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At least 181 records · Page 10

Exabiome: Advancing Microbial Science through Exascale Computing

The Exabiome project seeks to improve the understanding of microbiomes through the development of methods for accelerating metagenomic science using exascale computing. This article gives an overview of scientific impact of the three components of the project: metagenome assembly, protein family detection, and comparative analysis of metagenomes. Exabiome developed MetaHipMer, the only metagenome assembler capable of scaling to full exascale systems. MetaHipMer has enabled ground-breaking assemblies on the Frontier supercomputer, with many scientific benefits, such as the discovery of rare species and viral genomes. To investigate protein families, Exabiome developed two exascale tools, PASTIS and HipMCL. Together, these can utilize exascale resources to understand the functional diversity of billions of dark matter proteins and novel protein families. For comparative analysis, Exabiome developed kmerprof, a tool that can be used to compare huge metagenomes for many different scientific purposes, for example, grouping human microbiomes according to body location.

59 BASIC BIOLOGICAL SCIENCES↗

Metagenomes and Metagenome-Assembled Genomes from Microbial Communities in a Biological Nutrient Removal Plant Operated at Hamptons Road Sanitation District (HRSD) with High and Low Dissolved Oxygen Conditions

In this study, we aimed to evaluate Biological Nutrient Removal (BNR) and investigate microbial community changes as the dissolved oxygen is reduced in the aerated portions of wastewater treatment trains. We present a dataset of Metagenome-Assembled Genomes (MAGs) obtained from activated sludge collected from the Hamptons Road Sanitation District (HRSD) BNR plant at the beginning of operation, when the DO was high, and at the end of operation, when the DO was low.

Genomics↗

Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in Pseudomonas putida

ABSTRACT Lignocellulose is a renewable resource for the production of a diverse array of platform chemicals, including the biofuel isoprenol. Although this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here, we examined the growth and isoprenol production in a Pseudomonas putida strain pre-tolerized (“PT”) background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon sources. We found that PT displays impaired growth in minimal medium containing acetate and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation pathways, were key for higher isoprenol titers in the tolerized strains, whereas a different set of mechanisms were likely enabling tolerance phenotypes in media containing acetate. Among these, a coproporphyrinogen-III oxidase (HemN) was upregulated across all tolerized strains and in one isolate required for acetate tolerance. Utilizing a defined glucose and acetate mixture ratio reflective of lignocellulosic feedstocks for isoprenol production in P. putida allowed us to obtain insights into the dynamics and challenges unique to dual carbon source utilization that are obscured when studied separately. Together, this enabled the development of a P. putida bioconversion chassis able to use a more complex carbon stream to produce isoprenol. IMPORTANCE Acetate is a relatively abundant component of many lignocellulosic carbon streams and has the potential to be used together with sugars, especially in microbes with versatile catabolism such as P. putida . However, the use of mixed carbon streams necessitates additional optimization. Furthermore, the use of P. putida for the production of the biofuel target, isoprenol, requires the use of engineered strains that have additional growth and production constraints when cultivated in acetate and glucose mixtures. In this study, we generate acetate-tolerant P. putida strains that overcome these challenges and examine their ability to produce isoprenol. We show that acetate tolerance and isoprenol production, although independent phenotypes, can both be optimized in a given P. putida strain. Using proteomics and whole genome sequencing, we examine the molecular basis of both phenotypes and show that tolerance to acetate can occur via alternate routes and result in different impacts on isoprenol production.

de Siqueira, Guilherme M. V. (ORCID:00000002364563↗

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

ABSTRACT Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitute the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased the MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full-length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs. IMPORTANCE Spatially organizing biosynthetic pathway enzymes is a promising strategy to increase pathway throughput and yield. Bacterial microcompartments (MCPs) are proteinaceous organelles that many bacteria natively use as a spatial organization strategy to encapsulate niche metabolic pathways, providing significant metabolic benefits. Encapsulating heterologous pathways of interest in MCPs could confer these benefits to industrially relevant pathways. Here, we investigate the role of signal sequences, short domains that target proteins for encapsulation in MCPs, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterize two novel signal sequences on structural proteins, constituting the first Pdu signal sequences found on structural proteins rather than enzymes, and perform knockout studies to compare the impacts of enzymatic and structural signal sequences on MCP assembly. Our results demonstrate that enzymatic and structural signal sequences play critical but distinct roles in Pdu MCP assembly and provide design rules for engineering MCPs while minimizing disruption to MCP assembly.

