Henske et al. Biotechnol Biofuels (2017) 10:305 https://doi.org/10.1186/s13068-017-0997-4 Biotechnology for Biofuels RESEARCH Open Access Transcriptomic characterization of Caecomyces churrovis: a novel, non‑rhizoid‑forming lignocellulolytic anaerobic fungus John K. Henske1, Sean P. Gilmore1, Doriv Knop1, Francis J. Cunningham1, Jessica A. Sexton1, Chuck R. Smallwood2, Vaithiyalingam Shutthanandan2, James E. Evans2, Michael K. Theodorou3 and Michelle A. O’Malley1* Abstract Anaerobic gut fungi are the primary colonizers of plant material in the rumen microbiome, but are poorly studied due to a lack of characterized isolates. While most genera of gut fungi form extensive rhizoidal networks, which likely participate in mechanical disruption of plant cell walls, fungi within the Caecomyces genus do not possess these rhizoids. Here, we describe a novel fungal isolate, Caecomyces churrovis, which forms spherical sporangia with a limited rhizoidal network yet secretes a diverse set of carbohydrate active enzymes (CAZymes) for plant cell wall hydrolysis. Despite lacking an extensive rhizoidal system, C. churrovis is capable of growth on fbrous substrates like switchgrass, reed canary grass, and corn stover, although faster growth is observed on soluble sugars. Gut fungi have been shown to use enzyme complexes (fungal cellulosomes) in which CAZymes bind to non-catalytic scafoldins to improve bio- mass degradation efciency. However, transcriptomic analysis and enzyme activity assays reveal that C. churrovis relies more on free enzymes compared to other gut fungal isolates. Only 15% of CAZyme transcripts contain non-catalytic dockerin domains in C. churrovis, compared to 30% in rhizoid-forming fungi. Furthermore, C. churrovis is enriched in GH43 enzymes that provide complementary hemicellulose degrading activities, suggesting that a wider variety of these activities are required to degrade plant biomass in the absence of an extensive fungal rhizoid network. Overall, molecular characterization of a non-rhizoid-forming anaerobic fungus flls a gap in understanding the roles of CAZyme abundance and associated degradation mechanisms during lignocellulose breakdown within the rumen microbiome. Keywords: Anaerobic fungi, Neocallimastigomycota, Cellulase, Enzyme, Cellulosome Background application in the production of lignocellulose-derived Anaerobic gut fungi are robust degraders of plant bio- chemical products [6]. Most known genera (Anaeromy- mass in the guts of ruminants and other large monogas- ces, Buwchfawromyces, Neocallimastix, Oontomyces, tric herbivorous mammals [1]. Tey have also been Orpinomyces, Pecoramyces, Piromyces) of gut fungi have identifed using microscopy and molecular methodolo- an extensive network of penetrating rhizoids (tapering gies in the digestive tract of herbivorous reptiles [2]. Due mycelia) that aid, alongside enzymatic activity, in biomass to the large amount of biomass-degrading enzymes that colonization and deconstruction [7–11]. However, two these organisms secrete [3–5], they have potential for known genera within the clade of anaerobic fungi (Cae- comyces, Cyllamyces) do not produce rhizoidal networks, *Correspondence: [email protected] but form a limited system simply capable of attaching to 1 Department of Chemical Engineering, University of California, Santa plant biomass [7, 12]. However, like their rhizoidal coun- Barbara, CA 93106, USA Full list of author information is available at the end of the article terparts, non-rhizoid producing gut fungi are profcient © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Henske et al. Biotechnol Biofuels (2017) 10:305 Page 2 of 12 degraders of crude plant biomass. Tis raises the possi- Microscopic analysis suggested that C. churrovis is a bility that there are diferences between the diversity of monocentric fungus, and confrmed that it does not pos- enzymes employed by rhizoid-forming vs. non-rhizoid- sess an extensive rhizoidal network to penetrate biomass. forming fungi, and/or the mechanisms they use for enzy- Rather, C. churrovis forms a large, spherical sporangium matic degradation of lignocellulose. with holdfast structures to attach it to plant biomass While there is a general lack of genomic information and other solid substrates (Fig. 1). Although there is not for the Neocallimastigomycota, recently fve complete an extensive rhizoidal network that aids in biomass dis- genomes and transcriptomes have been published for ruption, these fungi still localize to, and