Experimentally Validated Reconstruction and Analysis of a Genome-Scale Metabolic Model of an Anaerobic Neocallimastigomycota Fungus

Abstract

Anaerobic gut fungi in the phylum Neocallimastigomycota typically inhabit the digestive tracts of large mammalian herbivores, where they play an integral role in the decomposition of raw lignocellulose into its constitutive sugar monomers. However, quantitative tools to study their physiology are lacking, partially due to their complex and unresolved metabolism that includes the largely uncharacterized fungal hydrogenosome. Modern omics approaches combined with metabolic modeling can be used to establish an understanding of gut fungal metabolism and develop targeted engineering strategies to harness their degradation capabilities for lignocellulosic bioprocessing. Here, we introduce a high-quality genome of the anaerobic fungus Neocallimastix lanati from which we constructed the first genome-scale metabolic model of an anaerobic fungus. Relative to its size (200 Mbp, sequenced at 62× depth), it is the least fragmented publicly available gut fungal genome to date. Of the 1,788 lignocellulolytic enzymes annotated in the genome, 585 are associated with the fungal cellulosome, underscoring the powerful lignocellulolytic potential of N. lanati. The genome-scale metabolic model captures the primary metabolism of N. lanati and accurately predicts experimentally validated substrate utilization requirements. Additionally, metabolic flux predictions are verified by 13C metabolic flux analysis, demonstrating that the model faithfully describes the underlying fungal metabolism. Furthermore, the model clarifies key aspects of the hydrogenosomal metabolism and can be used as a platform to quantitatively study these biotechnologically important yet poorly understood early-branching fungi. IMPORTANCE Recent genomic analyses have revealed that anaerobic gut fungi possess both the largest number and highest diversity of lignocellulolytic enzymes of all sequenced fungi, explaining their ability to decompose lignocellulosic substrates, e.g., agricultural waste, into fermentable sugars. Despite their potential, the development of engineering methods for these organisms has been slow due to their complex life cycle, understudied metabolism, and challenging anaerobic culture requirements. Currently, there is no framework that can be used to combine multi-omic data sets to understand their physiology. Here, we introduce a high-quality PacBio-sequenced genome of the anaerobic gut fungus Neocallimastix lanati. Beyond identifying a trove of lignocellulolytic enzymes, we use this genome to construct the first genome-scale metabolic model of an anaerobic gut fungus. The model is experimentally validated and sheds light on unresolved metabolic features common to gut fungi. Model-guided analysis will pave the way for deepening our understanding of anaerobic gut fungi and provides a systematic framework to guide strain engineering efforts of these organisms for biotechnological use.

Co‑cultivation of the anaerobic fungusCaecomyces churroviswithMethanobacterium bryantiienhances transcription of carbohydrate binding modules

“…70,71 A list of genes associated with the fungal hydrogenosome of the C. churrovis strain was constructed based on homology with known hydrogenosome components, shown in Supplemental Table 3. FASTA sequences from known hydrogenosomal components identified in the fungal strain Neocallimastix lanati 4 were aligned to filtered model proteins of C. churrovis using the blastp alignment program in MycoCosm. One or more genes within the C. churrovis genome aligned to all listed hydrogenosomal enzymes found in N. lanati.…”

“…As shown in Supplemental Table 3, 21 genes were homologous to both pyruvate formate lyases (PFLs) that were identified in the N. lanati genome. 4 This enzyme reversibly converts pyruvate and CoA into acetyl-CoA and formate, which plays a central role in anaerobic glucose fermentation. 72 It has been shown that this enzyme is functional in hydrogenosomes of the anaerobic fungal species Piromyces sp.…”

“…Anaerobic fungi are efficient degraders of recalcitrant lignocellulosic biomass that are found in the guts of herbivores. The high number of CAZymes (carbohydrate active enzymes) that anaerobic fungi produce has driven efforts to collect genomic and transcriptomic data for a variety of emerging anaerobic fungal species [1][2][3][4][5] . Gut fungi function within a community of biomass-degrading bacteria, protozoa, and methanogenic archaea linked by complex metabolic interactions and functional redundancy.…”

Modular dynamic biomolecular modelling with bond graphs: the unification of stoichiometry, thermodynamics, kinetics and data

“…Such models have the potential to ‘predict phenotype from genotype’ [1–3] and hence to ‘transform bioscience and medicine’ [4]. Critical to understanding the large-scale metabolism within cells is the stoichiometric approach [5–8], which has had notable successes including the genome-scale reconstruction of the metabolism of Escherichia coli [9–11] and Neocallimastigomycota fungus [12].…”

Isolation and Biochemical Characterization of Six Anaerobic Fungal Strains from Zoo Animal Feces

“…Anaerobic fungi gain energy through mixed acid fermentation, with acetate, formate, succinate, lactate, ethanol, and hydrogen being the main products [ 32 , 33 ]. Production of hydrogen, acetate, and formate occurs mainly in the hydrogenosomes, organelles which have evolved from mitochondria lacking in AF [ 34 ].…”