Anaerobic fermentation with microbial communities (microbiomes) is an emerging platform for conversion of lignocellulosic biomass to biofuels and bioproducts. The process relies on diverse anaerobic microbes that interact to deconstruct and convert lignocellulosic biomass into a range of products, such as volatile fatty acids (VFAs), which can be achieved by arresting methanogenesis during fermentation. However, defining the distinct functional roles played by various fungi and bacteria during anaerobic biodegradation remains poorly understood. Here, we performed parallel enrichment experiments from cow faeces, goat faeces, and anaerobic digester sludge, selecting for fungal or bacterial dominated communities that convert sorghum biomass into VFAs. Subsequently we reconstructed metabolic networks across these enrichments based on recovered bacterial metagenome-assembled genomes (MAGs) and fungal isolate genomes and profiled their metabolic activity using metatranscriptomics to identify potential functional niches. Our findings implicate diverse bacteria affiliated with the Bacteroidales and Lachnospiraceae in the direct conversion of lignocellulosic biomass to propionate and butyrate, respectively, whereas Neocallimastix -dominated fungal enrichments converted lignocellulose to lactate, acetate and formate. Analysis of carbohydrate-active enzymes (CAZymes) revealed fine-scale differences between microbes that expressed unique multi-functional enzymes linking two or more CAZymes together with distinct carbohydrate binding motifs, implicating lignocellulose structure as a key driver of selection and niche differentiation. Most of these multi-functional enzymes localized complementary degradation functions together, likely conferring synergistic degradation effects within and between microbiome members. We anticipate that these findings will help inform efforts to develop synthetic microbiomes with tailored functionality for low-cost conversion of lignocellulosic biomass to fuels and bio-based chemicals. ### Competing Interest Statement The authors have declared no competing interest. Office of Biological and Environmental Research, https://ror.org/0114b2m14, DE-AC02-05CH11231 Office of Science, DE-SC0022142

Although marine plastics fragment and sink into suboxic seafloor sediments, little is known regarding microbiological and material outcomes. Here, open glass microcosms containing hydrocarbon-venting marine sediments were amended with either polymer film (three types of ethylene vinyl alcohol or EVOH, a polyethylene terephthalate or PET, and cellulose acetate or CA) or glass control substrates, submerged in a natural seawater flow-through tank, and sampled immediately after deployment (T0) and at approximately 0.5, 1, 3, and 6 months of incubation (T1-T4). Recovered polymer substrates were analyzed for chemical composition, surface morphology, hydrophobicity, crystallinity, and glass transition, melting, and crystallization temperatures. All substrates were colonized, as per extractable DNA. By sequencing genes encoding 16S rRNA, substrate bacterial communities differed substantially from those in seawater and sediments, even at T0, and changed over time, particularly from T3 to T4 (ANOSIM test, all P = 0.001). Gammaproteobacteria were initially dominant, and Bacteroidota increased over time. No bacterial genera were relatively abundant on the EVOHs, and only one genus, Clostridium, was more abundant on PET. However, several taxa were more abundant on CA, including those from the classes Bacillota and Clostridia, and sulfate-reducing bacteria. According to changes in cellulose backbone spectral abundances of CA polymers, biodegradation was inferred. Evidence for EVOH biodegradation was scant, although abiotic hydrolysis was apparent. Altogether, the polymer substrates were readily colonized, but only CA-according to material alteration and changes to surrounding sediment bacterial communities-appeared to biodegrade, suggesting otherwise slow degradation or broad recalcitrance of the studied plastics in suboxic marine sediments.IMPORTANCEPlastic debris entering the ocean is a worldwide problem. Most plastics sink to the seafloor and become buried in marine sediments, yet ecosystem-associated fates of such accumulations are unknown. Additionally, plastics studied for environmental influence and biodegradation in marine ecosystems rarely include laminate polymers such as the ethylene vinyl alcohol copolymers (EVOH) in addition to core polymers such as polyethylene terephthalate (PET). The results of this study indicated CA biodegradation, weak evidence for EVOH biodegradation, and none for PET over the study time frames. Thus, many plastics are likely to persist in suboxic marine sediments.

ABSTRACT Hydrogenosomes are mitochondrion-derived organelles that produce ATP and H2 to support energy metabolism in anaerobic eukaryotes. H2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H2 production in anaerobic eukaryotes remain incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P)+ and link electron transfer to H2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H2 production in the AF Caecomyces churrovis. Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H2:NAD+ oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE and NuoF) in C. churrovis and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme known from anaerobic prokaryotes. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex for H2 production in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H2 production and altering fungal metabolism.IMPORTANCEH2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H2 production in Neocallimastix was first reported, the molecular mechanism for hydrogenosomal H2 production and redox balance remains unclear. We demonstrate that AF produce H2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H2-producing systems.

