Biology subjects

Carper, D. L.

Publications and source records attributed to Carper, D. L..

11 recordsLinked to original sources

bioRxiv2026

Engineered Pseudomonas putida reconfigures metabolic fluxes to support energy demands during muconate bioproduction from lignin-related aromatics

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

synthetic biology↗

bioRxiv2026

Cellular Stress Tolerance Governs Genetic Transformability in Recalcitrant Candida Species

Candida species are fungal pathogens whose rapidly increasing antifungal resistance poses a substantial public health challenge. High-throughput CRISPR-based screening could accelerate antifungal target discovery, yet its application in Candida has been limited by low DNA transformation efficiency. Chemical transformation exposes cells to environmental stresses to permit DNA uptake, but the physiological constraints on transformability remain poorly defined. Here, we show that genetic transformability in C. albicans is governed by the cellular capacity to withstand and recover from transformation-induced stress. Nutrient limitation markedly enhances transformation efficiency, while extracellular pH and lithium acetate chemistry strongly modulate this response. Systems-level proteomic analyses reveal that nutrient limitation and transformation chemistry prime oxidative stress tolerance, and transformation efficiency correlates with the expression of oxidative stress response proteins. Guided by these insights, we developed a generalizable fungal advanced chemical transformation (FACT) method that increases transformation efficiency across diverse Candida species and enables robust pooled CRISPR screening.

systems biology↗

bioRxiv2026

Structural and Oligomeric Characterization of Substrate- and Product-selective Nylon Hydrolases

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

biochemistry↗

bioRxiv2025

Process optimization for enhanced enzymatic nylon deconstruction

Plastics such as polyamides (PAs) possess unique physicochemical properties that make them indispensable in modern society. However, their energy-intensive production and challenging end-of-life management highlight the urgent need for efficient recycling or remanufacturing solutions. Enzymatic depolymerization offers a promising route toward circular recycling, yet remains constrained by limited enzyme characterization, lack of validation under industrially relevant conditions, and overall performance. Here we optimized the reaction conditions for three recently discovered nylon-degrading enzymes. One of them, Nyl12, achieved product titers with PA6 and PA66 that exceed previously reported values, without enzyme engineering or substrate pretreatment. We further demonstrated the scalability of the process and its application to complex PA-based materials used in microelectronic components. Analysis of substrate features, including surface area and particle size, revealed key parameters governing enzymatic activity and provided a framework for future pretreatment and process optimization efforts. In combination, these efforts provide a new benchmark for enzymatic nylon recycling.

biochemistry↗

bioRxiv2025

Proteome Reallocation Reveals Dynamics and Mechanisms of Phage Infection and Defense in Staphylococcus aureus

The coexistence of bacteria and phages is marked by dynamic interactions that determine infection outcomes. However, the mechanisms by which the host allocates its resources to cope with phage infection remain largely unknown. In this study, using longitudinal proteomics, we elucidated these interactions for the temperate staphylococcal phage{phi} NM1 and strains of Staphylococcus aureus either harboring its cognate prophage or lacking it. We demonstrated that infection of non-lysogenic S. aureus with{phi} NM1 induces a dramatic shutdown of host translation, reducing proteome allocation by over 20%. Quantitative analysis of the economics of{phi} NM1 infection revealed that the diversion of these cellular resources toward phage replication imposes a significant metabolic burden, thereby impairing cell growth. In contrast, lysogenic cells cope with phage infection and prevent culture collapse through a coordinated response of prophage-encoded defenses, host-encoded stress effectors, and reprogrammed cellular metabolism, thereby avoiding translation shutdown. Through coinfection with the wildtype phage and an engineered phage-like particle carrying a CRISPR-Cas phagemid, we revealed that synthetic DNA cargos evade host defenses and hijack the transcriptional machinery, altering infection outcomes. Without immunity, coinfection could collapse the non-lysogens more quickly than the native phage by overexpressing the cargo proteins, suppressing carbohydrate metabolism, and accelerating structural phage protein production through increased phage genome replication. Together, these findings provide a systems-level understanding of phage infection in S. aureus, uncovering the mechanisms for host takeover and prophage-mediated defense, with implications for next-generation phage therapy. SignificanceMultidrug-resistant bacteria pose a major threat to human health. Phage therapy offers a precise therapeutic approach by leveraging the specificity of phage infection and cargo delivery. However, as with conventional antibiotics, phage resistance can develop. Understanding the dynamic interactions between bacterial hosts and phages is therefore essential for predicting infection outcomes and designing precision phage therapies to suppress resistance. Using longitudinal proteomics, we elucidated the dynamics by which bacterial hosts reallocate cellular resources to cope with phage infection at the systems level. Coordinated defenses and reprogrammed metabolism are critical for host survival, but phage-delivered cargos can effectively bypass these barriers. These insights into the intricate interplay between the phage and host are crucial for designing next-generation precision phage therapies with greater lethality and less resistance.

