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DOE OSTI2022

Improved Mobilome Delineation in Fragmented Genomes

The mobilome of a microbe, i.e., its set of mobile elements, has major effects on its ecology, and is important to delineate properly in each genome. This becomes more challenging for incomplete genomes, and even more so for metagenome-assembled genomes (MAGs), where misbinning of scaffolds and other losses can occur. Genomic islands (GIs), which integrate into the host chromosome, are a major component of the mobilome. Our GI-detection software TIGER, unique in its precise mapping of GI termini, was applied to 74,561 genomes from 2,473 microbial species, each species containing at least one MAG and one isolate genome. A species-normalized deficit of ∼1.6 GIs/genome was measured for MAGs relative to isolates. To test whether this undercount was due to the higher fragmentation of MAG genomes, TIGER was updated to enable detection of split GIs whose termini are on separate scaffolds or that wrap around the origin of a circular replicon. This doubled GI yields, and the new split GIs matched the quality of single-scaffold GIs, except that highly fragmented GIs may lack central portions. Cross-scaffold search is an important upgrade to GI detection as fragmented genomes increasingly dominate public databases. TIGER2 better captures MAG microdiversity, recovering niche-defining GIs and supporting microbiome research aims such as virus-host linking and ecological assessment.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Decomposing a San Francisco estuary microbiome using long-read metagenomics reveals species- and strain-level dominance from picoeukaryotes to viruses

ABSTRACT Although long-read sequencing has enabled obtaining high-quality and complete genomes from metagenomes, many challenges still remain to completely decompose a metagenome into its constituent prokaryotic and viral genomes. This study focuses on decomposing an estuarine metagenome to obtain a more accurate estimate of microbial diversity. To achieve this, we developed a new bead-based DNA extraction method, a novel bin refinement method, and obtained 150 Gbp of Nanopore sequencing. We estimate that there are ~500 bacterial and archaeal species in our sample and obtained 68 high-quality bins (>90% complete, <5% contamination, ≤5 contigs, contig length of >100 kbp, and all ribosomal and tRNA genes). We also obtained many contigs of picoeukaryotes, environmental DNA of larger eukaryotes such as mammals, and complete mitochondrial and chloroplast genomes and detected ~40,000 viral populations. Our analysis indicates that there are only a few strains that comprise most of the species abundances. IMPORTANCE Ocean and estuarine microbiomes play critical roles in global element cycling and ecosystem function. Despite the importance of these microbial communities, many species still have not been cultured in the lab. Environmental sequencing is the primary way the function and population dynamics of these communities can be studied. Long-read sequencing provides an avenue to overcome limitations of short-read technologies to obtain complete microbial genomes but comes with its own technical challenges, such as needed sequencing depth and obtaining high-quality DNA. We present here new sampling and bioinformatics methods to attempt decomposing an estuarine microbiome into its constituent genomes. Our results suggest there are only a few strains that comprise most of the species abundances from viruses to picoeukaryotes, and to fully decompose a metagenome of this diversity requires 1 Tbp of long-read sequencing. We anticipate that as long-read sequencing technologies continue to improve, less sequencing will be needed.

Lui, Lauren M.↗
DOE OSTI2022

Functional characterization of prokaryotic dark matter: the road so far and what lies ahead

Eight-hundred thousand to one trillion prokaryotic species may inhabit our planet. Yet, fewer than two-hundred thousand prokaryotic species have been described. This uncharted fraction of microbial diversity, and its undisclosed coding potential, is known as the “microbial dark matter” (MDM). Next-generation sequencing has allowed to collect a massive amount of genome sequence data, leading to unprecedented advances in the field of genomics. Still, harnessing new functional information from the genomes of uncultured prokaryotes is often limited by standard classification methods. These methods often rely on sequence similarity searches against reference genomes from cultured species. This hinders the discovery of unique genetic elements that are missing from the cultivated realm. It also contributes to the accumulation of prokaryotic gene products of unknown function among public sequence data repositories, highlighting the need for new approaches for sequencing data analysis and classification. Increasing evidence indicates that these proteins of unknown function might be a treasure trove of biotechnological potential. Here, we outline the challenges, opportunities, and the potential hidden within the functional dark matter (FDM) of prokaryotes. We also discuss the pitfalls surrounding molecular and computational approaches currently used to probe these uncharted waters, and discuss future opportunities for research and applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Conversion of CO 2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO 2 , has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin-Benson-Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. Further, the metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ..delta..phcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genome-Resolved Metagenomics and Detailed Geochemical Speciation Analyses Yield New Insights into Microbial Mercury Cycling in Geothermal Springs