Background/Objectives: Anaerobic gut fungi (Neocallimastigomycota) are biotechnologically relevant, lignocellulose-degrading microbes with under-explored biosynthetic potential for secondary metabolites. Untargeted metabolomic profiling with gas chromatography–mass spectrometry (GC-MS) was applied to two gut fungal strains, Anaeromyces robustus and Caecomyces churrovis, to establish a foundational metabolomic dataset to identify metabolites and provide insights into gut fungal metabolic capabilities. Methods: Gut fungi were cultured anaerobically in rumen-fluid-based media with a soluble substrate (cellobiose), and metabolites were extracted using the Metabolite, Protein, and Lipid Extraction (MPLEx) method, enabling metabolomic and proteomic analysis from the same cell samples. Samples were derivatized and analyzed via GC-MS, followed by compound identification by spectral matching to reference databases, molecular networking, and statistical analyses. Results: Distinct metabolites were identified between A. robustus and C. churrovis, including 2,3-dihydroxyisovaleric acid produced by A. robustus and maltotriitol, maltotriose, and melibiose produced by C. churrovis. C. churrovis may polymerize maltotriose to form an extracellular polysaccharide, like pullulan. GC-MS profiling potentially captured sufficiently volatile products of proteomically detected, putative non-ribosomal peptide synthetases and polyketide synthases of A. robustus and C. churrovis. The triterpene squalene and triterpenoid tetrahymanol were putatively identified in A. robustus and C. churrovis. Their conserved, predicted biosynthetic genes—squalene synthase and squalene tetrahymanol cyclase—were identified in A. robustus, C. churrovis, and other anaerobic gut fungal genera. Conclusions: This study provides a foundational, untargeted metabolomic dataset to unmask gut fungal metabolic pathways and biosynthetic potential and to prioritize future efforts for compound isolation and identification.

Desert tortoises play ecologically significant roles, including plant seed dispersal and mineral cycling, and yet little is known about microbial members that are critical to their gut and overall health. Tortoises consume recalcitrant plant material, which their gut microbiota degrades and converts into usable metabolites and nutrients for the tortoise. Findings from tortoise gut microbiomes may translate well into biotechnological applications as these microbes have evolved to efficiently degrade recalcitrant substrates and generate useful products. In this study, we cultivated microbial communities from desert tortoise fecal samples following a targeted anaerobic enrichment for microbes involved in deconstruction and utilization of plant biomass. We employed 16S rRNA amplicon sequencing to compare cultivated communities to initial fecal source material and found high abundances of Firmicutes and Bacteroidota typically associated with biomass deconstruction in all cultivated samples. Significantly decreased microbial diversity was observed in the cultivated microbial communities, yet several key taxa thrived in lignocellulose enrichments, including Lachnospiraceae and Enterococcus. Additionally, cultivated communities produced short-chain fatty acids under anaerobic conditions, and their growth and metabolic output provide evidence of their viability in the initial fecal communities. Overall, this study adds to the limited understanding of reptilian herbivore microbiota, and offers a path towards biotechnological translation based on the ability of the cultivated communities to convert lignocellulose directly to acetate, propionate, and butyrate.

Anaerobic fermentation of lignocellulosic biomass offers a cost-effective route for sustainable biomanufacturing, yet constructing synthetic consortia for targeted bioproduction remains challenging. Inspired by herbivore rumen microbiome, we engineered bottom-up anaerobic fungal-bacterial consortia for direct conversion of lignocellulose to medium-chain fatty acids (MCFAs). Initial screening experiment identified a newly isolated fungal strain, Neocallimastix sp. FC1, in co-culture with the bacterial strain Megasphaera hexanoica , as an optimal co-culture pair, achieving 21.0 % (carbon-to-carbon basis) lignocellulose-to-MCFAs yield. Lactate, the main fermentation product of Neocallimastix sp. FC1 and primary substrate for M. hexanoica , was undetectable in co-culture suggesting tight cross-feeding. This triggered upregulation of reverse β-oxidation pathway enzymes in M. hexanoica , favoring the production of MCFAs (hexanoate and octanoate) over butyrate. These results suggest that securing high-performing lactate-producing fungi, while maintaining low lactate levels, could be a promising strategy to maximize lignocellulose-to-MCFA conversion. Technoeconomic analysis further supported the process’s economic feasibility and delineated the cost-yield ranges required for profitable implementation. ### Competing Interest Statement The authors have declared no competing interest. Office of Science, https://ror.org/00mmn6b08, DE-SC0022142 Office of Biological and Environmental Research, DE-AC02–05CH11231 Natural Sciences and Engineering Research Council, RGPIN-2021-02684, NSERC-CREATE 528163-201 Institute for Collaborative Biotechnologies, https://ror.org/04kz4p343, 2128271

