BioSciences

Redox-balance-based growth coupling links oxidoreductase activity to cellular fitness and can enable high-throughput enzyme selection and strain evolution. However, because growth reflects the fitness of the whole cell, metabolic adaptation can circumvent the intended selective pressure. Here, we investigated the evolutionary stability of an aerobic NADPH redox-balance selection strain of Escherichia coli engineered to accumulate NADPH during growth on glucose. Adaptive evolution first selected mutations predicted to relax cofactor specificity in NADH-dependent quinone oxidoreductases, allowing NADPH-derived reducing equivalents to enter respiratory electron flow. Removing major NADH-to-quinone oxidoreductases did not eliminate the capacity for evolutionary escape; in one successfully evolving lineage, adaptive evolution uncovered a latent lactate-mediated route for electron transfer into the quinone pool. These results reveal strong selective pressure to reconnect soluble nicotinamide redox metabolism with energy-conserving respiration and demonstrate that E. coli can access multiple pathways for restoring electron flux between these systems. Overall this work outlines design principles for aerobic, growth-coupled strains which rely upon redox balance, illustrating that successful designs must limit respiratory rewiring while preserving sufficient carbon and energy flux to support growth and pathway selection.

Most biorefineries rely on fermentation to produce a single molecule, such as ethanol. This exposes facilities to price volatility and limits their flexibility to respond to market shifts and critical domestic supply chain needs. Reorienting biomanufacturing toward greater flexibility necessitates the development of versatile production platforms and chemicals with multiple upgrading options to broaden the suite of products. This study presents a design that leverages biomass ensiling, ionic liquid pretreatment, industrial microbial hosts, and catalytic upgrading. The result is a platform to convert sorghum to organic acids, indigoidine (an example of a polymer precursor and alternative to a textile dye), and varying ratios of gasoline and jet fuel blendstocks. The facility can reach net average profits above $111 per bone-dry metric ton of biomass, but only if industrial-grade organic acids are recovered and indigoidine can be used as a one-to-one replacement for synthetic indigo dye at current market prices. Results further demonstrate that economic performance is strongly governed by product allocation decisions, particularly the balance between fuels and higher-value chemical products. Rather than relying on single-product optimization, this work highlights a portfolio-based production strategy that enables dynamic adjustment of product outputs in response to market conditions. Overall, the integrated use of flexible feedstock processing, multiproduct upgrading, and selective valorization pathways provides a framework for improving the economic resilience and adaptability of future lignocellulosic biorefineries.

Ecosystem retrogression drives nutrient depletion, reduced productivity, and profound reorganization of soil microbial communities. Using amplicon sequencing and genome-resolved metagenomics, we examined how cross-domain microbial networks and functional gene potential respond to long-term phosphorus and nitrogen limitation along the well-characterized Ecological Staircase chronosequence in Mendocino, California, USA. Microbial diversity and abundance declined sharply with terrace age for prokaryotes, predatory protists, and bacteriophages, whereas fungi and phototrophic protists increased in nutrient-depleted, acidic soils. These compositional shifts were accompanied by major changes in reconstructed microbial networks: relative modularity increased alongside robustness, indicating adaptive reorganization that may sustain ecosystem function under resource scarcity. Fungi emerged as central stabilizers in these restructured networks, carrying enriched genetic potential to degrade plant polymers and mobilize phosphorus and nitrogen. Despite a decline in overall phage diversity, the relative abundance of phages encoding phosphorus-mobilizing auxiliary metabolic genes increased, suggesting that viral contributions to host phosphorus metabolism may be enhanced under nutrient limitation. Together, these results demonstrate that ecosystem retrogression drives cross-domain microbial reorganization toward fewer but more interconnected lineages, characterized by greater integration of functional genetic potential. This reorganization enhances the potential for functional resilience under extreme nutrient limitation, revealing how microbial networks adapt to maintain the capacity for nutrient cycling and stability as soils age and fertility declines.

Efficient co-utilization of hexose and pentose sugars from lignocellulose is essential for microbial bioconversion, yet engineered catabolic pathways can be unstable or suboptimal in complex resource environments. Here, we use a Pseudomonas putida strain engineered to catabolize xylose and arabinose to examine how resource abundance, temporal availability, and subculturing shape evolutionary outcomes. Using an automated adaptive laboratory evolution (ALE) platform, we evolve the strain under simple single-substrate and complex multi-substrate selection pressures. These environments drive divergence between catabolic specialists and generalists. Weak or absent selection for xylose frequently leads to loss of xylose catabolism, whereas carbon-limited mixed-sugar environments promote stable retention and coordinated optimization of multiple catabolic pathways, enhancing growth and substrate utilization. Genomic, proteomic, and biochemical analyses show that pathway-specific fitness costs determine evolutionary stability. A generalist clone also shows improved indigoidine production from mixed sugars relative to the parental strain. Together, these findings show how resource dynamics shape fitness landscapes that govern catabolic specialization, generalization, evolutionary trade-offs, and engineering of bioconversion.

