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At least 973 records · Page 54
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

The HIGH CHLOROPHYLL FLUORESCENCE 244 homolog CrHCF244 is required for psbA (D1) translation in Chlamydomonas reinhardtii

Translation of psbA, the chloroplast gene that encodes the D1 subunit of PSII, is important for both PSII biogenesis and repair. The translation of psbA transcripts in the chloroplast is under the control of nuclear gene products. Using a forward genetic screen and whole-genome sequencing of the alga Chlamydomonas reinhardtii , we found a mutant defective in PSII activity and mapped the causative gene to be the homolog of Arabidopsis HIGH CHLOROPHYLL FLUORESCENCE 244 (HCF244) , namely CrHCF244 . We then demonstrated that CrHCF244 is required for psbA translation in the alga, consistent with the function of HCF244 in Arabidopsis, and found that AtHCF244 also partially complemented the algal mutant. These results experimentally support the functional conservation of the homologs in green algae and land plants. Intriguingly, the CrHCF244 mutant also exhibited a relatively high rate of suppressor mutants, pointing to the presence of alternative factor(s)/pathway(s) for D1 translational control. The establishment of CrHCF244 as a psbA translation factor in C. reinhardti i shows the similarities in psbA translation regulation in algae and plants. The future identification of the alternative factor(s) in this alga will provide insights on psbA translation in plants.

Arabidopsis↗
DOE OSTI2026

Poxvirus infection triggers remodeling of host m⁶A epitranscriptome and benefits from the m⁶A regulatory responses

Understanding how host gene regulation responds to viral infection is essential for developing effective antiviral strategies. Emerging evidence suggests that host transcripts undergo dynamic chemical modifications to counteract viral invasion. Conversely, viruses that rely on nuclear transcription exploit host RNA methyltransferases to enhance mRNA export and translation. Orthopoxviruses, however, complete their entire replication cycle within compartmentalized cytoplasmic “factories” utilizing enzymes encoded by their large double-stranded viral DNA genomes. The dynamic interplay between host and poxviral epitranscriptome remains poorly characterized. Using a temporally resolved model of Vaccinia virus (VV) infection, we investigated host-virus interactions through transcriptome and N6-methyladenosine (m⁶A) epitranscriptome whole genome sequencing. We found that host m⁶A modifications respond rapidly to VV infection, preceding the delayed transcriptional changes that emerge at later stages. Early m⁶A signatures included key innate immunity factors as well as host genes involved in transcriptional regulation, post-transcriptional modification, and protein ubiquitination. Functional assays validated two host factors with early m⁶A modification changes that are essential for VV infection: a m⁶A reader, YTHDF1, and a component of the SCF E3 ubiquitin ligase complex, FBXO31. The m⁶A gain on YTHDF1 enhanced its protein expression and promoted efficient VV replication. In addition, we identified previously unrecognized roles of FBXO31 and the SCF E3 ligase complex in supporting VV infection. Temporal profiling of the m⁶A epitranscriptome reveals how VV exploits host post-transcriptional regulatory pathways, specifically m⁶A RNA modification and protein ubiquitination. These findings highlight critical host factors co-opted during poxvirus infection and identify potential targets for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism’s energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T . pallidum lipoproteins, we proposed a “flavin-centric” metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T . pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T . pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T . pallidum , and we propose that the protein be referred to henceforth as TpPta.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2016

Integrating Large Scale Data Sets to Develop Predictive Hypotheses of Low-Dose Radiation-Induced Health Effects

Over one hundred years of radiation biology research has revealed much about the DNA damages induced by the deposition of energy from exposure to ionizing radiation and the subsequent cellular responses. However, there are still significant gaps in our understanding of how these might lead to detrimental health effects, particularly at low doses (100 mGy (milligray)). Recent advances in high throughput omics technologies enable interrogation of induced radiation effects at the genomic, proteomic and metabolomic levels. These include changes in gene expression, protein modifications, e.g., phosphorylation, acetylation, and methylation, and metabolic changes. We will discuss the integration of data obtained from multiple omics platforms to understand radiation dose, and dose rate effects in a complex human tissue model as a function of time. We will use as an example our results on the low dose responses in a 3D human skin model.

ionizing radiation↗
NASA NTRS2023

Evolutionary Flexibility and Rigidity in the Bacterial Methylerythritol Phosphate (Mep) Pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Bailey Marshall↗
DOE OSTI2026

Unraveling plant phenotype to genotype associations with daily hyperspectral traits in Populus trichocarpa

