Applied and Industrial Microbiology
Review
19 July 2022

Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycetes) Enables Lignocellulosic Biorefinery Innovation

Special Series: Using Microbes To Make Chemicals from Unconventional Substrates 

SUMMARY

Lignocellulosic biorefineries require innovative solutions to realize their full potential, and the discovery of novel lignocellulose-active enzymes could improve biorefinery deconstruction processes. Enzymatic deconstruction of plant cell walls is challenging, as noncarbohydrate linkages in hemicellulosic sidechains and lignin protect labile carbohydrates from hydrolysis. Highly specialized microbes that degrade plant biomass are attractive sources of enzymes for improving lignocellulose deconstruction, and the anaerobic gut fungi (Neocallimastigomycetes) stand out as having great potential for harboring novel lignocellulose-active enzymes. We discuss the known aspects of Neocallimastigomycetes lignocellulose deconstruction, including their extensive carbohydrate-active enzyme content, proficiency at deconstructing complex lignocellulose, unique physiology, synergistic enzyme complexes, and sizeable uncharacterized gene content. Progress describing Neocallimastigomycetes and their enzymes has been rapid in recent years, and it will only continue to expand. In particular, direct manipulation of anaerobic fungal genomes, effective heterologous expression of anaerobic fungal enzymes, and the ability to directly relate chemical changes in lignocellulose to fungal gene regulation will accelerate the discovery and subsequent deployment of Neocallimastigomycetes lignocellulose-active enzymes.

INTRODUCTION

Conversion of abundant and renewable plant biomass into replacements for petroleum-derived products is a promising avenue to sustainable commodity chemical production, but innovation is required to mature this concept to reality. Lignocellulose, the compound that makes up secondary cell walls in plants, is a promising feedstock replacement for fossil fuels since it is the most naturally abundant, renewable organic carbon pool in the terrestrial biosphere (1). Biorefineries that aim to upgrade lignocellulosic biomass into value-added chemicals are termed second-generation biorefineries, contrasting with their more simplistic, starch-based, first-generation counterparts (2). Second-generation biorefineries process nonfood crops that grow on marginal land (3) or make use of preexisting agricultural waste streams (4), alleviating competition between food production and bioproduct synthesis that plagued the first-generation concept. However, second-generation biorefineries are accompanied by their own unique challenges that were not inherent to the first-generation concept. Deconstruction of complex bonds in lignocellulose, which contrasts with the deconstruction of simple bonds in starch, is a central challenge for second-generation biorefineries because lignocellulose must first be disassembled before complementary processes upgrade small molecules into value-added target bioproducts (5, 6). The discovery of new enzymes that deconstruct lignocellulose is an essential component of lignocellulosic biorefinery innovation (7) and the focus of this review.
Feedstock deconstruction in second-generation biorefineries is complex, and biological deconstruction of lignocellulose requires a much more diverse array of enzymes than depolymerization of starch. Noncarbohydrate portions of secondary plant cell walls, namely, hemicellulosic sidechains and lignin, protect labile carbohydrates and are themselves valuable sources of carbon for the synthesis of bioproducts (810). The motivation for improving biological deconstruction of hemicellulose and lignin is therefore 2-fold; depolymerization of noncarbohydrate parts of the cell wall increases access to more easily converted carbohydrates and, second, allows parallel upgrading of a range of monomeric degradation products, including pentose sugars, hexose sugars, and monoaromatics (8, 11, 12). The resulting diversity of degradation products is an opportunity to coproduce a range of diverse commodity chemicals, whereas first-generation pipelines were typically focused on producing a single high-yield chemical (8). The prospect of discovering undescribed lignin-active enzymes is a particular focus of enzyme discovery efforts, as lignin is a highly recalcitrant compound, and very few biological routes for lignin depolymerization have been reported.
Anaerobic environments are potential sources of enzymes and organisms for industrial lignocellulose deconstruction, but the applications of the enzymes and organisms from this environment are not fully realized. Some anaerobic environments, such as herbivore guts, harbor highly active communities of anaerobic microorganisms that cooperatively degrade lignocellulose on rapid timescales and so might serve as sources for novel lignocellulose-degrading enzymes (13). However, prospecting for lignin-active enzymes in anaerobic environments is considered counterintuitive by some, as most lignin-active enzymes are oxidative and mechanistically require molecular oxygen (14). Despite the lack of discovered enzymes and described mechanisms, there is evidence that some modification of lignin does occur in anaerobic environments such as ruminant guts (1518). Further, some recent publications hypothesize that anaerobes may possess yet undescribed lignin depolymerization systems that employ radical chemistry, mirroring aerobic mechanisms (1923).
Neocallimastigomycetes, which are often called anaerobic gut fungi (AGF), are a class of lignocellulose deconstruction specialists that inhabit the guts of large herbivores (2426). While these organisms are unwieldy and enigmatic, a growing number of isolates have been sequenced and characterized (24, 2729), which has revealed an impressive array of carbohydrate-active enzymes (CAZymes) and synergistic enzyme complexes termed fungal cellulosomes. The unique physiology, biochemistry, and genomic content of the AGF make them excellent candidates for potentially harboring novel enzymes and provide an opportunity to meld biotechnological innovation and basic biological research (24, 30).
Here, we review lignocellulose deconstruction by anaerobic fungi, discussing opportunities for discovering novel lignocellulose-active enzymes and focusing on the potential for discovering novel enzymes that disrupt noncarbohydrate bonds in hemicellulose side chains and lignin. We first briefly summarize the general features of plant cell wall polymers and the enzymes that deconstruct them and highlight the importance of hemicellulosic side chains and lignin in the context of lignocellulose deconstruction. We then discuss the characterized enzymes known to be deployed by Neocallimastigomycetes and provide informed speculation about what other valuable, uncharacterized enzymes AGF might possess.

