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DOE OSTI2024

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Soil Microbes Trade-Off Biogeochemical Cycling for Stress Tolerance Traits in Response to Year-Round Climate Change

Winter air temperatures are rising faster than summer air temperatures in high-latitude forests, increasing the frequency of soil freeze/thaw events in winter. To determine how climate warming and soil freeze/thaw cycles affect soil microbial communities and the ecosystem processes they drive, we leveraged the Climate Change across Seasons Experiment (CCASE) at the Hubbard Brook Experimental Forest in the northeastern United States, where replicate field plots receive one of three climate treatments: warming (+5°C above ambient in the growing season), warming in the growing season + winter freeze/thaw cycles (+5°C above ambient +4 freeze/thaw cycles during winter), and no treatment. Soil samples were taken from plots at six time points throughout the growing season and subjected to amplicon (rDNA) and metagenome sequencing. We found that soil fungal and bacterial community composition were affected by changes in soil temperature, where the taxonomic composition of microbial communities shifted more with the combination of growing-season warming and increased frequency of soil freeze/thaw cycles in winter than with warming alone. Warming increased the relative abundance of brown rot fungi and plant pathogens but decreased that of arbuscular mycorrhizal fungi, all of which recovered under combined growing-season warming and soil freeze/thaw cycles in winter. The abundance of animal parasites increased significantly under combined warming and freeze/thaw cycles. We also found that warming and soil freeze/thaw cycles suppressed bacterial taxa with the genetic potential for carbon (i.e., cellulose) decomposition and soil nitrogen cycling, such as N fixation and the final steps of denitrification. These new soil communities had higher genetic capacity for stress tolerance and lower genetic capacity to grow or reproduce, relative to the communities exposed to warming in the growing season alone. Our observations suggest that initial suppression of biogeochemical cycling with year-round climate change may be linked to the emergence of taxa that trade-off growth for stress tolerance traits.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

A shift from inorganic to organic nitrogen-dominance shapes soil microbiome composition and co-occurrence networks

Soil microbiomes are characterized by their composition and networks, which are linked to soil nitrogen (N) availability. In nature, inorganic N dominates at one end and organic N dominates at the other end along soil N gradients; however, little is known about how this shift influences soil microbiome composition and co-occurrence networks, as well as their controls. To this end, we conducted an experiment with the host plant Solidago canadensis , which was subject to three N regimes: inorganic N-dominated, co-dominated by inorganic and organic N (CIO), and organic N-dominated. Organic N dominance exhibited stronger effects on the composition and co-occurrence networks of soil microbiomes than inorganic N dominance. The predominant control was plant traits for bacterial and fungal richness, and soil pH for keystone species. Relative to the CIO regime, inorganic N dominance did not affect fungal richness and increased keystone species; organic N dominance decreased fungal richness and keystone species. Pathogenic fungi and arbuscular mycorrhizal fungi were suppressed by organic N dominance but not by inorganic N dominance. These findings suggest that the shift from soil inorganic N-dominance to soil organic N-dominance could strongly shape soil microbiome composition and co-occurrence networks by altering species diversity and topological properties.

Xin, Yue↗

DOE OSTI2023

Community response of soil microorganisms to combined contamination of polycyclic aromatic hydrocarbons and potentially toxic elements in a typical coking plant