Geothermal systems emit substantial amounts of aqueous, gaseous, and methylated mercury, but little is known about microbial influences on mercury speciation. Here, we report results from genome-resolved metagenomics and mercury speciation analysis of acidic warm springs in the Ngawha Geothermal Field (< 55°C, pH < 4.5), Northland Region, Aotearoa New Zealand. Our aim was to identify the microorganisms genetically equipped for mercury methylation, demethylation, or Hg(II) reduction to volatile Hg(0) in these springs. Dissolved total and methylated mercury concentrations in two adjacent springs with different mercury speciation ranked among the highest reported from natural sources (250 to 16,000 ng liter -1 and 0.5 to 13.9 ng liter -1 , respectively). Total solid mercury concentrations in spring sediments ranged from 1,274 to 7,000 μg g -1 . In the context of such ultrahigh mercury levels, the geothermal microbiome was unexpectedly diverse and dominated by acidophilic and mesophilic sulfur- and iron-cycling bacteria, mercury- and arsenic-resistant bacteria, and thermophilic and acidophilic archaea. By integrating microbiome structure and metagenomic potential with geochemical constraints, we constructed a conceptual model for biogeochemical mercury cycling in geothermal springs. The model includes abiotic and biotic controls on mercury speciation and illustrates how geothermal mercury cycling may couple to microbial community dynamics and sulfur and iron biogeochemistry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Northern peatland microbial communities exhibit resistance to warming and acquire electron acceptors from soil organic matter

The response of microbial communities that regulate belowground carbon turnover to climate change drivers in peatlands is poorly understood. Here, we leverage a whole ecosystem warming experiment to elucidate the key processes of terminal carbon decomposition and community responses to temperature rise. Our dataset of 697 metagenome-assembled genomes (MAGs) represents the microbial community from the surface (10 cm) to 2 m deep into the peat column, with only 3.7% of genomes overlapping with other well-studied peatlands. Community composition has yet to show a significant response to warming after 3 years, suggesting that metabolically diverse soil microbial communities are resistant to climate change. Surprisingly, abundant and active methanogens in the genus Candidatus Methanoflorens, Methanobacterium , and Methanoregula show the potential for both acetoclastic and hydrogenotrophic methanogenesis. Nonetheless, the predominant pathways for anaerobic carbon decomposition include sulfate/sulfite reduction, denitrification, and acetogenesis, rather than methanogenesis based on gene abundances. Multi-omics data suggest that organic matter cleavage provides terminal electron acceptors, which together with methanogen metabolic flexibility, may explain peat microbiome composition resistance to warming.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Microbial species and intraspecies units exist and are maintained by ecological cohesiveness coupled to high homologous recombination

Abstract Recent genomic analyses have revealed that microbial communities are predominantly composed of persistent, sequence-discrete species and intraspecies units (genomovars), but the mechanisms that create and maintain these units remain unclear. By analyzing closely-related isolate genomes from the same or related samples and identifying recent recombination events using a novel bioinformatics methodology, we show that high ecological cohesiveness coupled to frequent-enough and unbiased (i.e., not selection-driven) horizontal gene flow, mediated by homologous recombination, often underlie these diversity patterns. Ecological cohesiveness was inferred based on greater similarity in temporal abundance patterns of genomes of the same vs. different units, and recombination was shown to affect all sizable segments of the genome (i.e., be genome-wide) and have two times or greater impact on sequence evolution than point mutations. These results were observed in bothSalinibacter ruber, an environmental halophilic organism, andEscherichia coli, the model gut-associated organism and an opportunistic pathogen, indicating that they may be more broadly applicable to the microbial world. Therefore, our results represent a departure compared to previous models of microbial speciation that invoke either ecology or recombination, but not necessarily their synergistic effect, and answer an important question for microbiology: what a species and a subspecies are.

