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At least 19 recordsLinked to original sources
PubMed2026

Rewiring Carbon Metabolism in Bacillus methanolicus via Heterologous Phosphoketolase Expression Enhances Biomass Yield From Methanol and Reduces CO2 Loss.

Methylotrophic microbes are attractive alternatives to traditional heterotrophic production platforms, yet their efficiency is constrained by carbon loss through pyruvate decarboxylation and the oxidative branch of the RuMP cycle. The phosphoketolase (PKT) pathway provides a carbon-conserving alternative by cleaving fructose-6-phosphate and/or xylulose-5-phosphate into acetyl-phosphate, which can subsequently be converted to acetyl-coA without pyruvate decarboxylation. The remaining carbon intermediates are recycled through central metabolism to regenerate RuMP cycle intermediates without direct CO2 release. Here, we engineered this strategy in Bacillus methanolicus, a thermophilic methylotroph with strong industrial potential. We first established a versatile expression toolkit comprising inducible and constitutive promoters, benchmarked using an sfGFP reporter. Leveraging this system, we heterologously expressed the phosphoketolase B (pktB) gene from Methylotuvimicrobium buryatense 5GB1C which increased methanol-to-biomass yields by 18%-24% relative to controls and reduced biogenic CO2 production by 9%-12%. Chromosomal integration of pktB preserved these gains, demonstrating stability without reliance on plasmid-based expression. Together, these results show that PKT-driven metabolic rewiring enhances substrate yields in B. methanolicus and provides a scalable strategy to improve methylotrophic bioprocesses. This work expands the metabolic engineering toolbox for methylotrophs and highlights carbon-conserving pathway design as a key lever for advancing single carbon (C1) biomanufacturing.

Bacillus↗
PubMed2026

Potential survival strategies of novel comammox and nitrite-oxidizing Nitrospira synthesizing osmoprotectants in a wastewater microbiome treating high-ammonia brackish landfill leachate.

BACKGROUND: In the late stages of landfill operation, leachate becomes brackish and contains high concentrations of ammonia with limited organic carbon. At leachate treatment facilities, it is typically subjected to nitrification followed by denitrification, with methanol supplied as an external electron donor. This unique environment may harbor novel microorganisms, including nitrifiers. Although a variety of microorganisms are involved in nitrification, their substrate specificity and salinity tolerance remain insufficiently understood. In this study, a genome-centric metagenome analysis was conducted on the microbiome from a leachate treatment facility at a closed landfill. RESULTS: A total of 68 metagenome-assembled genomes (MAGs) were reconstructed, including 64 putative novel species. Among these, two Nitrospira MAGs were recovered: a novel complete ammonia-oxidizing bacterium (comammox), Nitrospira LAS72 (88.72% completeness, 2.10% contamination), and canonical nitrite-oxidizing Nitrospira LAS18 (99.98% completeness, 2.29% contamination). Comparative genomic analysis with 260 publicly available Nitrospira genomes revealed that LAS18 represents a new sub-lineage within lineage VII of the Nitrospira genus. Two ammonia-oxidizing archaea (AOA), Candidatus Nitrosocosmicus LAS21 and Nitrosarchaeum LAS73, were also identified, while canonical ammonia-oxidizing bacteria were not detected. Given the brackish conditions (1.23% salinity) and the methanol-fed operation of the treatment facility, the genomic potential for osmotic stress adaptation and methanol metabolism was investigated. Comammox Nitrospira LAS72 harbors biosynthetic pathways for several compatible solutes (osmoprotectants), including glycine betaine, proline, trehalose, and L-glutamate. Moreover, comammox Nitrospira LAS72 possesses genetic potential for oxidizing formaldehyde, suggesting that it may exploit these methanol-derived intermediates as energy sources. These features indicate that LAS72 may withstand osmotic fluctuations through the production of various osmoprotectants and thrive under the unique conditions of a methanol-fed environment. CONCLUSIONS: The discovery of novel comammox Nitrospira and canonical Nitrospira forming a new sub-lineage within lineage VII of the Nitrospira genus in an ammonia-rich brackish environment provides the first genomic evidence for evolutionary adaptation among nitrifiers to saline, methanol-fed environments. These findings enhance our understanding of the ecological and evolutionary dynamics shaping nitrifier communities in complex treatment ecosystems. Video Abstract.