Both polycyclic aromatic hydrocarbons (PAHs) and potentially toxic elements (PTEs) of coking industries impose negative effects on the stability of soil ecosystem. Soil microbes are regarded as an essential moderator of biochemical processes and soil remediation, while their responses to PAHs-PTEs combined contamination are largely unknown. In the present study, soil microbial diversity and community composition in the typical coking plant under the chronic co-exposure of PAHs and PTEs were investigated and microbial interaction networks were built to reveal microbial co-occurrence patterns. The results indicated that the concentrations of PAHs in the soil inside the coking plant were significantly higher than those outside the plant. The mean concentration of ∑16PAHs was 2894.4 ng·g −1 , which is 5.58 times higher than that outside the plant. The average Hg concentration inside the coking plant was 22 times higher than the background value of Hebei province. The soil fungal community inside the coking plant showed lower richness compared with that of outside community, and there are significant difference in the bacterial and fungal community composition between inside and outside of coking plant ( p < 0.01). Predicted contribution of different environmental factors to each dominant species based on random forest identified 20 and 25 biomarkers in bacteria and fungi, respectively, that were highly sensitive to coking plant soil in operation, such as Betaproteobacteria，Sordariomycetes and Dothideomycetes . Bacterial and fungal communities were shaped by the soil chemical properties (pH), PTEs (Hg), and PAHs together in the coking plant soils. Furthermore, the bacterial and fungal interaction patterns were investigated separately or jointly by intradomain and interdomain networks. Competition is the main strategy based on the co-exclusion pattern in fungal community, and the competitive relationship inside the coking plant is more complex than that outside the plant. In contrast, cooperation is the dominant strategy in bacterial networks based on the co-occurrence pattern. The present study provided insights into microbial response strategies and the interactions between bacteria and fungi under long-term combined contamination.

Shen, Qihui↗

DOE OSTI2024

Continuous cropping of potato changed the metabolic pathway of root exudates to drive rhizosphere microflora

For potato production, continuous cropping (CC) could lead to autotoxicity buildup and microflora imbalance in the field soil, which may result in failure of crops and reduction in yield. In this study, non-targeted metabolomics (via liquid chromatography with tandem mass spectrometry (LC–MS/MS)) combined with metagenomic profiling (via high-throughput amplicon sequencing) were used to evaluate correlations between metabolomics of potato root exudates and communities of bacteria and fungi around potato plants to illustrate the impacts of CC. Potato plants were grown in soil collected from fields with various CC years (0, 1, 4, and 7 years). Metabolomic analysis showed that the contents and types of potential autotoxins in potato root exudates increased significantly in CC4 and CC7 plants (i.e., grown in soils with 4 and 7 years of CC). The differentially expressed metabolites were mainly produced via alpha-linolenic acid metabolism in plant groups CC0 and CC1 (i.e., no CC or 1 year CC). The metabolomics of the groups CC4 and CC7 became dominated by styrene degradation, biosynthesis of siderophore group non-ribosomal peptides, phenylpropanoid biosynthesis, and biosynthesis of various plant secondary metabolites. Continuous cropping beyond 4 years significantly changed the bacterial and fungal communities in the soil around the potato crops, with significant reduction of beneficial bacteria and accumulation of harmful fungi. Correlations between DEMs and microflora biomarkers were established with strong significances. These results suggested that continuous cropping of potato crops changed their metabolism as reflected in the plant root exudates and drove rhizosphere microflora to directions less favorable to plant growth, and it needs to be well managed to assure potato yield.

Xing, Yanhong↗

DOE OSTI2024

Fabricated devices for performing bacterial-fungal interaction experiments across scales

Diverse and complex microbiomes are found in virtually every environment on Earth. Bacteria and fungi often co-dominate environmental microbiomes, and there is growing recognition that bacterial-fungal interactions (BFI) have significant impacts on the functioning of their associated microbiomes, environments, and hosts. Investigating BFI in vitro remains a challenge, particularly when attempting to examine interactions at multiple scales of system complexity. Fabricated devices can provide control over both biotic composition and abiotic factors within an experiment to enable the characterization of diverse BFI phenotypes such as modulation of growth rate, production of biomolecules, and alterations to physical movements. Engineered devices ranging from microfluidic chips to simulated rhizosphere systems have been and will continue to be invaluable to BFI research, and it is anticipated that such devices will continue to be developed for diverse applications in the field. This will allow researchers to address specific questions regarding the nature of BFI and how they impact larger microbiome and environmental processes such as biogeochemical cycles, plant productivity, and overall ecosystem resilience. Devices that are currently used for experimental investigations of bacteria, fungi, and BFI are discussed herein along with some of the associated challenges and several recommendations for future device design and applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Evaluation of aflatoxin contamination in protein-rich pulses using a GFP-expressing Aspergillus flavus strain