Diatoms are a class of algae that play an essential role in global ecology and produce valuable chemicals. They are known for forming intricate nanostructured silica cell walls (frustules). The introduction of non-siliceous elements like aluminum into diatoms induces properties such as a lower dissolution rate of the frustule, increasing the specific surface area of the frustule and enhancing metabolism. Previous studies have focused primarily on characterizing physiological impacts, leaving the genetic response(s) to non-siliceous elements largely unexplored. This study investigates the transcriptional response of the pennate diatom, Entomoneis vertebralis to dissolved aluminum. Our findings reveal that in the presence of added 10 µM aluminum, biogenic silica content of the cell wall increases approximately twofold along with significant changes to core metabolism. An increase in transcription of genes encoding nitrate transporters has been observed despite the apparent downregulation of nitrogen assimilation pathways. Additionally, increased transcription of genes involved in carbon fixation were noted. Amino acid and protein motif analyses identified proteins that were differentially regulated shared amino acid compositions and motifs characteristic to silicification-associated genes. A unique structure-based analysis pipeline revealed that some of these proteins have a conserved structural core while being diverse in sequence, which could be features associated with biosilicification. Differential expression transcriptomics has provided insight into diatom metabolism when exposed to aluminum, highlighting potential targets for metabolic engineering. Furthermore, we identified potential silicification-associated genes using tools based on structure and amino acid composition, advancing our understanding of diatom silicification.

The genomes of anaerobic gut fungi (AGF) encode a diverse array of carbohydrate-active enzymes (CAZymes), yet exceedingly few of these enzymes have been experimentally validated or expressed in heterologous systems. Here, we developed a predictive bioinformatic pipeline to annotate novel putative CAZymes from anaerobic fungi and validate their activity through large-scale heterologous expression in Escherichia coli. A total of 173 fungal proteins from Piromyces finnis associated with biomass degradation were synthesized and expressed in E. coli, and 9.8% were soluble with expression levels exceeding 5% of the total proteome using high-throughput proteomic screening. Among these 17 heterologously expressed proteins, analysis with AlphaFold and FoldSeek predicted 13 multi-functional proteins containing catalytic domains fused with repetitive fungal dockerins, and half of the substrate predictions were experimentally validated. One promising enzyme, celsome_012, exhibited robust and specific activity against beechwood xylan at 37°C and pH 6.4, with titers that were also fivefold higher than those of other recombinant proteins screened here. Both Michaelis-Menten kinetics and the linearized Lineweaver-Burk equation yielded consistent values for Km, and its activation energy was estimated at 51.9 kJ/mol based on the Arrhenius model. This work supports the industrial translation of anaerobic fungal CAZymes due to their robust lignocellulolytic activity and provides a framework for prioritizing AGF proteins for efficient E. coli heterologous expression.IMPORTANCEEfficient breakdown of plant biomass is crucial for producing high-value bio-based products, but identifying enzymes that reduce deconstruction costs remains a challenge. In this study, we harnessed novel CAZymes encoded in the AGF genome through high-throughput proteomic screening for CAZyme expression to identify promising fungal enzymes suitable for large-scale production in E. coli. Additionally, we leveraged cutting-edge computational tools to predict enzyme structure and function, accelerating the screening process beyond traditional methods. Experimental validation confirmed the accuracy of these predictions and revealed a highly active novel xylanase, expanding the available enzyme toolbox for biomass conversion. Overall, this study represents a comprehensive large-scale screening campaign of putative AGF CAZymes, highlighting proteins amenable to E. coli overexpression, integrating advanced sequence and structural annotation, and identifying a robust, novel fungal xylanase for detailed biochemical characterization.

Here, we report the genome sequence of Thermobifida halotolerans DSM 44931, a bacterium that was originally isolated from a salt mine in the Yunnan Province of China. This genome was sequenced using Pacific Biosciences sequencing technology and was assembled into 2 contigs in 2 scaffolds. It has a total length of 5,506,851 bp and a GC content of 71.16%. Functional annotation of this genome provides further metabolic insight into this species.