Science & Technology - Other Topics↗
DOE OSTI2020

Accurate and complete genomes from metagenomes

Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Platform for efficient large-scale storage and analysis of multi-omics data in plant and microbial systems (Final Technical Report)

Genomic variation at the sequence level fundamentally affects the phenotypic state of all organisms at all stages of development, while dynamic processes such as changes in the epigenome (e.g. DNA methylation state) and transcriptome regulate the specific phenotype expressed at any given state of development based upon that genomic variation. In plants, DNA methylation is a particularly important mechanism for both regulating transcriptomic expression and for management of genomic variations that could be deleterious to the organism due to the presence of active retrotransposons in plant genomes. While DNA methylation is heritable, it is also dynamic through a given plant’s development and life cycle, particularly during the development from seed to mature specimen suggesting variations in DNA methylation could be critical regulators of biologically and commercially important phenotypes such as time to flowering; in addition, plant DNA methylation is more complex than that of animals, with methylation of CHG and CHH trinucleotides evident in addition to the better-known CG methylation. The complexity of plant DNA methylation and its interplay with genomic sequence variation, transcriptomics and other epigenomic factors demand a storage and analysis framework that can cope with the complexity both within a single specimen and with analyses that span many individuals and even many species, such as attempts to extend models from model organisms to commercially relevant species. In addition to complexity, the rapid development and proliferation of sequencing technology has led to an explosion of data volume that conventional storage and analysis solutions will likely be unable to cope with in the long run. We proposed to study these with suitable distributed storage and computation and therefore for the application of cloud computing to biological analyses; integrate with existing data sources and compatible with virtually any interface use case, from fully automated shell scripts to notebooks and do all these at scale in this STTR grant.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Predominance of Methanomicrobiales and diverse hydrocarbon–degrading taxa in the Appalachian coalbed biosphere revealed through metagenomics and genome–resolved metabolisms

Coalbed deposits are a unique subsurface environment and represent an underutilized resource for methane generation. Microbial communities extant in coalbed deposits are responsible for key subsurface biogeochemical cycling and could be utilized to enhance methane production in areas where existing gas wells have depleted methane stores, or in coalbeds that are unmined, or conversely be utilized for mitigation of methane release. Here we utilize metagenomics and metagenome-assembled genomes to identify extant microbial lineages and genome-resolved microbial metabolisms of coalbed produced water, which has not yet been explored in the Appalachian Basin. Our analyses resulted in the recovery of over 40 metagenome-assembled genomes (MAGs) from eight coalbed methane wells. The most commonly identified taxa among samples were hydrogenotrophic methanogens from the order Methanomicrobiales and these dominant MAGs were highly similar to one another. Conversely, low-abundance coalbed bacterial populations were taxonomically and functionally diverse, mostly belonging to a variety of Proteobacteria classes, and encoding various hydrocarbon solubilization and degradation pathways. Further, the data presented herein provides novel insights into Appalachian Basin coalbed microbial ecology, and our findings provide new perspectives on underrepresented Methanocalculus species and low-relative abundance bacterial assemblages in coalbed environments, and their potential roles in stimulation or mitigation of methane release.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

From soil to sequence: filling the critical gap in genome-resolved metagenomics is essential to the future of soil microbial ecology

Abstract Soil microbiomes are heterogeneous, complex microbial communities. Metagenomic analysis is generating vast amounts of data, creating immense challenges in sequence assembly and analysis. Although advances in technology have resulted in the ability to easily collect large amounts of sequence data, soil samples containing thousands of unique taxa are often poorly characterized. These challenges reduce the usefulness of genome-resolved metagenomic (GRM) analysis seen in other fields of microbiology, such as the creation of high quality metagenomic assembled genomes and the adoption of genome scale modeling approaches. The absence of these resources restricts the scale of future research, limiting hypothesis generation and the predictive modeling of microbial communities. Creating publicly available databases of soil MAGs, similar to databases produced for other microbiomes, has the potential to transform scientific insights about soil microbiomes without requiring the computational resources and domain expertise for assembly and binning.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Recovering new viruses from New Mexico soils