Background Mycotoxigenic fungi pose significant threats to food safety and marketability. Crop-specific differences in susceptibility to these fungi can influence contamination levels. Objectives The resistance or susceptibility of protein-rich pulse crops—chickpeas (Cicer arietinumL. cv. CDC Frontier), lentils (Lens culinarisMedik cv. Eston), peas (Pisum sativumL. cv. LeRoy), and corn (Zea maysL. cv. H97C) to infection byAspergillus flavuswere evaluated using a kernel screening assay (KSA). Methodology A. flavusstrain 70 (AF-70) expressing green-fluorescent protein (GFP) was used to quantify fungal spread and mycotoxin production. Fungal infection and toxin levels, including aflatoxins (AFB 1 , AFB 2 ), cyclopiazonic acid (CPA), and α-aflatrem, were monitored at 2-day intervals over a 10-day period post inoculation. Results Although all seeds were infected byA. flavus, corn produced significantly higher levels of AFB 1 and AFB 2 compared to pulses. However, pulses accumulated relatively higher levels of CPA and α‑aflatrem. Conclusion While pulses may be less susceptible to aflatoxin contamination than corn, the elevated concentrations of CPA and α‑aflatrem underscore the need for further toxicological evaluation and mechanistic studies. Future research should explore the underlying resistance mechanisms from field to storage to better ensure crop safety.

Microbiology↗

DOE OSTI2021

Understanding Multilevel Selection May Facilitate Management of Arbuscular Mycorrhizae in Sustainable Agroecosystems

Studies in natural ecosystems show that adaptation of arbuscular mycorrhizal (AM) fungi and other microbial plant symbionts to local environmental conditions can help ameliorate stress and optimize plant fitness. This local adaptation arises from the process of multilevel selection, which is the simultaneous selection of a hierarchy of groups. Studies of multilevel selection in natural ecosystems may inform the creation of sustainable agroecosystems through developing strategies to effectively manage crop microbiomes including AM symbioses. Field experiments show that the species composition of AM fungal communities varies across environmental gradients, and that the biomass of AM fungi and their benefits for plants generally diminish when fertilization and irrigation eliminate nutrient and water limitations. Furthermore, pathogen protection by mycorrhizas is only important in environments prone to plant damage due to pathogens. Consequently, certain agricultural practices may inadvertently select for less beneficial root symbioses because the conventional agricultural practices of fertilization, irrigation, and use of pesticides can make these symbioses superfluous for optimizing crop performance. The purpose of this paper is to examine how multilevel selection influences the flow of matter, energy, and genetic information through mycorrhizal microbiomes in natural and agricultural ecosystems, and propose testable hypotheses about how mycorrhizae may be actively managed to increase agricultural sustainability.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Specificity in plant-mycorrhizal fungal relationships: prevalence, parameterization, and prospects

Species interactions exhibit varying degrees of specialization, ranging from generalist to specialist interactions. For many interactions (e.g., plant-microbiome) we lack standardized metrics of specialization, hindering our ability to apply comparative frameworks of specificity across niche axes and organismal groups. Here, we discuss the concept of plant host specificity of arbuscular mycorrhizal (AM) fungi and ectomycorrhizal (EM) fungi, including the predominant theories for their interactions: Passenger, Driver, and Habitat Hypotheses. We focus on five major areas of interest in advancing the field of plant-mycorrhizal fungal host specificity: phylogenetic specificity, host physiology specificity, functional specificity, habitat specificity, and mycorrhizal fungal-mediated plant rarity. Considering the need to elucidate foundational concepts of specificity in this globally important symbiosis, we propose standardized metrics and comparative studies to enhance our understanding. We also emphasize the importance of analyzing global mycorrhizal data holistically to draw meaningful conclusions and suggest a shift toward single-species analyses to unravel the complexities underlying these associations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Plant root associated chitinases: structures and functions

Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc 3 . They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5–7 and 30–40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50–60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc 3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Legacy Effects of Intercropping and Nitrogen Fertilization on Soil N Cycling, Nitrous Oxide Emissions, and the Soil Microbial Community in Tropical Maize Production