LIGNOCELLULOSE IS A COMPLEX MATERIAL, AND ITS CONSTITUENT POLYMERS ARE BROKEN DOWN BY DIVERSE ENZYMES

Lignocellulose Architecture Protects Labile Carbohydrates with Side Chains and Lignin

Lignocellulose is a complex, heterogeneous composite of cellulose, hemicellulose, and lignin, and these three polymers have various complexities, heterogeneity, branching, aromaticity, hydrophobicity, and, by extension, degradability (11) (Fig. 1). Various lignocelluloses, in turn, have different ratios of constituent polymers, a property lending itself to variable total lignocellulose recalcitrance from plant to plant and even between tissue types within a single plant (8, 11). In a typical plant cell wall, cellulose is the most abundant polymer by mass, followed by lignin and then hemicellulose, but all three are typically present in significant quantities. Biological deconstruction of secondary plant cell walls therefore requires a diverse ensemble of enzymes working in concert to break a variety of chemical bonds.
FIG 1
FIG 1 Covalent linkages between hemicellulose and lignin protect labile cellulose and hemicellulose from enzymatic hydrolysis, highlighting the need for more diversity in biorefinery enzymes. Lignocellulose is a complex composite material that forms cell walls in higher plants. Its three components, lignin (5 to 35% dry weight), hemicellulose (10 to 50% dry weight), and cellulose (9 to 80% dry weight), are bundled together in interwoven macrofibrils that constitute the cell wall (11). Each macrofibril contains a core of cellulose microfibers which are noncovalently associated with one another and hemicelluloses. Hemicelluloses form intermediates between cellulose and lignin and are decorated with both carbohydrate and noncarbohydrate side chains. Although hemicelluloses’ interactions with cellulose are noncovalent, hemicelluloses are covalently bonded to lignin through aromatic side chains. Lignin is the most recalcitrant portion of the cell wall and is an aromatic polymer.
Cellulose is the single most abundant, labile, and homogenous lignocellulose component. Plants organize cellulose into bundles of β-1,4 linked chains which form the core of macrofibrils (Fig. 1) (11). Three enzyme activities degrade cellulose into glucose monomers: endoglucanase, exoglucanase/cellobiohydrolase, and β-glucosidase (Fig. 2) (31). Current industrial applications largely source cellulases from aerobic fungi and anaerobic bacteria, and these enzymes are widely available (32). These well-characterized enzymes are not a focus of this review, but we consider genomic cellulase content a potential indicator of yet undescribed lignocellulose-active enzymes (30).
FIG 2
FIG 2 Bond types in lignin are more diverse than carbohydrate bonds and require specific enzymatic, oxygen-dependent enzymes. Neocallimastigomycetes are powerful degraders of lignocellulose, but they are not known to encode lignin-active enzymes. On the other hand, these fungi encode a large diversity of carbohydrate-active enzymes and hemicellulose side chain-modifying enzymes. Anaerobic fungi access carbohydrates protected by lignin by cleaving lignin-side chain linkages, but it is also possible that Neocallimastigomycetes modify lignin by undocumented means and yet-undiscovered enzymes systems. LPMO, lytic polysaccharide monooxygenase.
Hemicelluloses are diverse carbohydrate polymers made of mixed pentose and hexose sugars. They are decorated with carbohydrate, acetyl, and aromatic side chains, a property that contrasts them with cellulose (Fig. 1). Hemicellulosic side chains are multipurpose, protecting hemicelluloses from hydrolysis, allowing branching, and covalently linking them to lignin (10, 11, 33, 34). The greater diversity of chemical bonds in hemicelluloses requires more enzymatic variety for complete deconstruction; endoxylanase, xylobiase, feruloyl/coumaroyl esterase, and acetyl xylan esterase activities are needed to degrade hemicellulose into its constituent sugar, aromatic, and acetyl components (Fig. 2) (10).
Lignin is the phenylpropanoid heteropolymer that makes up the most recalcitrant portions of plant cell walls (Fig. 1). Lignin contributes to lignocellulose recalcitrance by physically blocking CAZyme access to carbohydrate polymers and irreversibly binding and inactivating these enzymes (35). Since lignin can constitute up to 30% of the dry weight of plant cell walls and, at the same time, inhibit CAZymes, lignin depolymerization is the central challenge of plant cell wall deconstruction in both natural and industrial settings. Further, second-generation biorefineries seek to depolymerize and upgrade lignin itself, providing additional motivation to lignin deconstruction (8). Efforts to discover novel enzymes for enhancing lignin breakdown in biorefineries recently yielded a novel ligninase encoded by an aerobic fungus (36) and suggested anti-lignin activity in termite microbiomes (23).

