucose or directly taken up due to a wealth of putative cellobiose transporters (Seppälä, Solomon, Gilmore, Henske, & O'Malley, ), though trace amounts were detected in the hydrolysate (Table S2). We note that a small amount of sugar was released from the reed canary grass upon autoclaving the media—these are likely soluble sugar components or easily hydrolyzed components of hemicellulose.…”

Top-Down Enrichment Guides in Formation of Synthetic Microbial Consortia for Biomass Degradation

“…From previous work 5,6 , it is known that the anaerobic fungi are enriched in biomass degrading enzymes, but these enzymes are primarily exo-and endo-cellulases. They contain some enzymes for degradation of smaller cellodextran fragments, yet they also have transporters capable of taking up these longer cellulose fragments 19 . In the enriched consortium, the fungi act as the primary degraders of the plant biomass, taking up primarily glucose.…”

Biomass‐degrading enzymes are catabolite repressed in anaerobic gut fungi

“…Within this consortium of anaerobic microbes, anaerobic fungi are the least well‐characterized, but they are some of the most active microbes against lignin‐infused substrates, containing the largest repertoire of biomass‐degrading genes in sequenced fungi . The low abundance of gut fungi in a rumen supplied with high‐starch diets, and conversely high abundance under high‐fiber conditions suggests that these fungi are highly adapted to fiber degradation, which is of particular interest for the hydrolysis of recalcitrant biomass encountered in bioprocessing . However, the first genomes of the anaerobic fungi were sequenced only in the past 5 years due to the problems associated with fungal culture, and difficulties that stem from AT‐richness and extensive genomic repeats .…”

“…However, the first genomes of the anaerobic fungi were sequenced only in the past 5 years due to the problems associated with fungal culture, and difficulties that stem from AT‐richness and extensive genomic repeats . Although sequencing has revealed a treasure trove of biomass‐degrading enzymes, nearly 70% of protein encoding genes in some gut fungal strains still lack functional annotation and may be involved in biomass degradation . Furthermore, emerging technologies seek to employ isolated gut fungi directly in anaerobic bioprocessing, though it is unknown how culture conditions influence enzyme secretion, especially when fungi are deployed in anaerobic consortia …”