Johnson, Elizabeth R. (ORCID:0000000179236881)↗

A constraint-based framework for exploring the impact of multireaction dependencies on metabolic functions

Abstract Metabolism operates under physico-chemical constraints that result in multireaction dependencies. Understanding how multireaction dependencies affect metabolic phenotypes remains challenging, hindering their biotechnological applications. Here, we propose the concept of a forcedly balanced complex that allows to efficiently determine the effects of specific multireaction dependencies on metabolic network functions in constrained-based models. Using this concept, we found that the fraction of multireaction dependencies induced by forcedly balanced complexes in genome-scale metabolic networks followed power law with exponential cut-off. We identified forcedly balanced complexes that are lethal in cancer but have little effect on growth in healthy tissue models. In addition, these forcedly balanced complexes are largely specific to models of particular cancer types. Therefore, multireaction dependencies resulting from forced balancing of complexes represent an innovative means to control cancers that, we argue, can be implemented via transporter engineering. The presented constraint-based approaches pave the way for using multireaction dependencies in metabolic engineering for diverse biotechnological applications.

Küken, Anika↗

Systematic engineering for production of anti-aging sunscreen compound in Pseudomonas putida

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO 2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13 C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

59 BASIC BIOLOGICAL SCIENCES↗

Data Mining of Groundwater to Identify MAGs with Methane, Propane and Toluene Monooxygenases

Whole genome sequencing datasets, involving more than 600 groundwater samples, from nine countries, were analyzed to identify metagenome assembled genomes (MAGs) containing full operons for propane monooxygenase, soluble methane monooxygease, toluene monooxygenase and particulate ammonia/methane monooxygenase. The enzymes encoded by these genes are a focus of interest because of their ability to degrade common groundwater contaminants. Due to the large amount of data, sequence analyses involved more than 80 individual KBase narratives. The approach followed the KBase tutorial called "Metagenome-Assembled Genome Extraction from a Compost Microbiome Enrichment" The generated MAGs were exported from each individual narrative into separate summary KBase narratives for each monooxygenase. Three KBase narratives were generated for particulate ammonia/methane monooxygenase, due to the large number of MAGs identified.

59 BASIC BIOLOGICAL SCIENCES↗

Development of modular expression across phylogenetically distinct diazotrophs

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here, we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

59 BASIC BIOLOGICAL SCIENCES↗

Expanding the genetic toolkit: adenine and cytosine base editors for gene disruption in Aspergillus niger

Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A‧T to G‧C, and cytosine base editors (CBEs), which convert C‧G to T‧A, offer a safer alternative by enabling predictable, target-specific single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger . For the first time, we constructed an ABE in A. niger , achieving up to 80% editing efficiency and inducing predictable A-to-G mutations at the intended intron sites, disrupting gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Leveraging this expanded scope, we established gene disruption approaches by targeting start codons via ABE-mediated A-to-G conversions (ATG-to-GTG and ATG-to-ACG) and CBE-mediated C-to-T conversion (ATG-to-ATA). Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Collectively, the scope-expanding strategies increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.

Aspergillus↗

Poplar

SAND2025-00683O Poplar is a software tool that generates a phylogenetic tree from input gene and genome sequences. It integrates established tools to identify genes within genomes, group sequences, construct gene trees, and infer a species tree. Poplar processes nucleotide sequences, identifies similar sequences using Nucleotide BLAST, groups them with DBSCAN, aligns sequences with MAFFT, constructs gene trees with RAxML-NG, and infers a species tree using ASTRAL-Pro3. This pipeline provides a structured approach to phylogenetic analysis, facilitating the study of evolutionary relationships among species. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.

Krishnakumar, Raga↗

Advancing Protein Display on Bacterial Spores through an Extensive Survey of Coat Components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 types of proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SPs investigated, 26 formed functional fusions with β-glucuronidase (GUS)─a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed 1) SP promoter activation dynamics, 2) GUS activity on spores, 3) surface availability, and 4) protection from thermal and proteolytic degradation. Multicopy expression and pairwise coexpression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and the difficulty in predicting compatibility between different SP fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface-exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (small spore coat assembly protein A) as an effective spore display carrier. SscA supported enzyme activity at least 4-fold higher than any other SP, including the well-established anchor, CotY. We attribute this to its promoter, which demonstrated early and sustained activation relative to other SPs and its small size (∼3 kDa), which likely minimally interferes with enzyme folding, oligomerization, and activity. Labeling and genetic studies, its hydrophobic nature, and low surface availability suggest that SscA assembles within the inner spore coat, which makes it stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledge base to advance the biotechnological utility of B. subtilis spores.