Characterized Lignin-Active Enzymes Are Limited in Diversity and Exclusively Aerobic

Aerobic fungi are the most common source for industrially useful lignin-active enzymes, and most of the enzymes sourced from these organisms explicitly require molecular oxygen or oxygen-derived free radicals to carry out lignin deconstruction (37). Briefly, aerobic fungi in the subkingdom Dikarya encode manganese peroxidases, versatile peroxidases, β-etherases, and lignin peroxidases (14, 38). Additionally, laccases are a type of multicopper oxidase encoded by both aerobic fungi and aerobic bacteria (14, 38). These enzymes and carbohydrate-active lytic polysaccharide monooxygenases (LPMOs) operate via oxidative mechanisms that are not suited to the reducing conditions presented by anaerobic environments. There are currently no lignin-active enzymes sourced from anaerobes.
Anaerobic lignin deconstruction is controversial because of the absence of documented means for the process to occur; however, large amounts of lignocellulose are deconstructed rapidly in anaerobic environments such as herbivore guts. Lignin depolymerization in animal guts has been reported (15, 17, 18, 39, 40) but not widely accepted because the methods available when these studies were carried out did not directly interrogate lignin-lignin linkages. Some studies measured lignin monomers freed from lignocellulose or observed changes in the total lignin content of processed lignocellulose and attributed these changes to the removal of side chains from hemicelluloses (16, 41, 42). Microbes in herbivore guts, including AGF, are known to remove aromatic side chains from hemicellulose (43), but future efforts will determine whether any of the monoaromatics freed from lignocellulose by Neocallimastigomycetes are derived from lignin.
Methods that interrogate lignin-lignin bonds directly might be able to fully resolve whether anaerobic lignin deconstruction occurs in settings where lignocellulose is anaerobically disassembled. Recent advances in two-dimensional heteronuclear single quantum shift coherence nuclear magnetic resonance (2D-HSQC-NMR) spectroscopy have enabled direct interrogation of bond types in lignin and whole plant cell walls (44, 45). Advances in 2D-HSQC-NMR have recently yielded the discovery of a novel lignin-active enzyme and allowed observation of hypoxic lignin modification by consortia enriched from termite gut microbiomes (23, 36). Applying 2D-HSQC-NMR methods to interrogate the activity of Neocallimastigomycetes might help resolve the ambiguity surrounding their interactions with lignin and AGF, as lignocellulose deconstruction specialists would be a logical anaerobic system to apply these advanced methods.