Anaerobic gut fungi effectively degrade lignocellulose in the guts of large herbivores, but there remain a limited number of isolated, publicly available, and sequenced strains that impede our understanding of the role of anaerobic fungi within microbial communities. We isolated and characterized a new fungal isolate, Neocallimastix cameroonii var. constans, providing a transcriptomic and genomic understanding of its ability to degrade diverse carbohydrates. This anaerobic fungal strain was stably cultivated for multiple years in vitro among members of an initial enrichment microbial community derived from goat feces, and it demonstrated the ability to pair with other microbial members, namely, archaeal methanogens to produce methane from lignocellulose. Genomic analysis revealed a higher number of predicted carbohydrate-active enzymes encoded in the N. cameroonii var. constans genome compared to most other sequenced anaerobic fungi. The carbohydrate-active enzyme profile for this isolate contained 660 glycoside hydrolases, 160 carbohydrate esterases, 194 glycosyltransferases, and 85 polysaccharide lyases. Differential gene expression analysis showed the upregulation of thousands of genes (including predicted carbohydrate-active enzymes) when N. cameroonii var. constans was grown on lignocellulose (reed canary grass) compared to less complex substrates, such as cellulose (filter paper), cellobiose, and glucose. AlphaFold was used to predict functions of transcriptionally active yet poorly annotated genes, revealing feruloyl esterases that likely play an important role in lignocellulose degradation by anaerobic fungi. The combination of this strain's genomic and transcriptomic characterization, omics-informed structural prediction, and robustness in microbial co-culture make it a well-suited platform to conduct future investigations into bioprocessing and enzyme discovery.

AbstractAnaerobic gut fungi effectively degrade lignocellulose in the guts of large herbivores, but there remains a limited number of isolated, publicly available, and sequenced strains that impede our understanding of the role of anaerobic fungi within microbial communities. We isolated and characterized a new fungal isolate,Neocallimastix camerooniivar. constans, providing a transcriptomic and genomic understanding of its ability to degrade diverse carbohydrates. This anaerobic fungal strain was stably cultivated for multiple yearsin vitroamong members of an initial enrichment microbial community derived from goat feces, and it demonstrated the ability to pair with other microbial members, namely archaeal methanogens to produce methane from lignocellulose. Genomic analysis revealed a higher number of predicted carbohydrate-active enzymes encoded in theN. camerooniivar. constans genome compared to most other sequenced anaerobic fungi. The carbohydrate-active enzyme profile for this isolate contained 660 glycoside hydrolases, 160 carbohydrate esterases, 194 glycosyltrasferases, and 85 polysaccharide lyases. Differential gene expression analysis showed the upregulation of thousands of genes (including predicted carbohydrate-active enzymes) whenN. camerooniivar. constans was grown on lignocellulose (reed canary grass) compared to less complex substrates, such as cellulose (filter paper), cellobiose, and glucose. AlphaFold was used to predict functions of transcriptionally active yet poorly annotated genes, revealing feruloyl esterases that likely play an important role in lignocellulose degradation by anaerobic fungi. The combination of this strain’s genomic and transcriptomic characterization, omics-informed structural prediction, and robustness in microbial co-culture make it a well-suited platform to conduct future investigations into bioprocessing and enzyme discovery.

Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value‐added bioproduction.

Biological deconstruction of lignocellulose for sustainable chemical production offers an opportunity to harness evolutionarily specialized enzymes and organisms for industrial bioprocessing. While hydrolysis of cellulose and hemicellulose by CAZymes yields fermentable sugars, ligninolysis releases a heterogeneous mix of aromatic compounds that likely play a crucial role in shaping microbial communities and microbial metabolism. Here, we interrogated the metabolomic and transcriptomic response of a lignocellulolytic anaerobic fungus, Neocallimastix californiae, to a heterogeneous mixture of aromatic compounds derived from lignin. Through exposing the fungus to both a concentration it might experience in its native environment and an elevated concentration of alkaline lignin, we observe that N. californiae transforms vanillin and that supplying alkaline lignin at 0.125 g/L, alongside cellulose, enhances the growth and polysaccharide-degrading activity of N. californiae. Altogether, our results further suggest that vanillin consumption, increased polymer-degrading activity, increased metabolic activity, and transcriptomic remodeling of amino acid synthesis genes all coincide with increased melanin production by fungal cells. These observations challenge previous notions that aromatics from lignocellulose only inhibit the growth and polymer deconstruction capabilities of the biomass-degrading anaerobic fungi (Neocallimastigomycetes). This study demonstrates that anaerobic fungi have a complex relationship with aromatic chemicals derived from lignin and hemicellulose and shift their metabolism in response to the addition of lignocellulose-derived aromatics to their growth medium. Further, as no known pathways for the biochemical transformation of aromatics were detected in these organisms despite observed transcriptome remodeling in the presence of aromatics, we suggest they might encode novel biochemical routes for scavenging amino acid building blocks from aromatic monomers derived from hemicellulose side chains and lignin.