Ammonia↗
PubMed2026

Cobalt starvation affects multiple cellular processes in Desulfofundulus kuznetsovii TPOSR during alcohol oxidation.

Cobalt influences the methanol metabolism of Desulfofundulus kuznetsovii TPOSR, specifically by modulating the activity of one of its alcohol dehydrogenases (ADH), Adh1. However, the effects of cobalt on the broader proteome of strain TPOSR, as well as the utilization of alcohols besides methanol, remain unexplored. Here, proteomic analyses of strain TPOSR grown with and without cobalt on different alcohol substrates show that cobalt starvation impacts multiple cellular processes, including cobalamin biosynthesis, iron-sulphur cluster assembly and, most prominently, energy metabolism as indicated by altered abundances of hydrogenases and NAD(P)-dependent oxidoreductases. Despite the presence of six ADH-encoding genes in the genome, Adh1 is the dominant ADH during growth not only on methanol but also on several primary alcohols and diols (ethanol, 1-propanol, 1,2-propanediol, 1,3-propanediol, butanol, pentanol and heptanol). Enzymatic assays with purified Adh1 confirm activity with these substrates, except 1,3-propanediol, and show no activity toward secondary alcohols (2-propanol and 2-butanol). Comparative proteomics analyses of other sulphate-reducing microorganisms (SRMs), namely Desulfofundulus australicum and Solidesulfovibrio carbinolicus, further indicate that methanol and ethanol oxidation in SRMs is mediated by a single ADH/AOR pair. Together, these findings highlight the central role of cobalt in alcohol metabolism in strain TPOSR and identify conserved ADH/AOR enzymes as promising candidates for biotechnological applications.

Cobalt↗
PubMed2025

Heterologous expression and optimization of the antimicrobial peptide acidocin 4356 in Komagataella phaffii to target Pseudomonas aeruginosa.

Multidrug-resistant (MDR) pathogens, particularly Pseudomonas aeruginosa, pose a serious global health threat due to their increasing prevalence and limited therapeutic options. Antimicrobial peptides (AMPs) offer promising alternatives to traditional antibiotics, yet their large-scale application remains constrained by high production costs and technical challenges. This research sought to develop a yeast-based system for the cost-efficient synthesis of acidocin 4356 (ACD), an antimicrobial peptide proven effective against P. aeruginosa. A codon-optimized ACD gene was cloned into the pPICZα-A expression vector and integrated into the Komagataella phaffii (formerly Pichia pastoris) GS115 genome. Colony PCR confirmed successful integration, and specific transformants demonstrated expression of the 6 × His-ECS-rACD fusion protein, as verified by SDS-PAGE and dot blot analysis. After Ni-NTA chromatography and enterokinase digestion, rACD was found at ~ 20 kDa instead of 8.3 kDa, suggesting oligomerization or post-translational modifications. Response surface methodology determined the optimal temperature, pH, and methanol concentration for peptide synthesis. Under optimal circumstances (21 °C, pH 6.24, and 1.089% methanol), rACD synthesis increased by 34.12% over baseline conditions (30 °C, pH 6, 1% methanol). AlphaFold structural modeling identified three α-helices in high-confidence regions, implicated in bacterial membrane disruption. Antimicrobial assays demonstrated potent rACD activity against P. aeruginosa, yielding a 58.29% reduction in growth at 150 µg/mL and MIC50 and MIC90 values of 143.04 and 320.64 µg/mL, respectively. These findings underscore K. phaffii as a robust platform for AMP production and highlight rACD's therapeutic potential as an effective agent against MDR P. aeruginosa, warranting further investigation into its clinical and industrial applications. KEY POINTS: • Developing a novel K. phaffii strain for heterologous expression supports efficient rACD peptide production. • Optimized conditions boosted expression yield by 34.12% above the reference fermentation settings. • Recombinant acidocin suppressed Pseudomonas aeruginosa growth by 58%, indicating anti-MDR activity.