NEOCALLIMASTIGOMYCETES ARE ANAEROBIC LIGNOCELLULOSE DECONSTRUCTION SPECIALISTS WITH DIVERSE CAZYMES, UNIQUE MORPHOLOGY, AND UNCHARACTERIZED GENES

Neocallimastigomycetes Deconstruct Lignocellulose with Extensive CAZyme Content

Neocallimastigomycetes are highly proficient degraders of lignocellulose, and their strategies could inspire biorefinery innovation. The proficiency of AGF for deconstructing carbohydrate polymers is well established, and their ability to deconstruct both lignocellulose and purified cellulose has been documented previously (25, 46). Isolated anaerobic fungi and AGF-dominated microbial consortia are effective degraders of purified cellulose, eliminating as much as 80% of the dry mass of supplied cellulose (1% wt/vol) during a weeklong experiment (46, 47). Neocallimastigomycetes are also highly proficient degraders of lignocellulose, solubilizing more than 75% (by mass) of grassy lignocelluloses (1% wt/vol) in monococultures, cocultures, and AGF-dominated consortia (46). In other documented instances, some AGF, including Piromyces indianae and Neocallimastix frontalis, grow on and depolymerize angiosperm woods such as poplar, eucalyptus, birch, sweetgum, and aspen (48, 49). The percentage of dry mass solubilized from these woods by Neocallimastix frontalis ranged from 2 to 32%, indicating a possible effect of lignin type on the ability of anaerobic fungi to solubilize lignocellulose (49). Further, anaerobic fungi did not appear to deconstruct wood from gymnosperm trees, such as pine, under these conditions.
The CAZyme content and diversity encoded by Neocallimastigomycetes genomes partially explain the superior capacity of anaerobic fungi for lignocellulose deconstruction. Whole-genome sequencing and annotation of 7 gut fungal genomes published to date revealed that anaerobic fungi, on average, encode about 4-fold more CAZymes than comparable phylogenetic groups containing Trichoderma reesei and Aspergillus niger, the current sources of industrial cellulolytic enzyme cocktails (20, 2527) (Fig. 3). The bulk of this difference can be attributed to the massive amount of carbohydrate esterase (CE), commonly involved in hemicellulose side chain modification, and glycoside hydrolase (GH) encoded by AGF genomes (Fig. 3). Among cellulolytic fungi, AGF encode the most diverse array of enzyme families; 43 GH families, 28 glycosyl transferase families, 9 CE families, 6 polysaccharide lyase (PL) families, and 19 carbohydrate-binding module (CBM) families are represented in the genomes of Neocallimastigomycetes (27, 29, 48, 50, 51).
FIG 3
FIG 3 The number of CAZymes in a fungal genome varies, but anaerobic gut fungi have the most, and some of this numerical superiority is derived from hemicellulose side chain-modifying enzymes. Anaerobic gut fungi (Neocallimastigomycetes) have more CAZymes than other fungi when compared to phylogenetic groups containing commonly deployed industrial fungi such as Saccharomyces cerevisiae (Saccharomycotina) and various species of Aspergillus (Eurotiomycetes). They also have higher average CAZyme contents than the class containing mushrooms and wood-decaying fungi (Agaricomycetes) and a class of fungi commonly associated with organic matter remineralization (Sordariomycetes). Abbreviations for the types of CAZyme are as follows: GH, glycoside hydrolase; GT, glycosyl transferase; PL, polysaccharide lyase; CE, carbohydrate esterase; CBM, carbohydrate binding module; and AA, auxiliary activities. Notably, the numerical superiority of Neocallimastigomycetes CAZymes is partially due to a disproportionate amount of encoded CE genes, which are implicated in modifying hemicellulose side chains. In contrast, Neocallimastigomycetes are currently understood to be deficient in AA enzymes, which include oxidative lignin-active enzymes and lytic polysaccharide monooxygenases. Data in this figure were retrieved from JGI’s Mycocosm (https://mycocosm.jgi.doe.gov/mycocosm/home).

Neocallimastigomycetes Morphology and Lifecycle Enhance their Lignocellulolytic Ability