The structural biology method of X-ray footprinting mass spectrometry (XFMS) is available at two national synchrotron beamlines in the USA: one at the Advanced Light Source (ALS) on the West Coast and the other at the National Synchrotron Light Source II on the East Coast. XFMS is a solution-state technique that utilizes oxidative modifications of proteins at micromolar concentrations in aqueous buffer to extract structural information. X-rays are employed to generate hydroxyl radicals in situ, which covalently modify specific protein side chains. These modifications are subsequently quantified using liquid chromatography and mass spectrometry. Ratiometric changes in modification levels between two protein states (e.g. with and without ligand) generate a relative solvent accessibility map of the protein pairs, which serves to reveal structural features. Up until recently, the XFMS capability was available as part of a shared program at the ALS without a dedicated beamline. In this article, we describe the commissioning of ALS beamline 3.3.1, dedicated to XFMS, including the installation of a new focusing mirror, the design and construction of a new endstation with automated sample handling and exposure capabilities, and the use of accurate empirical dose calculations using Gafchromic film. Finally, we showcase the new beamline capabilities using two protein systems.

Metabolic engineering to produce molecules not naturally synthesized by the host often requires directed evolution to improve pathway enzyme performance. Growth-coupled selection can dramatically increase directed-evolution throughput, and manipulation of redox balance has proven effective for tying reductase fitness to microbial growth. However, most redox-balance selections require feeding the reductase substrate because of stoichiometric constraints. This is impractical for many biosynthetic pathways either due to practical limitations on cost or complexity of bulk substrate synthesis, or the lack of an ability to transport substrate into cells, for example intracellular acyl-CoA/ACP intermediates. Here we define stoichiometric constraints that make substrate feeding necessary for many acetyl-CoA-derived reduction pathways in NADPH-imbalanced hosts. We overcome these constraints with a dual-feedstock strategy in which glucose provides reducing power while acetate supplies additional acetyl-CoA without directly perturbing redox balance. In an engineered Escherichia coli selection strain, acetate co-feeding enabled growth coupling of acetaldehyde, 3-hydroxybutyrate, and mevalonate production and produced a linear correlation between product formation and growth. We then used this selection to evolve a class II HMG-CoA reductase (HMGR) from Delftia acidovorans toward NADPH utilization, enriching variants with improved NADPH-dependent activity. Finally, propionate co-feeding enabled growth coupling of propionyl-CoA reduction, supporting the generality of carbon co-feeding for selecting enzymes in pathways involving acyl-chain elongation and reduction.

Thirdhand smoke (THS) is a reservoir of toxic compounds that persist in indoor environments and on surfaces long after active smoking has ceased. Major THS chemicals, including nicotine, tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, and heavy metals, can induce DNA damage, epigenetic alterations, oxidative stress, and chronic inflammation. These biological changes contribute to one or more canonical hallmarks of cancer. In this minireview, we propose an integrated mechanistic framework that incorporates the hallmarks of cancer into environmental exposure risk assessment. This framework offers a system-level approach to carcinogenicity assessment that transcends the limitations of conventional single-endpoint toxicological assays. As a case application, we systematically analyze how THS chemicals disrupt specific cancer hallmarks and delineate the molecular and cellular mechanisms through which persistent tobacco smoke residues promote carcinogenesis. These insights establish a mechanistically grounded framework for assessing the environmental cancer hazard associated with THS.

BackgroundPopulation-scale radiation exposure assessment during radiological emergencies is hindered by the slow and costly nature of current methods, creating a need for rapid, affordable screening tools. Radiation biodosimetry using peripheral blood counts is a promising approach, but estimating low-dose exposures and exposure at extended time points remains challenging, especially when accounting for inter-individual differences in radiation sensitivity.MethodsWe analyze complete blood count (CBC) profiles from a retrospective cohort of 1151 male and female BALB/cJ and C57BL/6 J mice exposed to total-body X-ray radiation at doses ranging from 0.05 to 4 Gy. CBCs are collected 1 to 150 days post exposure. We develop a predictive model of radiation exposure using a sparse representation learning strategy to identify the most informative CBC parameters. Model performance is evaluated through exhaustive cross-validation and validated in a double-blind prospective cohort of 431 animals. To evaluate robustness in a genetically diverse population, we further test the model on CBC data from a Collaborative Cross (CC) cohort of 1720 animals representing 35 CC strains, 24 h and 28 days after sham or 1 Gy total-body X-ray exposure.ResultsExhaustive cross-validation shows good performance of the Sparse CBC model, with AUC, accuracy and sensitivity exceeding 80%. Similar performance is observed in the prospective cohort. In the CC cohort, performance is modest. Importantly, model performance varies across CC strains, suggesting that host genetic background significantly influences predictive accuracy.ConclusionsOur findings demonstrate that the Sparse CBC model effectively leverages CBC data to estimate radiation exposure across multiple mouse cohorts, including genetically diverse CC populations. While CBC-based predictions provide a complementary tool for exposure assessment, model performance varies with genetic backgrounds.

Artificial metalloenzymes (ArMs) expand the suite of synthetically valuable, new-to-nature biocatalytic reactions. Integrating these enzymes into biosynthetic pathways enables reactions not found in nature to occur in living cells with the intermediates or products of the metabolic pathways. However, the integration of reactions catalyzed by ArMs into complex metabolic pathways is constrained by the lack of methods to assemble these ArMs in organisms that are commonly used for metabolic engineering. We report the assembly of an iridium-containing artificial metalloenzyme (Ir-ArM) in Streptomyces albus, a Gram-positive bacterial chassis widely used for the heterologous expression of natural products. In this engineered organism, the Ir-ArM assembles in the cytoplasm and catalyzes abiological carbene transfer to the unactivated, disubstituted double bond of an exogenously added terpene with turnover numbers (TONs) that are two times higher than those for the same reaction catalyzed within E. coli cells harboring Ir-ArM and 20 times higher than the TONs for the same reaction catalyzed by the purified holoprotein itself.