Hyperspectral remote sensing is a powerful, high-throughput phenotyping tool that quantifies physiologically and structurally relevant wavelengths across diverse genotypes and over varying temporal scales. In this study, we combined tower-based continuous hyperspectral sensing with genome-wide association studies to analyze 1423 wavebands (400-900 nm) and derivative vegetation indices across 505 genotypes and the genetic architecture of hyperspectral phenotypes over time in Populus trichocarpa Torr. & Gray grown under field conditions. Wavelengths related to chlorophyll and carotenoid absorption spectra exhibited the strongest genetic variation resulting in 98 significant SNP associations. Notably, we found substantial overlap in genetic association between the blue and red spectral regions, indicative of carotenoids and chlorophyll, respectively, and identified more than 10 candidate genes associated with chloroplast function, underpinning photosynthetic activity. Furthermore, fluctuations in associations for vegetative indices, such as the chlorophyll:carotenoid index (CCI), across the growing season reveal a temporally dynamic genetic architecture of physiological traits associated with fall senescence of this temperate tree species. Finally, we also observed correlations (spearman rho = 0.3, p < 1x10 −8 ) between individual wavebands or vegetative indices and growth rate, assessed as the relative change of tree height over the growing season. The growth rate prediction was substantially improved by a regularization multivariate model (spearman rho>0.5, p < 1x10 −16 ), reinforcing the value of hyperspectral measurements for predicting traits linked to tree productivity. These findings highlight the potential of high-throughput, rapid, hyperspectral genome wide association studies GWAS to uncover physiologically meaningful genetic variation and offer promising insights for future acceleration for plant breeding.

09 BIOMASS FUELS↗
DOE OSTI2022

Phylogenomic structure and speciation in an emerging model: the Sphagnum magellanicum complex (Bryophyta)

Sphagnum magellanicum is one of two Sphagnum species for which a reference-quality genome exists to facilitate research in ecological genomics. Phylogenetic and comparative genomic analyses were conducted based on resequencing data from 48 samples and RADseq analyses based on 187 samples. Here, we report that there are four clades/species within the S. magellanicum complex in eastern North America and that the reference genome belongs to Sphagnum divinum . The species exhibit tens of thousands (RADseq) to millions (resequencing) of fixed nucleotide differences. Two species, however, referred to informally as S. diabolicum and S. magni because they have not been formally described, are differentiated by only 100 (RADseq) to 1000 (resequencing) of differences. Introgression among species in the complex is demonstrated using D-statistics and f 4 ratios. One ecologically important functional trait, tissue decomposability, which underlies peat (carbon) accumulation, does not differ between segregates in the S. magellanicum complex, although previous research showed that many closely related Sphagnum species have evolved differences in decomposability/carbon sequestration. Phylogenetic resolution and more accurate species delimitation in the S. magellanicum complex substantially increase the value of this group for studying the early evolutionary stages of climate adaptation and ecological evolution more broadly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

ggtaxplot v 0.0.1

ggtaxplot is an R package designed to process and visualize taxonomic data through a taxonomic river plot. This package is ideal for researchers and data scientists who need to visualize taxonomic data. ggtaxplot function processes data and generates a taxonomic river plot, allowing users to visualize the distribution of taxa across different samples.

Coclet, Clement [Lawrence Berkeley National Labora↗
DOE OSTI2023

Water mass age structures the auxiliary metabolic gene content of free-living and particle-attached deep ocean viral communities

Viruses play important roles in the ocean’s biogeochemical cycles. Yet, deep ocean viruses are one of the most under-explored fractions of the global biosphere. Little is known about the environmental factors that control the composition and functioning of their communities or how they interact with their free-living or particle-attached microbial hosts. We analysed 58 viral communities associated with size-fractionated free-living (0.2–0.8 μm) and particle-attached (0.8–20 μm) cellular metagenomes from bathypelagic (2150–4018 m deep) microbiomes obtained during the Malaspina expedition. These metagenomes yielded 6631 viral sequences, 91% of which were novel, and 67 represented high-quality genomes. Taxonomic classification assigned 53% of the viral sequences to families of tailed viruses from the order Caudovirales. Computational host prediction associated 886 viral sequences to dominant members of the deep ocean microbiome, such as Alphaproteobacteria (284), Gammaproteobacteria (241), SAR324 (23), Marinisomatota (39), and Chloroflexota (61). Free-living and particle-attached viral communities had markedly distinct taxonomic composition, host prevalence, and auxiliary metabolic gene content, which led to the discovery of novel viral-encoded metabolic genes involved in the folate and nucleotide metabolisms. Water mass age emerged as an important factor driving viral community composition. We postulated this was due to changes in quality and concentration of dissolved organic matter acting on the host communities, leading to an increase of viral auxiliary metabolic genes associated with energy metabolism among older water masses. These results shed light on the mechanisms by which environmental gradients of deep ocean ecosystems structure the composition and functioning of free-living and particle-attached viral communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A comparative genomics approach identifies contact-dependent growth inhibition as a virulence determinant

