Michelle A. O'Malley

• PhD
• Professor (Assistant) at University of California, Santa Barbara

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 in...

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 i...

Anaerobic microbial communities are often highly degradative, such as those found in the herbivore rumen and large‐scale anaerobic digesters. Since the microbial communities in these systems degrade recalcitrant organic polymers, we hypothesize that some microbes in anaerobic environments may be involved in man‐made plastic association, deformation...

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 r...

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 degra...

Bacterial mRNA sequencing is inefficient due to the abundance of ribosomal RNA that is challenging to deplete. While commercial kits target rRNA from common bacterial species, they are frequently inefficient when applied to divergent species, including those from environmental isolates. Similarly, other methods typically employ large probe sets tha...

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 re...

Membrane‐embedded transporters impart essential functions to cells as they mediate sensing and the uptake and extrusion of nutrients, waste products, and effector molecules. Promiscuous multidrug exporters are implicated in resistance to drugs and antibiotics and are highly relevant for microbial engineers who seek to enhance the tolerance of cell...

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (Ce...

Anaerobic fungi produce biomass-degrading enzymes and natural products that are important to harness for several biotechnology applications. Although progress has been made in the development of methods for extracting nucleic acids for genomic and transcriptomic sequencing of these fungi, most studies are limited in that they do not sample multiple...

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...

Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic en...

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnolo...

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clos...

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis rev...

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear crit...

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 carbo...

Small genes (<150 nucleotides) have been systematically overlooked in phage genomes. We employ a large-scale comparative genomics approach to predict >40,000 small-gene families in ∼2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enric...

Anaerobic gut fungi (AGF) are lignocellulose degraders that naturally form biofilms in the rumen of large herbivores and in standard culture techniques. While biofilm formation enhances biomass degradation and carbohydrate active enzyme (CAZyme) production in some bacteria and aerobic fungi, gene expression and metabolism in AGF biofilms have not b...

The directed evolution of proteins comprises a search of sequence space for variants that improve a target phenotype, yet identification of desirable variants is inherently limited by library size and screening ability. Selections that couple protein phenotype to cell viability accelerate identification of promising variants by depleting libraries...

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that m...

Background Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are...

Lignocellulose processing yields a heterogeneous mixture of substances, which are poorly utilized by current industrial strains. For efficient valorization of recalcitrant biomass, it is critical to identify and engineer new membrane proteins that enable the broad uptake of hydrolyzed substrates. Whereas glucose consumption rarely presents a bottle...

Anaerobic fungi are outnumbered by bacteria by 4 orders of magnitude in the herbivore rumen. Despite their numerical disadvantage, they are resilient members of the rumen microbiome.

Anaerobic fungi are a potential biotechnology platform to produce biomass-degrading enzymes. Unlike model fungi such as yeasts, stress responses that are relevant during bioprocessing have not yet been established for anaerobic fungi. In this work, we characterize both the heat shock and unfolded protein responses of four strains of anaerobic fungi...

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that m...

Microbiomes are complex and ubiquitous networks of microorganisms whose seemingly limitless chemical transformations could be harnessed to benefit agriculture, medicine, and biotechnology. The spatial and temporal changes in microbiome composition and function are influenced by a multitude of molecular and ecological factors. This complexity yields...

Anaerobic fungi ( Neocallimastigomycota ) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes...

Changes in the sequence of an organism’s genome, i.e., mutations, are the raw material of evolution. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as diversity-generating retroelements (DGRs). DGRs have been characterized from cultivated bacteria and bacteriophages, and perform error-prone reverse...

Significance Anaerobic gut fungi are important members of the gut microbiome of herbivores, yet they exist in small numbers relative to bacteria. Here, we show that these early-branching fungi produce a wealth of secondary metabolites (natural products) that may act to regulate the gut microbiome. We use an integrated 'omics'-based approach to clas...

The herbivore digestive tract is home to a complex community of anaerobic microbes that work together to break down lignocellulose. These microbiota are an untapped resource of strains, pathways and enzymes that could be applied to convert plant waste into sugar substrates for green biotechnology. We carried out more than 400 parallel enrichment ex...

In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we ide...

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofue...

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 unresolv...

The anaerobic gut fungi (Neocallimastigomycota), first described almost 50 years ago, hold extraordinary potential for biotechnology. Anaerobic fungi could contribute to bioenergy and bio-based chemical production via their ability to degrade lignocellulose, may enhance animal health and production, and are now being revealed to have interesting bi...

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well docum...

Measuring the growth rate of non‐model anaerobic microbes typically requires the use of time‐consuming and often destructive manual measurements. Here, an Arduino based automatic pressure evaluation system (A‐APES) was developed to automatically measure the rate of fermentation gas production as a proxy for microbial growth in anaerobic systems. Th...