Anaerobic gut fungi possess filamentous morphology, complex lifecycles, and anaerobic metabolism, rendering them unique among characterized lignocellulolytic organisms (24, 29, 52). Many mature AGF develop an extensive rhizoid network, not dissimilar in appearance to the roots of higher plants (Fig. 4). These microscopic roots intercalate lignocellulose and likely enable the delivery of tailored enzyme payloads to relevant polymers in lignocellulose (Fig. 4). A recent investigation of protein localization across anaerobic fungal life stages suggested that lignocellulose-active enzymes are localized to the rhizoids in mature AGF cells and also found zoospores enriched in domains associated with lignocellulose hydrolysis (52). The presence of CAZyme domains in rhizoids could allude to the role of fungal microrhizoids in increasing the surface area of fungal cells so that fungi can more precisely localize enzymes to their target polymers (Fig. 4). Zoospores might be enriched in CAZyme domains to aid in the colonization of and encystment on newly ingested lignocellulose.
FIG 4
FIG 4 Neocallimastigomycetes deliver lignocellulose-active enzymes to lignocellulose polymers with microrhizoid networks. Anaerobic gut fungi colonize intact lignocellulosic biomass (left) and deliver enzymes to the component polymer through their extensive microrhizoid networks (middle). Eventually, gut fungi depolymerize cellulose to the greatest extent, hemicellulose to a large extent, and free aromatics from hemicellulose side chains (right). The most complete knowledge to date suggests anaerobic fungi colonize lignocellulose and modify cellulose, hemicellulose, and hemicellulose sidechains, but not lignin. Fungi represented in the image are not to scale and are represented in a much larger format than the polymers they act on.

Enzyme Synergy, Facilitated by Cellulosomes, Enhances Polymer Deconstruction

Cellulosomes are large lignocellulolytic enzyme complexes found in both Neocalli mastigomycetes and anaerobic bacteria. These complexes colocalize lignocellulose-active enzymes through noncovalent interactions between enzyme-fused dockerin domains and a central, cohesin-containing scaffoldin, and this colocalization is thought to partly explain the enhanced biomass hydrolysis exhibited over free enzymes (29). Endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase, feruloyl/coumaroyl esterase, xylanase, mannanase, and acetyl xylan esterase activities are biochemically verified to be present in anaerobic fungal cellulosomes (5355). Direct comparison of the fungal cellulosome and free enzymes isolated from culture supernatants of Piromyces sp. strain E2 suggests cellulosomal synergy. Cellulosomes achieved complete conversion of 2% (wt/vol) Avicel to glucose, while free enzymes accomplished only 25% conversion over 12 days (56). Another study comparing cellulosome and free enzyme systems of Neocallimastix frontalis observed a similar advantage in cotton solubilization (57, 58).
Fungal cellulosomes and those of anaerobic bacteria are similar but with several key differences. The dominant component of Neocallimastigomycetes cellulosomes, similar to all known cellulosome-producing bacteria, is a GH48 exocellulase (24, 59). An endoglucanase in the GH9 family is also among the dominant fungal cellulosome components in fungi from the genus Piromyces (60), and GH10/GH11 endoxylanases and GH5 endoglucanases are abundant in fungal cellulosomes from the genera Neocallimastix and Anaeromyces (29, 54). Fungal cellulosomes diverge from bacterial cellulosomes in their inclusion of GH6-containing proteins, which most commonly act as cellobiohydrolases, and the presence of GH1/3 family β-glucosidases and GH43 β-xylosidases, which cleave cellobiose or xylobiose into their monomers (24, 29, 61). The inclusion of β-glucosidases, unique to fungal cellulosomes, causes these enzyme complexes to produce glucose as a sole product from crystalline cellulose hydrolysis (56).
How enzymes achieve increased efficacy in cellulosomes is still unclear, but designer cellulosomes, using bacterial dockerins and cohesins, illuminate the disproportionate benefits of colocalizing some lignocellulolytic activities. The core synergism in lignocellulose degradation by bacterial cellulosomes appears to occur between GH48 exocellulases and GH9 endoglucanases (62). In one study, the trifunctional complexes of recombinant Clostridium cellulovorans enzymes were generally 2- to 6-fold less active those of than native cellulosomes from C. cellulovorans, but the GH48 and GH9 subunits contributed ~75% of the overall enzymatic activity regardless of the third subunit when tested against crystalline cellulose. Colocalization of enzymes also seems to increase efficacy against complex lignocellulosic substrates. When tested on wheat straw, complexation of GH48 and GH9 with a GH10/CE1 multifunctional enzyme enhanced degradation 4-fold over a mixture of the free enzymes (62). Similar investigations into the core enzymatic synergies underlying anaerobic fungal cellulosome activity have not been done using pure native or reconstituted cellulosomes with defined composition. These are difficult to obtain, as methods for purifying homogenous fungal cellulosomes are not well established and many fungal cellulosome proteins express poorly or are not similarly glycosylated in model organisms, which may affect function (29, 63). Concerted efforts to characterize the biochemistry of pure fungal cellulosomes will improve the mechanistic understanding of lignocellulose hydrolysis by cellulosomes and may potentially yield better enzyme cocktails, enhancing the economic sustainability of industrial bioprocesses.