systems biology↗

bioRxiv2025

Genome shuffling enables quantitative trait locus mapping in Bacillus subtilis

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion in Bacillus subtilis to generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size ([~]10 kb), and these associations were validated through targeted genetic swaps. This B. subtilis QTL population can easily be used to map additional phenotypes, and the general approach for QTL mapping is applicable in a wide range of bacteria. One-Sentence SummaryBacterial genome shuffling mimics sexual recombination and enables genetic mapping of complex phenotypes to causal alleles.

microbiology↗

bioRxiv2025

Microbial Modulation of Host Plant Proteasome Activity Improves Heat Stress Tolerance

Global climate change marked by rising temperatures and increasingly frequent, severe heat waves, threatens plant health, productivity, and ecosystem function. Beneficial plant-microbe associations are increasingly recognized for their role in mitigating abiotic stress, yet the molecular mechanisms underlying microbially-conferred thermotolerance remain poorly understood. Here, we developed a population-based assay using Sphagnum peat mosses, a key species in peatland ecosystems that sequester 33-50% of the worlds soil carbon as recalcitrant peat, to perform microbiome transfers and assess microbial contributions to heat resilience. Strain-based assays in Sphagnum and Arabidopsis identified Variovorax as a key thermotolerance-enhancing microbe and identified ECM29, a proteasome-associated protein, as critical for this interaction. In Arabidopsis, Variovorax sp. CF313 association led to ECM29 preferentially binding the RPT1 subunit of the 19S regulatory particle, inhibiting 20S proteasome gate opening and slightly reducing proteasomal activity. This results in the accumulation of ubiquitinated proteins and induces HSP70 transcription, priming the host for heat shock stress even in the absence of prior heat exposure. These findings establish ECM29 as a key regulator of proteasome activity in response to microbial interactions, highlighting a previously unknown mechanism by which microbes enhance plant thermotolerance. More broadly, this work highlights the importance of leveraging natural systems and ecologically relevant models to identify strategies for enhancing plant and ecosystem resilience to a changing climate. Significance StatementGlobal climate change poses severe threats to plant health and productivity, with cascading impacts on ecosystems. Beneficial plant-microbe interactions offer promising strategies to mitigate abiotic stress, yet the molecular mechanisms often remain unclear. This study uncovers a novel thermotolerance mechanism mediated by Variovorax modulating the plant host proteasome. Using a QTL derived from Sphagnum peat mosses, and tested within a genetically tractable Arabidopsis model, we identified ECM29, a highly conserved protein across diverse plant species, as a key regulator of proteasome activity. ECM29 primes plants for heat stress by inducing HSP70 transcription through proteasome modulation. Additionally, we demonstrated that ECM29 is required for the host to receive microbially conferred thermotolerance, but is not necessary for innate priming to heat stress. These findings highlight the potential of leveraging natural systems and ecologically relevant species to develop innovative genetic models, advancing plant resilience and productivity in a warming world.

