Biology subjects

Woolston, B. M.

Publications and source records attributed to Woolston, B. M..

9 recordsLinked to original sources

bioRxiv2026

13C-Metabolic Flux Analysis of Substrate Co-Metabolism in the Methylotrophic Acetogen Eubacterium limosum.

Acetogens are promising microbial catalysts for the sustainable production of fuels and chemicals, metabolizing a range of renewable single-carbon (C1) compounds with high energetic efficiency. The major challenge is their energy-limited anaerobic lifestyle, which results in slow growth and a limited product spectrum on individual C1 substrates. We previously showed in the acetogen Eubacterium limosum that co-metabolism of glucose-methanol or CO-methanol mixtures results in substantial improvements in growth and acetate productivity. To investigate the underlying mechanism(s), here we conducted 13C metabolic flux analysis (13C-MFA) of E. limosum grown on a glucose-methanol mixture, as well as in single-substrate controls. The results suggest that the main benefit of glucose cofeeding is not provision of additional ATP, but rather a reduction of flux through the ferredoxin-dependent and kinetically challenging reactions of the Wood-Ljungdahl pathway. Subsequent RNAseq analysis showed no significant differences in the expression level of genes involved in methanol metabolism between cofeeding and methanol-only controls, suggesting this reduction of flux is controlled post-transcriptionally. Most surprisingly, we found that under all growth conditions, flux through pyruvate:ferredoxin oxidoreductase (PFOR) was in the oxidative direction, with cysteine (added as a reducing agent) providing all required gluconeogenic carbon flux under CO2-fixing conditions. This work provides critical insights into the metabolic network of an emerging model acetogen that will facilitate future metabolic engineering efforts to channel flux to value-added products, and highlights the under-appreciated role of cysteine as a carbon source in acetogenic fermentations.

systems biology↗

bioRxiv2026

Metabolic engineering of Escherichia coli to modulate hydrogen sulfide levels in the mammalian gut

Hydrogen sulfide (H2S) is a microbiota-derived metabolite in the gastrointestinal tract implicated in a number of diseases. Its volatility and reactivity make experimentally controlling H2S concentration in vivo difficult, limiting our ability to interrogate its dose-dependent effects on host physiology. Engineered bacteria present a compelling solution, yet most probiotic metabolic engineering approaches have focused on in vitro optimization, failing to account for the complex intestinal environment. Here, we engineered Escherichia coli strains to produce or consume H2S in specific intestinal regions by incorporating knowledge of the local metabolic environment and resident microbial activities into the design process. Analysis of human-derived ex vivo cultures revealed that glutathione (GSH) is inefficiently converted to H2S, suggesting GSH as a relatively stable substrate for engineered sulfide production. We thus engineered a GSH-dependent H2S producer, which increased levels 21-fold ex vivo. To target the nutrient-rich, microbially sparse environment of the small intestine, we optimized a H2S producer that uses L-cysteine as a sulfur source, demonstrating a 7-fold increase in H2S levels in mice. Finally, to develop strains capable of sequestering H2S, we leveraged the availability of fumarate and nitrate as electron acceptors in the large intestine by engineering a strain expressing sulfide:quinone oxidoreductase (Sqr). This enables oxidation of H2S to intracellular polysulfides and achieves higher consumption rates than alternative sequestration strategies reliant on resource-intensive GSH production. Together, this work developed engineered microbes as precision tools to modulate H2S levels and showcases a generalizable framework for region-targeted design of engineered probiotics.

synthetic biology↗

bioRxiv2025

Exploring C1 substrate cofeeding in Eubacterium limosum with AneVO, a low-cost anaerobic parallel bioreactor platform

Acetogenic bacteria have emerged as attractive biocatalysts for renewable biochemical production, using the highly efficient Wood-Ljungdahl pathway to convert a range of sustainable single-carbon (C1) feedstocks. The major challenge is their energy-constrained anaerobic lifestyle, which results in slow growth and limits the product spectrum. To overcome this limitation, here we investigate substrate co-metabolism in the acetogen Eubacterium limosum, cofeeding either carbon monoxide (CO) or glucose alongside the primary C1 substrate (methanol or formate). To increase experimental throughput, we developed AneVO, a parallel mini bioreactor system based on eVOLVER that enables benchtop anaerobic batch and fed-batch cultivation, along with continuous delivery of anaerobic gas blends. With all substrate pairs tested, E. limosum grew faster and reached 52-254% higher cell densities with cofeeding, while maintaining or improving the volumetric uptake rate of the main C1 substrate. Product formation also improved, with an increase in volumetric acetate productivity from glucose cofeeding of 2.2-fold with formate and 2.4-fold with methanol, and 3-fold from CO cofeeding with methanol. Together these results validate AneVO as a low-cost platform for convenient benchtop cultivation of strict anaerobic microbes in multiple growth modes, and present a strategy for enhancing C1 bioconversion rates in E. limosum, an emerging model acetogen.

microbiology↗

bioRxiv2024

Development of a recombineering system for the acetogen Eubacterium limosum with Cas9 counterselection for markerless genome engineering

Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system allows recombination to reach an efficiency of up to 100%, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck Eubacterium limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/588731v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1366103org.highwire.dtl.DTLVardef@11b1f0borg.highwire.dtl.DTLVardef@1931600org.highwire.dtl.DTLVardef@1896e4c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Prevotella are major contributors of sialidases in the human vaginal microbiome.

Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis. These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment. Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis. Despite their implications for sexual and reproductive health, sialidases from other vaginal bacteria have not been characterized. Here, we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin. These sialidases are highly conserved across clades of Prevotella from different geographies, hinting at their importance globally. Finally, we find that Prevotella sialidases, including mucin-degrading enzymes from Prevotella timonensis, are highly prevalent and abundant in human vaginal metagenomes and metatranscriptomes, Together, our results identify Prevotella as a critical source of sialidases in the vaginal microbiome, improving our understanding of this detrimental bacterial activity. Significance StatementSialidase activity in the vaginal microbiome is increased in bacterial vaginosis and strongly associated with other adverse health outcomes. Sialidase enzymes release sialic acid from host-derived glycans in the vaginal environment, altering their structures and functions. However, biochemical studies of vaginal bacterial sialidases have been limited to one genus, Gardnerella. In this work, we identify and characterize multiple active sialidase enzymes in vaginal bacteria of the genus Prevotella. We find that Prevotella sialidases are more prevalent and abundant in vaginal microbial communities than Gardnerella sialidases. Our work highlights Prevotella bacteria as an underappreciated source of sialidase activity with important implications for both our understanding of vaginal health and therapeutic development.

microbiology↗

bioRxiv2023

Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions

Oxygen (O2) tension plays a key role in tissue function and pathophysiology. O2-controlled cell culture, in which the O2 concentration in an incubators gas phase is controlled, is an indispensable tool to study the role of O2 in vivo. For this technique, it is presumed that the incubator setpoint is equal to the O2 tension that cells experience (i.e., pericellular O2). We discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0.0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O2 tensions, we developed a reaction-diffusion model that predicts pericellular O2 tension a priori. This model revealed that the effect of cellular O2 consumption is greatest in smaller volume culture vessels (e.g., 96-well plate). By controlling pericellular O2 tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs. hypoxia. MCF7 also expressed higher levels of HIF2A, CD73, NDUFA4L2, etc. and lower levels of HIF1A, CA9, VEGFA, etc. in response to hypoxia vs. anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs. anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O2 tension in cell culture incubators is insufficient to control O2 in cell culture and introduce the concept of pericellular O2-controlled cell culture.

bioengineering↗

bioRxiv2023

Engineered bacteria titrate hydrogen sulfide and induce concentration-dependent effects on host in a gut microphysiological system

Hydrogen sulfide (H2S) is a gaseous microbial metabolite whose role in gut diseases is debated, largely due to the difficulty in controlling its concentration and the use of non-representative model systems in previous work. Here, we engineered E. coli to titrate H2S controllably across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells. The chip was designed to maintain H2S gas tension and enable visualization of co-culture in real-time with confocal microscopy. Engineered strains colonized the chip and were metabolically active for two days, during which they produced H2S across a sixteen-fold range and induced changes in host gene expression and metabolism in an H2S concentration-dependent manner. These results validate a novel platform for studying the mechanisms underlying microbe-host interactions, by enabling experiments that are infeasible with current animal and in vitro models.

bioengineering↗

bioRxiv2021

Characterization of vaginal microbial enzymes identifies amylopullulanases that support growth of Lactobacillus crispatus on glycogen

The healthy human vaginal microbiota is generally dominated by lactobacilli, and the transition to a more diverse community of anaerobic microbes is associated with health risks. Glycogen released by lysed epithelial cells is believed to be an important nutrient source in this environment. However, the mechanism by which vaginal bacteria metabolize glycogen is unclear, with evidence implicating both microbial and human enzymes. Here, we biochemically characterize six glycogen-degrading enzymes (GDEs) from vaginal bacteria that support the growth of amylase-deficient L. crispatus on glycogen. We reveal variations in the pH tolerance and susceptibility to inhibition between enzymes from different organisms. Analysis of vaginal microbiome datasets show these enzymes are expressed in all Community State Types. Finally, we confirm the presence and activity of bacterial GDEs in cervicovaginal fluid. This work establishes that bacterial GDEs can participate in the breakdown of glycogen, providing insight into metabolism that may shape the vaginal microbiota.

biochemistry↗

bioRxiv2021

Cysteine dependence in Lactobacillus iners constitutes a novel therapeutic target to modify the vaginal microbiota

Vaginal microbiota composition affects several important reproductive health outcomes. Lactobacillus crispatus-dominant bacterial communities have favorable associations whereas anaerobe-dominant communities deficient of lactobacilli are linked to poor outcomes, including bacterial vaginosis (BV). Lactobacillus iners, the most abundant vaginal species worldwide, has adverse associations compared to L. crispatus, but standard metronidazole treatment for BV promotes L. iners-dominance, likely contributing to post-treatment relapse. L. iners is under-studied because it fails to grow in standard Lactobacillus media in vitro. Here we trace this in vitro phenotype to a species-specific cysteine requirement associated with limitations in cysteine-related transport mechanisms and show that vaginal cysteine concentrations correlate with Lactobacillus abundance in vivo. We demonstrate that cystine uptake inhibitors selectively impede L. iners growth and that combining an inhibitor with metronidazole thus promotes L. crispatus dominance of defined BV-like communities. These findings identify a novel target for therapeutic vaginal microbiota modulation to improve reproductive health.

microbiology↗