Maize-forage grasses intercropping systems have been increasingly adopted by farmers because of their capacity to recycle nutrients, provide mulch, and add C to soil. However, grasses have been shown to increase nitrous oxide (N 2 O) emissions. Some tropical grasses cause biological nitrification inhibition (BNI) which could mitigate N 2 O emissions in the maize cycle but the reactions of the N cycle and the microbial changes that explain the N 2 O emissions are little known in such intercropping systems. With this in mind, we explored intercropping of forage grasses ( Brachiaria brizantha and Brachiaria humidicola ) with distinct BNI and yield potential to increase N cycling in no-till maize production systems compared to monocrop with two N rates (0 and 150 kg ha −1 ) applied during the maize season. These grasses did not strongly compete with maize during the period of maize cycle and did not have a negative effect on grain yield. We observed a legacy of these grasses on N mineralization and nitrification through the soil microbiome during maize growth. We observed that B. humidicola , genotype with higher BNI potential, increased net N mineralization by 0.4 mg N kg −1 day −1 and potential nitrification rates by 1.86 mg NO 3 -N kg −1 day −1 , while B. brizantha increased the soil moisture, fungi diversity, mycorrhizal fungi, and bacterial nitrifiers, and reduced saprotrophs prior to maize growth. Their legacy on soil moisture and cumulative organic inputs (i.e., grass biomass) was strongly associated with enhanced mineralization and nitrification rates at early maize season. These effects contributed to increase cumulative N 2 O emission by 12.8 and 4.8 mg N 2 O-N m −2 for maize growing after B. brizantha and B. humidicola , respectively, regardless of the N fertilization rate. Thus, the nitrification inhibition potential of tropical grasses can be outweighed by their impacts on soil moisture, N recycling, and the soil microbiome that together dictate soil N 2 O fluxes.

Canisares, Lucas P.↗

DOE OSTI2022

Advances on Bacterial and Fungal Biofilms for the Production of Added-Value Compounds

In recent years, abundant research has been performed on biofilms for the production of compounds with biotechnological and industrial relevance. The use of biofilm platforms has been seen as a compelling approach to producing fine and bulk chemicals such as organic acids, alcohols, and solvents. However, the production of recombinant proteins using this system is still scarce. Biofilm reactors are known to have higher biomass density, operational stability, and potential for long-term operation than suspended cell reactors. In addition, there is an increasing demand to harness industrial and agricultural wastes and biorefinery residues to improve process sustainability and reduce production costs. The synthesis of recombinant proteins and other high-value compounds is mainly achieved using suspended cultures of bacteria, yeasts, and fungi. This review discusses the use of biofilm reactors for the production of recombinant proteins and other added-value compounds using bacteria and fungi.

Carvalho, Fábio M.↗

DOE OSTI2021

A Sesquiterpene Synthase from the Endophytic Fungus Serendipita indica Catalyzes Formation of Viridiflorol

Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica, a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene (SiTPS). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS-over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2026

Transformative Impacts of Laser-Induced Breakdown Spectroscopy on Environmental and Biological Research at Oak Ridge National Laboratory