Pseudomonas aeruginosa↗
PubMed2026

Inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production.

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation or gravity sedimentation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY® VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

Flocculation↗
PubMed2024

One-carbon unit supplementation fuels purine synthesis in tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control of formate production. Safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade in MC38 tumors, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

Animals↗
PubMed2025

Diversity and distribution of the lanthanome in aerobic methane-oxidising bacteria.

BACKGROUND: Lanthanides (Ln) play important and often regulatory roles in the metabolism of methylotrophs, including methanotrophs, particularly through their involvement in methanol oxidation. However, the diversity, distribution, and ecological relevance of Ln-associated proteins (the lanthanome) in aerobic methane-oxidising bacteria (MOB) remain underexplored. This study investigates the lanthanome using genome, plasmid, and proteome data, alongside metatranscriptome data from methane-rich lake sediments. RESULTS: We surveyed 179 genomes spanning Proteobacterial, Verrucomicrobial, and Actinobacterial MOBs to examine the distribution of Ln-dependent methanol dehydrogenases (MDHs) and Ln transport proteins. Distinct lineage-specific patterns were observed: XoxF5 was the most widespread MDH variant in Proteobacteria, while XoxF2 was restricted to Verrucomicrobia. Transporter systems also showed distinct patterns, with LanM restricted to Alphaproteobacteria, LanPepSY and LanA confined to Gammaproteobacteria, and LutH-like receptors broadly distributed across all lineages. Homologues of these genes were also detected on plasmids, indicating potential for horizontal gene transfer. In Lake Washington sediment metatranscriptomes, lanthanome transcripts were detected, with Proteobacteria as dominant contributors. Notably, a large fraction of xoxF transcripts were affiliated with non-MOB Methylophilaceae, consistent with known cooperative interactions with MOB. Using Methylosinus trichosporium OB3b as a model, we assessed methane oxidation and proteomic responses to soluble CeCl3 and a mixed-lanthanide ore. Lag phases were prolonged in the presence of lanthanides, particularly with ore, but methane oxidation rates converged across treatments after acclimation. Proteomic analysis revealed extensive condition-specific responses, with 724 proteins differentially expressed in Ore treatment compared to 60 under CeCl3. XoxF3 and XoxF5 were upregulated while MxaF and its accessory proteins were downregulated, consistent with the "lanthanide switch". Notably, LanM was not expressed despite being encoded, whereas LutH-like receptor was downregulated under both treatments, likely reflecting regulatory control to prevent excess metal uptake. Additional upregulation of a TonB-dependent receptor and ABC transporter suggests a potential lanthanophore-mediated uptake strategy. CONCLUSION: This study highlights the diversity and ecological activity of Ln-binding and transport systems in MOBs, their plasmid localisation and potential mobility, and their distinct regulation under different Ln sources. The strong proteomic response to complex ore underscores the physiological flexibility of MOBs in coping with natural lanthanide forms. These findings provide a framework for ecological studies and candidate targets for biotechnological applications in methane bioconversion and sustainable lanthanide recovery from complex materials.

Horizontal gene transfer↗
PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗
PubMed2025

Lanthanide-dependent isolation of phyllosphere methylotrophs selects for a phylogenetically conserved but metabolically diverse community.