Abstract The animal gut microbiome is a complex system of diverse, predominantly anaerobic microbiota with secondary metabolite potential. These metabolites likely play roles in shaping microbial community membership and influencing animal host health. As such, novel secondary metabolites from gut microbes hold significant biotechnological and therapeutic interest. Despite their potential, gut microbes are largely untapped for secondary metabolites, with gut fungi and obligate anaerobes being particularly under-explored. To advance understanding of these metabolites, culture-based and (meta)genome-based approaches are essential. Culture-based approaches enable isolation, cultivation, and direct study of gut microbes, and (meta)genome-based approaches utilize in silico tools to mine biosynthetic gene clusters (BGCs) from microbes that have not yet been successfully cultured. In this mini-review, we highlight recent innovations in this area, including anaerobic biofoundries like ExFAB, the NSF BioFoundry for Extreme & Exceptional Fungi, Archaea, and Bacteria. These facilities enable high-throughput workflows to study oxygen-sensitive microbes and biosynthetic machinery. Such recent advances promise to improve our understanding of the gut microbiome and its secondary metabolism. Key points • Gut microbial secondary metabolites have therapeutic and biotechnological potential • Culture- and (meta)genome-based workflows drive gut anaerobe metabolite discovery • Anaerobic biofoundries enable high-throughput workflows for metabolite discovery Graphical abstract

Diversity-generating retroelements (DGRs) are used by bacteria, archaea, and viruses as a targeted mutagenesis tool. Through error-prone reverse transcription, DGRs introduce random mutations at specific genomic loci, enabling rapid evolution of these targeted genes. However, the function and benefits of DGR-diversified proteins in cellular hosts remain elusive. We find that 82% of DGRs from one of the major monophyletic lineages of DGR reverse transcriptases are encoded by multicellular bacteria, which often have two or more DGR loci in their genomes. Using the multicellular purple sulfur bacterium Thiohalocapsa sp. PB-PSB1 as an example, we characterized nine distinct DGR loci capable of generating 10282 different combinations of target proteins. With environmental metagenomes from individual Thiohalocapsa aggregates, we show that most of PB-PSB1's DGR target genes are diversified across its biogeographic range, with spatial heterogeneity in the diversity of each locus. In Thiohalocapsa PB-PSB1 and other bacteria hosting this lineage of cellular DGRs, the diversified target genes are associated with NACHT-domain anti-phage defenses and putative ternary conflict systems previously shown to be enriched in multicellular bacteria. We propose that these DGR-diversified targets act as antigen sensors that confer a form of adaptive immunity to their multicellular consortia, though this remains to be experimentally tested. These findings could have implications for understanding the evolution of multicellularity, as the NACHT-domain anti-phage systems and ternary systems share both domain homology and conceptual similarities with the innate immune and programmed cell death pathways of plants and metazoans.

Marine macroalgae produce abundant and diverse polysaccharides which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of recalcitrant polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, California, USA. Based on 16S rRNA gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimaitellaeota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and iota-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases ( nifDKH ) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a group of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672) is proposed to be the type strain of a novel species in Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.Importance The growth and preservation of marine macroalgae is considered as a carbon dioxide reduction strategy. However, we still have limited knowledge about the eventual fate of carbon stored in macroalgae. Diverse heterotrophic microbial communities in the ocean specialize on these complex polymers, for example, members in the Kiritimatiellaeota phylum. However, only four type strains within the phylum have been cultured and characterized to date, and there is limited knowledge about the metabolic capabilities and functional role of this phylum in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum with the capability to utilize macroalgal polysaccharides agar and iota-carrageenan. It is also the first strain in the Kiritimatiellaeota phylum to be reported with nitrogen fixing ability.

Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.

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.

Polymer production is rapidly increasing, but there are no large-scale technologies available to effectively mitigate the massive accumulation of these recalcitrant materials. One potential solution is the development of a carbon-neutral polymer life cycle, where microorganisms convert plant biomass to chemicals, which are used to synthesize biodegradable materials that ultimately contribute to the growth of new plants. Realizing a circular carbon life cycle requires the integration of knowledge across microbiology, bioengineering, materials science, and organic chemistry, which itself has hindered large-scale industrial advances. This review addresses the biodegradation status of common synthetic polymers, identifying novel microbes and enzymes capable of metabolizing these recalcitrant materials and engineering approaches to enhance their biodegradation pathways. Design considerations for the next generation of biodegradable polymers are also reviewed, and finally, opportunities to apply findings from lignocellulosic biodegradation to the design and biodegradation of similarly recalcitrant synthetic polymers are discussed.