This manuscript will present an advancement of transformative research that has been conducted at Oak Ridge National Laboratory (ORNL) over a 25-year period (2000–2025) on a variety of environmental and biological matrices. These investigations derived a fundamental understanding of how elemental detection and analysis of these matrices led to the knowledge and discovery of natural processes in plants and the environment. Each project led to the initiation of a new research area which unearthed awesome and novel breakthroughs. Highlights are listed below: 1. The preliminary research at ORNL centered on the detection of aerosols utilizing Laser-induced Breakdown Spectroscopy (LIBS) technology. The Clean Air Act Amendment (CAAA) of 1990 highlighted the importance of identifying hazardous air pollutants (HAPs) due to their impact on environmental and human health, thereby underscoring the need to detect various toxic elements. Research in aerosol chemistry aimed to identify these harmful elements released by factories during periods of increased emissions in their manufacturing processes. LIBS emerged as the most effective method for real-time, in situ measurements of metal species in both gaseous and aerosol phases. 2. An understanding of the presence of total carbon in soils gives perspective on how to develop carbon sequestration strategies. The recognition that carbon sinks can evolve back to carbon sources to emit back to the atmosphere was an important consideration. Also, the concentration of carbon in soil indicates the health of land areas for growing crops successfully. 3. The direct detection of most of the elements in a wood sample in a single emission spectrum, without sample preparation, encouraged the research to use the LIBS technique for preservative treated wood coupled with use of multivariate statistical methodology. Additionally, it encouraged the researchers to try to differentiate natural woods from different parts of the country, and it was successfully demonstrated that LIBS coupled with MVA analysis could differentiate wood of different species from each other and of similar species grown in different environments based on their elemental spectra. This was a breakthrough since it revealed a systematic approach to connect elemental scarcity and abundance to either drought or typical rainfall conditions for the hardwood trees grown in specific areas. 4. Furthermore, the research progressed to reveal physiological and developmental processes contributing to biomass production such that the variation in leaf elemental composition increases our understanding of terrestrial nutrient cycles, as well as tracking the transfer of toxic elements from soils to living organisms. 5. Recently another breakthrough viz., ionomics initiated the correlation of elements to specific genes, uncovering the function that the element performed in the plant. More recently, this has been extended from plants to fungi as well as fungi growing in symbiotic relations with plants.

09 BIOMASS FUELS↗

DOE OSTI2021

Using MALDI-FTICR-MS Imaging to Track Low-Molecular-Weight Aromatic Derivatives of Fungal Decayed Wood

Low-molecular-weight (LMW) aromatics are crucial in meditating fungal processes for plant biomass decomposition. Some LMW compounds are employed as electron donors for oxidative degradation in brown rot (BR), an efficient wood-degrading strategy in fungi that selectively degrades carbohydrates but leaves modified lignins. Previous understandings of LMW aromatics were primarily based on “bulk extraction”, an approach that cannot fully reflect their real-time functions during BR. Here, we applied an optimized molecular imaging method that combines matrix-assisted laser desorption ionization (MALDI) with Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to directly measure the temporal profiles of BR aromatics as Rhodonia placenta decayed a wood wafer. We found that some phenolics were pre-existing in wood, while some (e.g., catechin-methyl ether and dihydroxy-dimethoxyflavan) were generated immediately after fungal activity. These pinpointed aromatics might be recruited to drive early BR oxidative mechanisms by generating Fenton reagents, Fe2+ and H2O2. As BR progressed, ligninolytic products were accumulated and then modified into various aromatic derivatives, confirming that R. placenta depolymerizes lignin. Together, this work confirms aromatic patterns that have been implicated in BR fungi, and it demonstrates the use of MALDI-FTICR-MS imaging as a new approach to monitor the temporal changes of LMW aromatics during wood degradation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Genomic Diversity and Phenotypic Variation in Fungal Decomposers Involved in Bioremediation of Persistent Organic Pollutants

Fungi work as decomposers to break down organic carbon, deposit recalcitrant carbon, and transform other elements such as nitrogen. The decomposition of biomass is a key function of wood-decaying basidiomycetes and ascomycetes, which have the potential for the bioremediation of hazardous chemicals present in the environment. Due to their adaptation to different environments, fungal strains have a diverse set of phenotypic traits. This study evaluated 320 basidiomycetes isolates across 74 species for their rate and efficiency of degrading organic dye. We found that dye-decolorization capacity varies among and within species. Among the top rapid dye-decolorizing fungi isolates, we further performed genome-wide gene family analysis and investigated the genomic mechanism for their most capable dye-degradation capacity. Class II peroxidase and DyP-type peroxidase were enriched in the fast-decomposer genomes. Gene families including lignin decomposition genes, reduction-oxidation genes, hydrophobin, and secreted peptidases were expanded in the fast-decomposer species. This work provides new insights into persistent organic pollutant removal by fungal isolates at both phenotypic and genotypic levels.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Mutations in the Second Alternative Oxidase Gene: A New Approach to Group Aspergillus niger Strains

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes—A. calidoustus and A. implicatus—as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices

Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery.

60 APPLIED LIFE SCIENCES↗