Bacillus subtilis↗

Towards engineering hybrid incompatibility in plants

The potential for gene flow between genetically modified organisms (GMOs) and non-GMO relatives poses a significant challenge to the development and regulatory approval of GMO crops (Wedger et al., 2024), for example, the spread of herbicide resistance transgenes from crops such as rice or sorghum to cross-pollinating weedy species. Addressing this concern, we developed Engineered Genetic Incompatibility (EGI) (Maselko et al., 2017), a system that establishes species-like barriers to gene flow between otherwise sexually compatible populations. EGI employs Programmable Transcriptional Activators (PTAs) to drive lethal over- and/or ectopic expression of tightly regulated genes following undesired hybridization events (Figure 1a,b). A benign mutation of the target promoter in the EGI organism protects it from ill effects of the PTA, which acts as a sentinel for the wild-type (WT) promoter sequence. Given numerous potential PTA targets, multiple mutually incompatible subpopulations are feasible (Maselko et al., 2020). EGI has been demonstrated in yeast as a proof-of-concept (Maselko et al., 2017) and in insects as a strategy for genetic biocontrol of pests (Maselko et al., 2020; Upadhyay et al., 2022). EGI in plants would provide a strategy to halt gene flow between engineered crops and their domestic and wild relatives without altering normal cultivation or propagation practices. Here, we present promising results towards the demonstration of EGI in plants and highlight technical challenges that still need to be overcome.

CRISPRa↗

Brochure for the DOE Office of Science Workshop on Envisioning Frontiers in AI and Computing for Biological Research

In February of 2025 a joint ASCR/BER workshop was held to identify key transformational research directions for understanding biology using artificial intelligence (AI), digital twins and high-performance (HPC) computational methods to facilitate scientific discovery and innovation in support of the Department of Energy mission. AI technologies offer exciting new groundbreaking methods to analyze large volumes of complex biological data, thereby greatly accelerating the ability to understand, predict, and design biological processes for beneficial purposes. In the laboratory, the bridging of AI-enabled automated experimental technologies, HPC and digital twins will provide potent tools for researchers to explore the fundamental nature of biology and harness its inherent metabolic potential for a variety of beneficial purposes. The focus of this workshop was on how high-performance computational methods can impact this objective by exploring digital twins, foundational models, and data-driven approaches with applications to advance automated laboratory experiments, modeling of complex living systems and engineering new functions into plants and microbial systems relevant to DOE mission. Workshop attendees with expertise in plant science, microbiology, mathematics, computer science, and AI assessed the current state of the science, trends, and AI challenges at the interface of plant and microbial systems biology and computational science to identify opportunities for high-impact research. This collaborative effort capitalized on ASCR's advancements in applied mathematics, computer science, and Exascale systems, and BER's expertise in basic genomics-enabled research on DOE relevant plant and microbial systems. The workshop culminated in four key priority research directions to guide future research and development within DOE Office of Science programs.

59 BASIC BIOLOGICAL SCIENCES↗

Leveraging CRISPR Cas9 RNPs and Cre- loxP in Picochlorum celeri for generation of field deployable strains and selection marker recycling

As new highly productive strains of algae are discovered and developed to meet the energy, chemical, and food requirements of the future, genetic engineering of those strains in a manner that yields deployable transformants is paramount. This study introduces the novel CRoxP ($\underline{\textrm{C}}$$\textrm{as9}$ $\underline{\textrm{R}}$$\textrm{NPs}$ coupled with an inducible $\underline{\textrm{CR}}$$\textrm{e}$-$\textrm{l}\underline{\textrm{oxP}}$) system for rapid generation of marker- and transgene-free strains of Picochlorum celeri. The CRoxP system allows reuse of selection markers without Cas9 expression in vivo, eliminating many of the bottlenecks associated with conventional CRISPR Cas9 use for precise genome editing. In P. celeri, transformants were generated with a turnaround time as short as 21 days between transformation and being ready for another round of transformation with the same selection marker by using the CRoxP system. As a use-case for CRoxP, depigmented strains of P. celeri were generated by multiplexed Cas9 disruption of major LHCII genes followed by either a second round of LHCII targeting, or knockout of an LHCI gene. One transformant tested in flask culture (R6) exhibited similar biomass production to the wild type with 46% less Chl a + b on a biomass basis. In photobioreactors and under diel light simulating a solar day, a transformant (LhcBM31) exhibited 34 g AFDW m –2 d –1 with 54% less Chl a + b on a biomass basis vs. wild type.