Changes in the sequence of an organism's genome, i.e. mutations, are the raw material of evolution1. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as Diversity-generating retroelements (DGRs). DGRs introduce mutations in specific target genes, and were characterized from several cultivated bacteri...

Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold. Through templating key protein domains (cohesin and dockerin), designer cellulosomes have been engineered from bacterial motifs to alter the activity, stability, and degradation efficiency of...

Epistasis emerges when the effects of an amino acid depend on the identities of interacting residues. This phenomenon shapes fitness landscapes, which have the power to reveal evolutionary paths and inform evolution of desired functions. However, there is a need for easily implemented, high-throughput methods to capture epistasis particularly at di...

Anaerobic fungi (Neocallimastigomycota) are emerging non-model hosts for biotechnology due to their wealth of biomass-degrading enzymes, yet tools to engineer these fungi have not yet been established. Here, we show that the anaerobic gut fungi have the most GC depleted genomes among 443 sequenced organisms in the fungal kingdom, which has ramifica...

Consortium-based approaches are a promising avenue toward efficient bioprocessing. However, many complex microbial interactions dictate community dynamics and stability that must be replicated in synthetic systems. The rumen and/or hindguts of large mammalian herbivores harbor complex communities of biomass-degrading fungi and bacteria, as well as...

Industrial biotechnology has the potential to decrease reliance on petroleum for fuel and bio-based chemical production and also enable valorization of waste streams. Anaerobic microorganisms thrive in resource-limited environments and offer an array of novel bioactivities in this regard that could revolutionize biomanufacturing, However, they have...

Anaerobic gut fungi are biomass degraders that form syntrophic associations with other microbes in their native rumen environment. Here, RNA-Seq was used to track and quantify carbohydrate active enzyme (CAZyme) transcription in a synthetic consortium composed of the anaerobic fungus Anaeromyces robustus with methanogen Methanobacterium bryantii. A...

Membrane-embedded transporters are crucial for the stability and performance of microbial production strains. Apart from engineering known transporters derived from model systems, it is equally important to identify transporters from nonconventional organisms that confer advantageous traits for biotechnological applications. Here, we transferred ge...

Nonmodel fungi are increasingly used in biotechnology, spanning medical, industrial, and even agricultural applications. Long-read sequencing technologies have led to a rapid rise in the number of high-quality sequenced fungal genomes and transcriptomes available for study. This information, coupled with bioinformatic analyses, allows access to a s...

Early-diverging anaerobic fungi (order: Neocallimastigomycota), lignocelluolytic chytrid-like fungi central to fiber degradation in the digestive tracts of large herbivores, are attractive sources of cellulases and hemicellulases for biotechnology. Enzyme expression is tightly regulated and coordinated through mechanisms that remain unelucidated to...

Anaerobic fungi are among the most active plant‐degrading microbes in nature. Increased insight into the mechanisms and environmental cues that regulate fungal hydrolysis would better inform bioprocessing strategies to depolymerize lignocellulose. Here, we compare the response of three strains of anaerobic fungi (Piromyces finnis, Anaeromyces robus...

The rapid development of molecular biology and bioinformatics has fueled renewed interests in anaerobic fungi from the phylum Neocallimastigomycota. This chapter presents well-established methods for isolation, routine cultivation, and cryopreservation of anaerobic fungi. Moreover, detailed nucleic acid extraction protocols are provided, which shou...

White rot fungi possess a powerful ability to degrade recalcitrant lignin within plant biomass. Pycnoporus cinnabarinus PB 94 accomplishes lignin degradation through the combined activity of laccases, peroxidases, and their supporting enzymes. Assembly of the de novo transcriptome for PB 94 resulted in identification of 45,286 transcripts, includin...

Lignocellulose is an abundant and renewable resource that holds great promise for sustainable bioprocessing. However, unpretreated lignocellulose is recalcitrant to direct utilization by most microbes. Current methods to overcome this barrier include expensive pretreatment steps to liberate cellulose and hemicellulose from lignin. Anaerobic gut fun...

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 rhiz...

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve p...

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which...

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that...

Background The metabolism of archaeal methanogens drives methane release into the environment and is critical to understanding global carbon cycling. Methanogenesis operates at a very low reducing potential compared to other forms of respiration and is therefore critical to many anaerobic environments. Harnessing or altering methanogen metabolism h...

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis¹. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds 1. The versatile protein assembly...

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages....

The capability to graft synthetic polymers onto the surfaces of live cells offers the potential to manipulate and control their phenotype and underlying cellular processes. Conventional grafting-to strategies for conjugating preformed polymers to cell surfaces are limited by low polymer grafting efficiency. Here we report an alternative grafting-fr...