Emerging evidence suggests the Pseudomonas aeruginosa accessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. In this work, we utilized a mouse bacteremia model to quantify the virulence of 100 individual P. aeruginosa bloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulent P. aeruginosa isolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that this P. aeruginosa CDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems in P. aeruginosa pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Enrichment of root-associated Streptomyces strains in response to drought is driven by diverse functional traits and does not predict beneficial effects on plant growth

The genus Streptomyces has consistently been found enriched in drought-stressed plant root microbiomes, yet the ecological basis and functional variation underlying this enrichment at the strain and isolate level remain unclear. Using two 16S rRNA sequencing methods with different levels of taxonomic resolution, we confirmed drought-associated enrichment (DE) of Streptomyces in field-grown sorghum roots and identified five closely related but distinct amplicon sequence variants (ASVs) belonging to the genus with variable drought enrichment patterns. From a culture collection of sorghum root endophytes, we selected 12 Streptomyces isolates representing these ASVs for phenotypic and genomic characterization. Whole-genome sequencing revealed substantial variation in gene content, even among closely related isolates, and exometabolomic profiling showed distinct metabolic responses to media supplemented with drought- versus well-watered root tissue. Traits linked to drought survival, including osmotic stress tolerance, siderophore production, and carbon utilization, varied widely among isolates and were not phylogenetically conserved. Using a broader panel of 48 Streptomyces, we demonstrate that DE scores, determined through mono-association experiments in gnotobiotic sorghum systems, showed high variability and lacked correlation with plant growth promotion. Pangenome-wide association identified orthogroups involved in osmolyte transport (e.g., proP) and membrane biosynthesis (e.g., fabG) as positively associated with DE, though most associations lacked phylogenetic signal. Collectively, these results demonstrate that Streptomyces DE is not a conserved genus-level trait but is instead strain-specific and functionally heterogeneous. Furthermore, DE in the root microbiome was shown not to predict beneficial effects on plant growth. This work underscores the need to resolve functional traits at the strain level and highlights the complexity of microbe-host-environment interactions under abiotic stress.

Fonseca-Garcia, Citlali↗
DOE OSTI2024

Chloroplast Stress Signals: Control of Retrograde Signaling, Chloroplast Turn-Over, and Cell Fate Decisions

Chloroplasts (photosynthetic plastids) are semiautonomous organelles that contain their own small genomes. The proteomes of chloroplasts, however, are a mixture of plastid and nuclear-encoded proteins. Chloroplasts perform photosynthesis, which is prone to damaging the organelles, leading to the production of reactive oxygen species (ROS) that damage the cell under environmental stresses. Thus, for the cell to maintain proper chloroplast function, efficient photosynthesis, and avoid ROS damage, it relies on complex crosstalk between the chloroplast, the nucleus, other organelles within the cell, and the cytoplasm in between. This communication involves retrograde signals from chloroplasts to control nuclear gene expression, programmed cell death (PCD), and chloroplast degradation. Here we review these signals with an emphasis on the roles of the ROS singlet oxygen ( 1 O 2 ) and plastid gene expression. We cover (1) recent work on understanding how multiple 1 O 2 signaling pathways can be initiated within stressed chloroplasts, (2) how individualized post-translational regulatory systems allow chloroplasts to control their proteomes and degradation, and (3) how chloroplast signals ultimately control cell fate decisions, such as PCD, senescence, and vacuole-mediated degradation of chloroplasts (chloroplast quality control). Overall, this chapter discusses how chloroplasts can act as environmental sensors for the cell and allow plants to acclimate to stress and thrive in dynamic environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Beyond Component Optimization: Systems Level Biodesign for Lanthanide Recovery

Global demand for lanthanides (Ln) is projected to rise sharply over the next decade, while geographically concentrated supply chains and the low concentrations and matrix complexity of secondary feedstocks limit the reach of conventional hydro- and pyrometallurgical separation. Engineered biological systems offer a selective, low-energy alternative, and component-level advances in Ln-binding proteins, AI-designed selective scaffolds, and cell-surface display platforms now rival synthetic chelators in affinity and selectivity. These components, however, remain functionally isolated. Currently, there are no engineered chassis coupling recognition, intracellular trafficking, accumulation, and controlled release into an end-to-end pipeline. Here, we outline how new biodesign strategies and chassis selection must move beyond bioleaching to encompass the full recovery pathway. Achieving this requires integrating AI/ML-guided design, genome-scale build tools, high-throughput phenotyping, and biophysical transport modeling within a Design–Build–Test–Learn cycle tuned to recognition, trafficking, accumulation, and release.