09 BIOMASS FUELS↗

Tapping the treasure trove of atypical phages

With advancements in genomics technologies, a vast diversity of ‘atypical’ phages, that is, with single-stranded DNA or RNA genomes, are being uncovered from different ecosystems. Though these efforts have revealed the existence and prevalence of these nonmodel phages, computational approaches often fail to associate these phages with their specific bacterial host(s), while the lack of methods to isolate these phages has limited our ability to characterize infectivity pathways and new gene function. In this review, we call for the development of generalizable experimental methods to better capture this understudied viral diversity via isolation and study them through gene-level characterization and engineering. Establishing a diverse set of new ‘atypical’ phage model systems has the potential to provide many new biotechnologies, including potential uses of these atypical phages in halting the spread of antibiotic resistance and engineering of microbial communities for beneficial outcomes.

59 BASIC BIOLOGICAL SCIENCES↗

Discovering methylated DNA motifs in bacterial nanopore sequencing data with MIJAMP

Abstract Bacterial DNA methylation is involved in diverse cellular functions, including modulation of gene expression, DNA repair, and restriction–modification systems for defense against viruses and other foreign DNA. Restriction systems hinder efforts to engineer organisms to produce fuels and chemicals from waste and renewable feedstocks by degrading DNA during transformation. Methylome analysis allows identification of motifs within a bacterial chromosome that may be targeted by native restriction enzymes. Further expression of the corresponding methyltransferases in Escherichia coli allows plasmid DNA to be protected from restriction in the target organism, thereby drastically enhancing transformation efficiency. Nanopore sequencing can detect methylated bases, but software is needed to transform modified base coordinates into methylated motifs. Here, we develop MIJAMP (MIJAMP Is Just A MethylBED Parser), a software package that was developed to discover methylated motifs from the output of ONT’s Modkit or other data in the methylBED format. MIJAMP employs a human-driven refinement strategy that empirically validates all motifs against genome-wide methylation data, thus eliminating incorrect motifs. MIJAMP also reports methylation data on specific, user-defined motifs. Using MIJAMP, we determined the methylated motifs both in a control strain (wild-type E. coli) and in Synecococcus sp. strain PCC7002, laying the foundation for improved transformation in this organism. MIJAMP is available at https://code.ornl.gov/alexander-public/mijamp/. One Sentence Summary: Here we describe software written to discover DNA methylation motifs from nanopore sequencing data.

59 BASIC BIOLOGICAL SCIENCES↗

Mechanisms of Polyethylene Terephthalate Pellet Fragmentation into Nanoplastics and Assimilable Carbons by Wastewater Comamonas

Comamonadaceae bacteria are enriched on poly(ethylene terephthalate) (PET) microplastics in wastewaters and urban rivers, but the PET-degrading mechanisms remain unclear. Here, we investigated these mechanisms with Comamonas testosteroniKF-1, a wastewater isolate, by combining microscopy, spectroscopy, proteomics, protein modeling, and genetic engineering. Compared to minor dents on PET films, scanning electron microscopy revealed significant fragmentation of PET pellets, resulting in a 3.5-fold increase in the abundance of small nanoparticles (<100 nm) during 30-day cultivation. Infrared spectroscopy captured primarily hydrolytic cleavage in the fragmented pellet particles. Solution analysis further demonstrated double hydrolysis of a PET oligomer, bis(2-hydroxyethyl) terephthalate, to the bioavailable monomer terephthalate. Supplementation with acetate, a common wastewater co-substrate, promoted cell growth and PET fragmentation. Of the multiple hydrolases encoded in the genome, intracellular proteomics detected only one, which was found in both acetate-only and PET-only conditions. Homology modeling of this hydrolase structure illustrated substrate binding analogous to reported PET hydrolases, despite dissimilar sequences. Mutants lacking this hydrolase gene were incapable of PET oligomer hydrolysis and had a 21% decrease in PET fragmentation; re-insertion of the gene restored both functions. Thus, we have identified constitutive production of a key PET-degrading hydrolase in wastewater Comamonas, which could be exploited for plastic bioconversion.

54 ENVIRONMENTAL SCIENCES↗

MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.

59 BASIC BIOLOGICAL SCIENCES↗