colonize, the rhizoid-forming anaerobic fungi—two representatives cellulose-rich surface of plant biomass. Figure 1 shows from the Piromyces genus, one each from Anaeromy- sections of plant material almost entirely covered in C. ces and Neocallimastix [13], and one from Orpinomyces churrovis sporangia. Sporangia are present within a range [14] (recently reclassifed as Pecoramyces [10]). Interest- of sizes, some near 20 µm in diameter, while others are ingly, this wealth of sequencing data has revealed that greater than 50 µm in diameter. Furthermore, Fig. 1B anaerobic fungi can draw from two modes of biomass- highlights the lytic life cycle of gut fungi, as a fraction degradation via the secretion of freely difusive enzymes of colonized mature zoosporangia rupture and collapse as well as via fungal cellulosomes (complexes of enzymes over time, releasing the cellular contents and motile zoo- tethered together for synergistic action) [13]. However, spores. Tese zoospores likely chemotax towards a car- at the present time, no high-resolution transcriptomic or bon source (e.g., biomass) and initiate the formation of genomic information has been reported for non-rhizoid- new monocentric sporangia. forming isolates. Tis precludes insight into the enzy- While morphological characterization indicates that matic machinery of non-rhizoid-forming fungi, or the the isolated fungal strain is likely a member of the Cae- mode of biomass degradation that they favor. comyces genus, anaerobic fungi are often pleomorphic Here, we describe a novel species of non-rhizoid- and require phylogenetic analysis of conserved genomic forming fungi belonging to the Caecomyces genus iso- sequences to confrm classifcation. Te internal tran- lated from the fecal pellets of a Navajo Churro sheep scribed spacer (ITS) regions are commonly used to deter- collected in 2015. While other Caecomyces isolates mine the genera of newly isolated fungi [18–21]. Te ITS1 have been described using morphological and phyloge- and ITS2 regions for C. churrovis (GenBank #MF460993) netic analyses [15, 16], including some analysis of the were amplifed and sequenced using PCR primers JB206 cellulolytic enzyme activity [17], extensive genomic or and JB205 [20], and subjected to phylogenetic analysis. transcriptomic sequencing has not been completed. By Given the abundance of ITS1 sequences deposited in assembling the frst sequenced transcriptome for an GenBank, we relied primarily on comparative alignment anaerobic gut fungus within the Caecomyces genus, C. with this region for preliminary identifcation and classi- churrovis, our analysis enabled us to identify the range of fcation. While other neighboring DNA sequences, such CAZymes available to a non-rhizoidal genus and test the as the ITS2 [20] and large subunit (28S) [22], have been null hypothesis that additional degradation mechanisms, used for phylogenetic analysis, restricting our analysis mechanical or enzymatic, are not required for dissolu- to ITS1 enabled comparison with the maximum num- tion of plant biomass. Tis isolated fungal strain was ber of GenBank submissions. From this analysis (Fig. 2), assessed for its ability to grow on a range of substrates, the isolated strain C. churrovis clearly clusters with other and demonstrated a greater preference for soluble sub- Caecomyces fungi. Subsequently, additional phylogenetic strates compared to other rhizoid-forming strains that analysis was completed using only sequences from fungi have been analyzed to date [5]. Transcriptome assem- within the Caecomyces genus to ensure that this isolated bly and analysis identifed a broad range of CAZymes strain was signifcantly divergent from previously char- within the genome, including a relative abundance of acterized strains to constitute its classifcation as a novel carbohydrate esterase and hemicellulase (GH 11/12, 43) species. Generation of the phylogenetic tree isolated C. transcripts. Comparison to other sequenced gut fungal churrovis from other Caecomyces strains with a bootstrap isolates also revealed a greater reliance on free enzymes value of 98, indicating that this node occurred in 98% of rather than enzymes bound in fungal cellulosome trees generated during the bootstrap analysis (Additional complexes. fle 1: Figure S1). Tus, the ITS1 region of C. churrovis was signifcantly diferent compared to other Caecomyces Results and discussion strains with ITS1 sequences available on GenBank, sug- Isolation and molecular classifcation of C.