Enzymes for Hemicellulose Side Chain Removal Increase the Efficacy of Cellulosomes

Since hemicelluloses are more amorphous than cellulose, colocalization of lignocellulose-active enzymes was once considered less beneficial for hemicellulose degradation. Evidence shows that incorporating enzymes with endoxylanase, carbohydrate esterase, and xylobiase activities into synthetic cellulosomes increases the rate and extent of wheat straw degradation compared to free enzymes (62, 64, 65). The beneficial inclusion of a hemicellulose side chain-modifying enzyme (CE1) highlights the advantage of colocalizing enzymes that remove acetyl groups and aromatic moieties from the hemicellulose backbone, increasing xylanase access to the polymer backbone (66). Enzymes that remove acetyl groups and aromatic ester linkages from hemicellulose are abundant in Neocallimastigomycetes genomes, and anaerobic fungi are documented to depolymerize glucan and mixed glucan/xylan polymers (29, 67).
Additional efforts to describe side chain-modifying enzymes from anaerobic fungi are needed, but at least one of these recombinant enzymes has been studied in the context of cellulosomal synergy. The enzyme EstA is a recombinant dockerin-containing feruloyl esterase from Piromyces equi; one study found that supplementing EstA with a xylanase from Trichoderma viride boosted the release of ferulic acid from destarched wheat bran over 100-fold, illustrating how esterase and xylanase activities synergize to degrade a lignocellulosic substrate (10). On average, there are approximately 35 CE1 enzymes, which commonly possess activity against ester bonds linking aromatic side chains to hemicellulose, in each annotated AGF genome (Fig. 3) (50).

NEOCALLIMSTIGOMYCETES AS BIOTECH PLATFORMS: FROM ENZYME DISCOVERY TO METABOLIC ENGINEERING OF ANAEROBIC FUNGI

Unannotated Genes of Neocallimastigomycetes Might Further Explain their Lignocellulolytic Power

The AGF are powerful degraders of lignocellulose, and their extensive uncharacterized gene content might explain the unknown mechanisms by which they achieve their lignocellulolytic ability. Despite the diversity and abundance of AGF lignocellulose-active enzymes, summarized in this review, 60 to 75% of all open reading frames in AGF genomes are not functionally annotated (30, 50). The very adenine thymine (AT)-rich genomes of Neocallimastigomycetes, ranging up to 80% AT content in some regions, are highly dissimilar to those of other fungi and other anaerobic microbes, presenting a gene characterization challenge (28, 68). There are, additionally, long stretches of AT repeats in the genomes of Neocallimastigomycetes which not only increase the difficulty of functional gene annotation but introduce technical challenges in sequencing and genome assembly. However, AGF unannotated gene content is an opportunity to discover entirely novel enzymes and biochemistries which operate without oxygen, and this search is ongoing.
Initial assembly and annotation of AGF transcriptomes and genomes illuminated the abundance of recognizable CAZymes previously discussed, but because these annotation strategies depend on a priori knowledge of gene sequences, they are only able to annotate homologs of described genes confidently. More advanced statistical methods combined with highly controlled experiments are therefore required to interrogate yet-uncharacterized AGF genes. Differential expression, which statistically interrogates gene expression dynamics under different regulatory regimes, has so far been invaluable to identifying entirely novel biomass-active enzymes in AGF genomes (69). Catabolite repression patterns, mediated by antisense transcripts, might be one mode by which AGF conserve resources when there is an abundance of glucose in the local environment (24, 29, 70, 71). Future studies will need to use differential expression to examine gene induction responses to other stimuli, and these techniques will be essential to interrogating potential interactions of AGF with lignin (72, 73).
Future efforts to describe uncharacterized AGF genes will rely on applying differential expression in combination with advanced chemistry methods to link gene expression to chemical reactions facilitated by fungal enzymes. 2D-HSQC-NMR spectroscopy could be particularly valuable for finding novel lignin-active enzymes, as 2D-HSQC-NMR directly interrogates the types and relative abundances of chemical bonds in lignocellulose. Employing 2D-HSQC-NMR in tandem with differential expression might allow tying specific gene expression to the cleavage of interunit linkages in lignin. Additionally, the structurally complex Neocallimastigomycetes are excellent candidates for characterization with spatially resolved proteomic methods such as matrix-assisted laser desorption–time of flight mass spectrometry (MALDI-TOF MS). Applying MALDI-TOF MS to AGF could help resolve which proteins are localized to specific structures, such as sporangia, microrhizoids, organelles, and zoospores.