plant biology↗

bioRxiv2025

Enzymatic synthesis of diverse oligoamide polymer precursors

Nylons, a major class of synthetic polyamides, are widely used due to their excellent mechanical strength, thermal stability, and chemical resistance. Conventional nylon production relies on the polymerization of lactams or stoichiometric nylon salts. However, applying these approaches to unconventional precursors such as bio-derived glutaric acid produces polymers with low molecular weight and limited applications. To address these challenges, we demonstrated that chemically-synthesized nylon diads enable the production of higher-molecular-weight polyamides compared with traditional salts. We then identified a biosynthetic approach using amide synthetases to convert unprotected bifunctional substrates into nylon-relevant diads. Using a cofactor regeneration system, enzymatic diad synthesis was scaled to produce sufficient material for laboratory-scale characterization and solid-state polymerization. Amide synthetases demonstrated broad substrate scope, catalyzing the regioselective assembly of diverse nylon-relevant diacids, diamines, and {omega}-amino acids. This strategy offers a novel route to synthesize challenging nylon monomers and advances production of bioderived nylons.

biochemistry↗

bioRxiv2024

High-density CRISPRi screens reveal adaptive transcriptional gradients in cyanobacteria

Cyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the biochemical basis of environmental adaptation in these microbes, it remains poorly understood and challenging to re-wire. This study uses a high-density, genome-wide CRISPR interference (CRISPRi) screen to examine the influence of gene-specific transcriptional variation on the growth of Synechococcus sp. PCC 7002 under environmental extrema. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Notably, transcriptional repression of a gene for a core subunit of the NDH-1 complex, which is important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. In general, most genes with fitness-improving knockdowns were distinct to each light color, evidencing unique stress responses and alleviation mechanisms. Multi-target transcriptional repression produced nonadditive effects. Findings reveal diverse mechanisms of environmental adaptation in cyanobacteria and provide a new approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells. SIGNIFICANCE STATEMENTCyanobacteria are the most abundant photosynthetic organisms on Earth, where they endure a striking variety of environmental fluctuations. This study examines the biochemical basis of environmental adaptation in Synechococcus sp. PCC 7002, an important model strain, by modulating the expression of every gene in its genome. Results show that partial, but not complete, reduction in the expression of a subset of influential genes can improve growth under cold monochromatic conditions. Optimal expression levels differ between red and blue light and shift with multi-gene adjustments. Findings show how minor transcriptional adjustments can yield major improvements in growth under environmental extrema and provide a powerful systems-level approach for studying--and fine-tuning--the adaptive capacity of microbes.

microbiology↗

bioRxiv2023

Functional analysis of Salix purpurea genes support roles for ARR17 and GATA15 as master regulators of sex determination

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea. Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP-Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea, contributing to a regulatory system that is notably different from that of its sister genus Populus. Evidence was also obtained for the roles of two transcription factors, an AP2/ERF family gene and a homeodomain-like transcription factor, in downstream regulation of sex dimorphism.

plant biology↗

bioRxiv2020

Sphagnum peat moss thermotolerance is modulated by the microbiome

Sphagnum peat mosses is a major genus that is common to peatland ecosystems, where the species contribute to key biogeochemical processes including the uptake and long-term storage of atmospheric carbon. Warming threatens Sphagnum mosses and the peatland ecosystems in which they reside, potentially affecting the fate of vast global carbon stores. The competitive success of Sphagnum species is attributed in part to their symbiotic interactions with microbial associates. These microbes have the potential to rapidly respond to environmental change, thereby helping their host plants survive under changing environmental conditions. To investigate the importance of microbiome thermal origin on host plant thermotolerance, we mechanically separated the microbiome from Sphagnum plants residing in a whole-ecosystem warming study, transferred the component microbes to germ-free plants, and exposed the new hosts to temperature stress. Although warming decreased plant photosynthesis and growth in germ-free plants, the addition of a microbiome from a thermal origin that matched the experimental temperature completely restored plants to their pre-warming growth rates. Metagenome and metatranscriptome analyses revealed that warming altered microbial community structure, including the composition of key cyanobacteria symbionts, in a manner that induced the plant heat shock response, especially the Hsp70 family and jasmonic acid production. The plant heat shock response could be induced even without warming, suggesting that the warming-origin microbiome provided the host plant with thermal preconditioning. Together, our findings show that the microbiome can transmit thermotolerant phenotypes to host plants, providing a valuable strategy for rapidly responding to environmental change.

plant biology↗