Here, we utilized metagenomic and size-filtered virome sequencing to recover 4,157 medium, high, or complete quality viral genomes from soils taken from three high elevation sites in New Mexico, USA. Among recovered viral genomes, 90% were from size-filtered samples, indicating the importance of this enrichment in assessments of complex viromes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Microbial inoculants for soil restoration: A Risk-Proportional Stewardship Framework Integrating Strain-Resolved Genomics and Adaptive Governance

Global soil degradation and increasing reliance on chemical inputs threaten agricultural sustainability, driving interest in microbial inoculants as tools for soil restoration. These biological products have the potential to enhance nutrient cycling, improve soil structure, and support plant resilience, but their environmental release raises important safety and stewardship considerations. Here, we propose a risk-proportional framework for the responsible deployment of microbial inoculants grounded in release-based stewardship. The framework integrates genome-resolved strain identification, exclusionary hazard screening, bioassay-based risk triage, ecological testing under realistic conditions, and monitored field deployment. Drawing on evidence from microbial ecology and invasion biology, we highlight how inoculants can alter resident microbial communities, influence ecosystem function, and, in some cases, facilitate gene flow, underscoring the need for risk assessment. We further outline a federated, genome-informed data infrastructure to support traceability, cross-jurisdiction learning, and adaptive management. Together, this approach provides a scalable and scientifically grounded pathway to balance innovation and safety, enabling microbial technologies to contribute to soil restoration and climate-resilient agriculture.

Edlund, Anna [OATH Inc]↗
DOE OSTI2026

Bioenergy Cropping Reduces the Spatiotemporal Scaling of Soil Bacterial Biodiversity

Widespread bioenergy cropping can transform landscapes, strongly affecting biodiversity. However, the impact of bioenergy cropping on the spatiotemporal scaling of soil biodiversity remains virtually unknown, despite its profound implications for the functioning of the ecological community. Here, we investigated how bioenergy cropping influenced the spatiotemporal scaling of soil bacterial biodiversity in marginal soils (sandy loam and clay loam soils) in Oklahoma, USA. We detected strong, significant species-time-area relationships (STARs) and phylogenetic-time-area relationships (PTARs) in bacterial communities and their lineages, suggesting that STARs and PTARs exist in microbial ecology within the studied system. Also, spatiotemporal scaling rates (the slopes of STAR and PTAR models) varied substantially among bacterial lineages and were positively correlated with their 16S rRNA gene copy numbers, a genomic trait indicative of microbial growth potentials. Strikingly, bioenergy cropping significantly reduced spatiotemporal scaling rates by 6.8%-14.1%, with a more pronounced reduction observed in sandy loam soils, where those rates were significantly lower than in clay loam soils. The heterogeneity of soil phosphorus and carbon resulted in variations in bacterial spatiotemporal scaling rates. Collectively, our findings suggest that bioenergy cropping may alleviate rapid shifts in soil biodiversity across space and time, thereby stabilizing soil biodiversity and supporting its role as part of sustainable land management and climate mitigation strategies.

bacterial diversity↗
DOE OSTI2022

Community Structure and Microbial Associations in Sediment-Free Methanotrophic Enrichment Cultures from a Marine Methane Seep

We report Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b (“Candidatus Methanocomedenaceae”) and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides, Anaerolineales, and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Codon bias, nucleotide selection, and genome size predict in situ bacterial growth rate and transcription in rewetted soil

In soils, the first rain after a prolonged dry period represents a major pulse event impacting soil microbial community function, yet we lack a full understanding of the genomic traits associated with the microbial response to rewetting. Genomic traits such as codon usage bias and genome size have been linked to bacterial growth in soils—however, often through measurements in culture. Here, we used metagenome-assembled genomes (MAGs) with 18 O-water stable isotope probing and metatranscriptomics to track genomic traits associated with growth and transcription of soil microorganisms over one week following rewetting of a grassland soil. We found that codon bias in ribosomal protein genes was the strongest predictor of growth rate. We also found higher growth rates in bacteria with smaller genomes, suggesting that reduced genome size enables a faster response to pulses in soil bacteria. Faster transcriptional upregulation of ribosomal protein genes was associated with high codon bias and increased nucleotide skew. We found that several of these relationships existed within phyla, indicating that these associations between genomic traits and activity could be generalized characteristics of soil bacteria. Finally, we used publicly available metagenomes to assess the distribution of codon bias across a pH gradient and found that microbial communities in higher pH soils—which are often more water limited and pulse driven—have higher codon usage bias in their ribosomal protein genes. Together, these results provide evidence that genomic characteristics affect soil microbial activity during rewetting and pose a potential fitness advantage for soil bacteria where water and nutrient availability are episodic.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Defining the Minimal Set of Microbial Genes Required for Valorization of Lignin Biomass (Final Report)