The Roles of Redox Reactions in Neocallimastigomycetes’ Biology Are Unknown

Neocallimastigomycetes do not encode the lignocellulose-active redox enzymes that typically complement hydrolysis-based lignocellulose degradation by aerobic fungi, but the redox chemistry of anaerobic fungi is underdescribed. Classic redox enzymes such as LPMOs, cellobiose dehydrogenases, and oxidative lignin-active enzymes, which fall into the CAZyme auxiliary activity classification, are notably absent in AGF genomes (Fig. 3) (74). The exclusion of these enzymes from AGF genomes might seem logical since they are oxygen-dependent. However, oxygen intrusion in the rumen, via rumination, is possible, and AGF tolerate oxidative stress by encoding an average of 15 copies of glutathione peroxidase and a single superoxide dismutase per genome (https://mycocosm.jgi.doe.gov) (75). Further, anaerobic fungi are known to possess enigmatic redox-active organelles known as hydrogenosomes that are suspected to be involved in energy conservation, but it is yet unclear what benefit they impart (72, 76). Future efforts to describe the redox chemistry of AGF should consider the role of redox reactions in lignocellulose hydrolysis as well as energy conservation, as redox reactions are an essential aspect of lignocellulose hydrolysis for analogous, aerobic organisms.

Advancements in Anaerobic Fungal Gene Manipulation Will Help Ascribe Function to Uncharacterized Neocallimastigomycetes Genes

Heterologous expression is a natural way to attempt characterization of novel lignocellulose deconstruction enzymes, as they can be produced by a model host, purified, and then exposed to their target substrate, directly linking them to measured catalytic activity. Beyond leveraging gene regulation patterns in response to stimuli, the rapid advancement of methods for expressing AGF proteins in model hosts is one promising avenue for characterizing uncharacterized lignocellulose-active enzymes. These methods have already been successfully deployed to describe anaerobic fungal membrane proteins, such as fluoride exporters and sugar transporters (77, 78). However, Neocallimastigomycetes also suffer from the inconvenience of being undercharacterized organisms with AT-biased genomes, which can make the heterologous expression of AGF proteins a significant challenge. Due to their odd codon usage (68), expression of AGF genes can, in some cases, present toxicity issues to platform organisms that were partially but not completely resolved by adapting codon usage to the host biases (79).
Employing direct genome engineering techniques to create gene deletion or gene introduction strains could be particularly essential to resolving outstanding questions about the life cycle and central metabolic pathways of anaerobic fungi (73). Electroporation has been used to engineer chytrid fungi, relatively closely related to Neocallimastigomycetes, but has not yet been successfully used for engineering anaerobic fungi (80). Similarly, deploying transkingdom gene transfer methods, such as Agrobacterium tumefaciens-mediated gene transfer, could be promising given the apparent prevalence of horizontally transferred genes in anaerobic fungal genomes (29). However, because anaerobic fungi grow optimally at relatively high temperatures (39°C) and without oxygen, the disconnect between their growth conditions and those of Agrobacterium tumefaciens (30°C, aerobic) must be resolved before these methods can be deployed to manipulate anaerobic fungal genomes.

CONCLUSION

The Neocallimastigomycetes are specialized lignocellulose degraders, but realizing their full potential for biotechnology will require further descriptions of their basic biology to explain how they achieve their lignocellulolytic capacity. Recent advances indirectly engineering anaerobic fungal genomes and heterologously expressing enzymes promise to move the field rapidly toward the technological advancements in describing anaerobic fungal biology and, given the amount of forethought ascribed to deploying these organisms in biotechnological settings, the deployment of lessons learned from these fungi in second-generation biorefineries might rapidly follow. Anaerobic fungi will likely deliver nonstandard lignocellulose-active enzymes for deployment in biorefineries, and while Neocallimastigomycetes might harbor a variety of undiscovered, useful enzymes, their potential to harbor lignin-active enzymes is one of the most pressing questions, as the absence of anaerobic lignin-active enzymes in ruminant guts offers a potential paradox between the completeness of lignocellulose deconstruction and the accepted paradigm that lignin deconstruction cannot occur without oxygen.