Biodesign↗
DOE OSTI2026

MAL33 drives natural variation in maltose metabolism in Saccharomyces eubayanus

Maltose is one of the most abundant sugars in brewer’s wort, and its efficient utilization is critical for successful fermentation. However, maltose consumption varies naturally among Saccharomyces eubayanus strains isolated from different host trees, such as Quercus and Nothofagus. To identify the genetic determinants underlying these phenotypic differences, we performed bulk segregant analysis (BSA) and quantitative trait loci (QTL) mapping using an F 2 offspring derived from QC18 (Quercus-associated) and CL467.1 (Nothofagus-associated) strains. QTL mapping identified two significant genomic regions on subtelomeric loci of chromosomes V-R and XVI-L, each containing complete MAL loci composed of MAL32 (encoding maltase), MAL31 (transporter), and MAL33 (transcriptional activator) genes. Comparative polymorphism analyses identified mutations in MAL32 and MAL33 of QC18, including frameshift mutations resulting in premature stop codons. Functional validation demonstrated that the heterologous expression of MAL33 ChrV from CL467.1 fully restored maltose utilization in QC18, indicating the functional presence of MAL33 cis-regulatory sequences and MAL32 and MAL31 genes in QC18. While structural protein predictions identified truncation and impaired functionality in the maltose-responsive activation domain of Mal33p from QC18, overexpression of QC18’s own MAL33 ChrV allele also improved maltose metabolism, suggesting dosage-dependent transcriptional limitations rather than complete functional loss. These results indicate that allelic variations in the maltose-responsive activation domain of Mal33p result in differences in maltose consumption between strains. Here, we hypothesized that reduced maltose metabolism in QC18 is an adaptive response to the distinct sugar composition in Quercus robur bark, contrasting with the starch-rich environment of Nothofagus pumilio. These findings highlight subtelomeric MAL gene diversity as a reservoir of genetic variation, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.

evolutionary plasticity↗
DOE OSTI2022

Chromosome-Level Genome Assembly of Mentha longifolia L. Reveals Gene Organization Underlying Disease Resistance and Essential Oil Traits

Mentha longifolia (L.) Huds., a wild, diploid mint species, has been developed as a model for mint genetic and genomic research to aid breeding efforts that target Verticillium wilt disease resistance and essential oil monoterpene composition. Here, we present a near-complete, chromosome-scale mint genome assembly for M. longifolia USDA accession CMEN 585. This new assembly is an update of a previously published genome draft, with dramatic improvements. A total of 42,107 protein-coding genes were annotated and placed on 12 chromosomal scaffolds. One hundred fifty-three genes contained conserved sequence domains consistent with nucleotide binding site-leucine-rich-repeat (NBS-LRR) plant disease resistance genes. Homologs of genes implicated in Verticillium wilt resistance in other plant species were also identified. Multiple paralogs of genes putatively involved in p-menthane monoterpenoid biosynthesis were identified and several cases of gene clustering documented. Heterologous expression of candidate genes, purification of recombinant target proteins, and subsequent enzyme assays allowed us to identify the genes underlying the pathway that leads to the most abundant monoterpenoid volatiles. Here, the bioinformatic and functional analyses presented here are laying the groundwork for using marker-assisted selection in improving disease resistance and essential oil traits in mints.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials

Abstract Suberin is a hydrophobic biopolymer of significance in the production of biomass-derived materials and in biogeochemical cycling in terrestrial ecosystems. Here, we describe suberin structure and biosynthesis, and its importance in biological (i.e., plant bark and roots), ecological (soil organic carbon) and economic (biomass conversion to bioproducts) contexts. Furthermore, we highlight the genomics and analytical approaches currently available and explore opportunities for future technologies to study suberin in quantitative and/or high-throughput platforms in bioenergy crops. A greater understanding of suberin structure and production in lignocellulosic biomass can be leveraged to improve representation in life cycle analysis and techno-economic analysis models and enable performance improvements in plant biosystems as well as informed crop system management to achieve economic and environmental co-benefits.

09 BIOMASS FUELS↗
DOE OSTI2025

PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification

Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization.

42 ENGINEERING↗