Project Goals: Lignin is the second most abundant biopolymer on earth and represents a critically underutilized biomass resource for hydrocarbon feedstocks. Despite substantial effort, there is still no efficient process to convert lignin to useable carbon-based platform chemicals and materials. The goal of this project is identify a minimal set of microbial enzymes necessary for lignin breakdown and sufficient for the synthesis of valuable chemical intermediates from lignin isolated as a byproduct of lignocellulosic ethanol production. These genes will be then used to engineer functional whole cell biocatalysts for tunable lignin metabolism. To date, although a number of enzymes have been associated with lignin degradation, most have been tested in isolation (as individual enzymes) and on drastically different substrates -- often dyes that are not related to lignin. In contrast, lignin utilization in nature likely occurs by microbial consortia with multiple enzymes acting synergistically. We propose to examine two separate stages of lignin breakdown carried out by the microbes that do it best: (1) early breakdown of native polymeric lignin into soluble fragments by a set of sequenced wood-rotting fungal species, and (2) downstream metabolism of these soluble lignin fragments to useful chemical intermediates by a panel of sequenced soil saprophytes. Our approach involves testing sets of genes that will be assayed combinatorially in the context of a heterologous expression host. The resulting engineered strains will be systematically assayed using soluble lignin fragments, synthetic defined polymeric lignin, and finally lignin directly sourced from lignocellulosic processing streams. In addition to resulting in a functional whole cell biocatalyst for lignin utilization, we anticipate that this approach will allow us to address key unanswered questions about lignin metabolism in nature, including: (1) Why does the Trametes versicolor genome contain 25 different class II peroxidases? (2) What is the role of laccases in lignin metabolism? Why do some aggressive lignin degraders have many laccases (e.g. >7 in T. versicolor) while others have none (e.g. P. chrysosporium)? (3) How is peroxide provided in a controlled manner to drive peroxidase activity without causing the enzyme inhibition that is so often observed in vitro? (4) What strategies do microbial lignin degraders use to avoid the problem of repolymerization during active lignin degradation? and (5) Can microbial lignin metabolism be diverted for high level production of defined aromatics? A final critical question is whether combining key minimal sets of enzymes from a wide range of organisms will result in engineered strains capable of highly efficient, streamlined pathways for lignin utilization that can be tuned for a specific carbon output. This effort will leverage DOE investments in microbial genome sequencing, and secure a critical channel for lignin biomass utilization that will also help to render lignocellulosic a viable feedstock for the production of renewable liquid biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineering Citrobacter freundii using CRISPR/Cas9 system

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) system is a useful tool to edit genomes quickly and efficiently. However, the use of CRISPR/Cas9 to edit bacterial genomes has been limited to select microbial chassis primarily used for bioproduction of high value products. Thus, expansion of CRISPR/Cas9 tools to other microbial organisms is needed. Here, our aim was to assess the suitability of CRISPR/Cas9 for genome editing of the Citrobacter freundii type strain ATCC 8090. We evaluated the commonly used two plasmid pCas/pTargetF system to enable gene deletions and insertions in C. freundii and determined editing efficiency. The CRISPR/Cas9 based method enabled high editing efficiency (~91%) for deletion of galactokinase (galk) and enabled deletion with various single guide RNA (sgRNA) sequences. To assess the ability of CRISPR/Cas9 tools to insert genes, we used the fluorescent reporter mNeonGreen, an endopeptidase (yebA), and a transcriptional regulator (xylS) and found successful insertion with high efficiency (81-100%) of each gene individually. These results strengthen and expand the use of CRISPR/Cas9 genome editing to C. freundii as an additional microbial chassis.

59 BASIC BIOLOGICAL SCIENCES↗