ACKNOWLEDGMENTS

We acknowledge funding from the Office of Biological and Environmental Research of the U.S. Department of Energy grant DE-SC0020420 (M.A.O.), the Institute for Collaborative Biotechnologies grants W911NF-09–D-0001 and W911NF-19-2-0026 (M.A.O.), and the Office of Biological and Environmental Research of the U.S. Department of Energy Joint BioEnergy Institute (JBEI, http://www.jbei.org) contract DE-AC02–05CH11231 (Lawrence Berkeley National Laboratory).
We used BioRender to create Fig. 1, Fig. 2, and Fig. 4 and GraphPad Prism v9.3.1 to create Fig. 3.
We acknowledge Patrick Lane of ScEYEnce Studios for his help finalizing figures for publication.

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Author Bios

Thomas S. Lankiewicz
Department of Chemical Engineering, University of California, Santa Barbara, California, USA
Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, California, USA
Joint BioEnergy Institute, Emeryville, California, USA
Thomas S. Lankiewicz is a Ph.D. candidate in the Department of Ecology, Evolution, and Marine Biology at the University of California, Santa Barbara. He studies the biotechnological applications of anaerobic microbes for producing commodity chemicals from plant cell walls. Thomas’s thesis work focuses primarily on the interactions of anaerobic fungi with lignin, the recalcitrant, noncarbohydrate fraction of plant material. He received a bachelor’s degree in biology from Grinnell College in Grinnell, IA, and a master’s degree in marine biosciences from the University of Delaware in Newark, DE. Thomas is chiefly interested in how an increased understanding of biology, especially microbiology and microbial ecology, can inform synthetic biology and help humanity have its best chance of achieving a sustainable future.
Stephen P. Lillington
Department of Chemical Engineering, University of California, Santa Barbara, California, USA
Stephen P. Lillington is a Ph.D. candidate in the Department of Chemical Engineering at the University of California, Santa Barbara, where his research applies complementary modeling and experimental methods to engineer protein machinery from anaerobic microbes for applications in bioprocessing and biomanufacturing. He received a bachelor’s degree in chemical engineering from Northwestern University in Chicago, IL. Broadly, Stephen’s research interests involve using a molecular-scale lens and modeling-guided approaches to engineer biomolecular technologies as solutions for grand challenges in environmental sustainability and human health.
Department of Chemical Engineering, University of California, Santa Barbara, California, USA
Joint BioEnergy Institute, Emeryville, California, USA
Michelle A. O’Malley is a professor in the Department of Chemical Engineering at the University of California, Santa Barbara, and the associate director of UCSB’s Bioengineering Program. Her research group harnesses the biotech potential of unusual microorganisms for sustainable chemical production, bioremediation, and natural product discovery. O’Malley earned a B.S. in chemical engineering and biomedical engineering from Carnegie Mellon University, a Ph.D. in chemical engineering from the University of Delaware, and was a USDA-NIFA postdoctoral fellow in the Department of Biology at MIT. O’Malley was named one of the 35 “Top Innovators Under 35” in the world by MIT Technology Review and one of the 10 “Scientists to Watch” by Science News and is the recipient of the Presidential Early Career Award for Scientists and Engineers (PECASE). Her work has also been recognized with the Allan P. Colburn Award from the AIChE, the ASM Award for Early Career Applied and Biotechnological Research, a DOE Early Career Award, an NSF CAREER award, and a Camille Dreyfus Teacher-Scholar Award. She was elected to the American Institute of Medical and Biological Engineers in 2020.

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cover image Microbiology and Molecular Biology Reviews
Microbiology and Molecular Biology Reviews
Volume 86Number 421 December 2022
eLocator: e00041-22
PubMed: 35852448

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Published online: 19 July 2022

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Keywords

  1. Neocallimastigomycetes
  2. anaerobic fungi
  3. biorefinery
  4. enzymes
  5. hemicellulose sidechains
  6. lignin
  7. lignocellulose

Contributors

Authors

Thomas S. Lankiewicz
Department of Chemical Engineering, University of California, Santa Barbara, California, USA
Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, California, USA
Joint BioEnergy Institute, Emeryville, California, USA
Stephen P. Lillington
Department of Chemical Engineering, University of California, Santa Barbara, California, USA
Department of Chemical Engineering, University of California, Santa Barbara, California, USA
Joint BioEnergy Institute, Emeryville, California, USA

Notes

The authors declare no conflict of interest.

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