Background Engineered 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...

Proteases regulate many biological processes through their ability to activate or inactive their target substrates. Because proteases catalytically turnover proteins and peptides, they present unique opportunities for use in biotechnological and therapeutic applications. However, many proteases are capable of cleaving multiple physiological substra...

The human adenosine A2a receptor (A2aR) tunes its function by forming homo-oligomers and hetero-oligomers with other GPCRs, but the biophysical characterization of these oligomeric species is limited. Here, we show that upon reconstitution into an optimized mixed micelle system, and purification via an antagonist affinity column, full length A2aR e...

Notes on 113 fungal taxa are compiled in this paper, including 11 new genera, 89 new species, one new subspecies, three new combinations and seven reference specimens. A wide geographic and taxonomic range of fungal taxa are detailed. In the Ascomycota the new genera Angustospora (Testudinaceae), Camporesia (Xylariaceae), Clematidis, Crassiparies (...

Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precu...

Mining gut fungi to break down biomass The recalcitrance of plant biomass remains a formidable bottleneck in the production of biofuels and other chemicals from renewable sources. Enzymes from microbial communities found within ruminants and hindgut fermenters, however, show considerable promise to break down plant material into simple sugars effic...

Anaerobic gut fungi reside in the digestive tract of large herbivores where they enable the digestion of resilient plant biomass into fermentable sugars. It is likely that the membrane envelope of these important but woefully understudied organisms is involved in their cellulolytic lifestyle. Our studies suggest that these fungal membranes contain...

Proteases are attractive as therapeutics given their ability to catalytically activate or inactivate their targets. However, therapeutic use of proteases is limited by insufficient substrate specificity, since off-target activity can induce undesired side-effects. In addition, few methods exist to enhance the activity and specificity of human prote...

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high...

Extraction of sugar is the rate-limiting step in converting unpretreated biomass into value-added products through microbial fermentation. Both anaerobic fungi and anaerobic bacteria have evolved to produce large multi-cellulase complexes referred to as cellulosomes, which are powerful machines for biomass deconstruction. Characterization of bacter...

G protein‐coupled receptor ( GPCR ) oligomers are promising targets for the design of new highly selective therapeutics. GPCRs have historically been attractive drug targets for their role in nearly all cellular processes, and their localization at the cell surface makes them easily accessible to most small molecule therapeutics. However, GPCRs hav...

Complex microbiomes within the digestive tract of herbivores are increasingly of interest for the discovery of novel lignocellulose degrading enzymes for the production of biofuels. Anaerobic fungi, in particular, are prolific digesters of crude lignocellulose through a combination of powerful secreted enzymes and invasive growth. However, due to d...

Microbial production platforms promise to be a sustainable alternative to today’s petrochemical industry. By harnessing the breadth and flexibility of cellular metabolism to process available energy sources, ‘living foundries’ can be engineered that convert renewable sugar feedstocks directly into a number of enantiopure commodity and high-value sp...

Anaerobic gut fungi are an early branching family of fungi that are commonly found in the digestive tract of ruminants and monogastric herbivores. It is becoming increasingly clear that they are the primary colonizers of ingested plant biomass, and that they significantly contribute to the decomposition of plant biomass into fermentable sugars. As...

It is becoming increasingly clear that microbes within microbial communities, for which cultured isolates have not yet been obtained, have an immense, untapped reservoir of enzymes that could help address grand challenges in human health, energy, and sustainability. Despite the obstacles associated with culturing these microbes, recent advances in...

Anaerobic gut fungi are the primary colonizers and decomposers of plant biomass in monogastric herbivores and ruminants. Their ability to hydrolyze complex lignocellulosic materials is of great promise to cellulosic bioenergy development, since enzymatic breakdown of biomass is a stringent bottleneck. It is hypothesized that cellulolytic activity w...

Lignocellulosic biomass is a renewable resource capable of supplying one third of the domestic liquid fuel demand. However, processes to extract fermentable sugars from this biomass involve expensive pretreatment and enzymatic digestion steps that rely on a limited number of characterized cellulases. Therefore, there is a critical need to identify...

The diverse chemistries of cellular metabolism make microbial systems an attractive solution to grand challenges in bioenergy, sustainability and human health. Among these are successes in the production of high-valued bulk and specialty chemicals such as isoprene and artemisinic acid (an antimalarial agent). The full economic potential of such sys...

Anaerobic gut fungi are among the most active and efficient plant-degrading microbes found in nature. Increased insight into the enzymatic mechanisms responsible for fungal hydrolysis would better inform sustainable chemical production strategies that rely on lignocellulosic depolymerization. Despite the powerful degradation capacity of gut fungi,...