Lanthanides have emerged as important metal cofactors for biological processes. Lanthanide-associated metabolisms are well-studied in leaf symbiont methylotrophic bacteria, which utilize reduced one-carbon compounds such as methanol for growth. Yet, the importance of lanthanides in plant-microbe interactions and on microbial physiology and colonization in plants remains poorly understood. To investigate this, 344 pink-pigmented facultative methylotrophs were isolated from soybean leaves by selecting for bacteria capable of methanol oxidation with lanthanide cofactors, but none were obligately lanthanide-dependent. Phylogenetic analyses revealed that all strains were nearly identical to each other and are part of the extorquens clade of Methylobacterium, despite variability in genome and plasmid sizes. Strain-specific identification was enabled by the higher resolution provided with rpoB compared to 16S rRNA as marker genes. Despite the low strain-level diversity, the metabolic capabilities of the collection diverged greatly. Strains encoding identical lanthanide-dependent alcohol dehydrogenases displayed significantly different growth rates and/or final ODs from each other on alcohols in the presence and absence of lanthanides. Several strains also lacked well-characterized lanthanide-associated genes thought to be important for phyllosphere colonization. Additionally, 3% of our isolates were capable of growth on sugars and 23% were capable of growth on aromatic acids, substantially expanding the range of substrates utilized by Methylobacterium extorquens in the phyllosphere. Our findings suggest that the expansion of metabolic capabilities, as well as differential usage of lanthanides and their influence on metabolism, among closely related strains point to evolution of niche partitioning strategies to promote colonization of the phyllosphere.

Journal Article↗
PubMed2023

One-carbon unit supplementation fuels tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate the contributions to purine nucleotides of salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic T cells) synthesize purines de novo. Purine synthesis requires two 1C units, which come from serine catabolism and circulating formate. Shortage of 1C units is a potential bottleneck for anti-tumor immunity. Elevating circulating formate drives its usage by tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling control of formate-production kinetics. In MC38 tumors, safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

Preprint↗
PubMed2026

Phytochemical Characterization and Effects of Randia monantha Benth. Leaf and Fruit Extracts on the Viability of Human Cancer Cell Lines.

Randia monantha, commonly known as crucetillo, is a medicinal plant traditionally used in Mexico to treat conditions such as diabetes, venomous animal bites, and cancer; however, its effects on cancer cell viability remain underexplored. In this study, hexane, ethyl acetate, and methanolic extracts from the leaves and fruits of Randia monantha were phytochemically characterized and evaluated for their effects on cell viability in HeLa cervical cancer and DU-145 prostate cancer cell lines using the MTT assay. Phytochemical analyses revealed differences in metabolite composition across solvents of varying polarity and plant tissues. Methanolic leaf extracts contained higher levels of terpenoids and polyphenols and produced the greatest reduction in HeLa cell viability, with an estimated IC50 of 4.87 µg/mL after 48 h of treatment. In contrast, DU-145 cells exhibited a different response pattern depending on the extract evaluated. Correlation analysis suggested that flavonoids were associated with reduced HeLa cell viability. Overall, leaf extracts produced greater reductions in cancer cell viability than fruit extracts, highlighting leaves as a promising but comparatively underexplored source of bioactive compounds in Randia monantha.

Humans↗
PubMed2026

Revealing Hidden Variables in DESI-Based Spatial Metabolomics: Solvent Composition and Tissue Type as Critical Drivers.

In the development of a desorption electrospray ionization (DESI) workflow for spatial metabolomics, we investigated the impact of two commonly used solvent systems, 90% acetonitrile (ACN) and 90% methanol (MeOH), on the spatial metabolomic profiling of various murine tissues. The performance of both solvents was evaluated across several metabolite classes (central carbon metabolites, amino acids, and fatty acids). Although the ACN-based solvent system led to higher signal intensities for small polar metabolites involved in glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid metabolism, the MeOH-based solvent system provided superior signal intensities for fatty acids. These findings demonstrate that the solvent composition differentially influences metabolite extraction and ionization processes in DESI and should be carefully matched to the biological questions and metabolite classes of interest. As a proof-of-principle, the ACN solvent system was applied to a pilot study based on a rat model of renal ischemic injury, revealing region-specific metabolic changes between normoxic and ischemic conditions. Together, these results demonstrate the importance of solvent selection in DESI-based spatial metabolomics and showcase the ability of this approach to uncover spatially resolved metabolic adaptations associated with tissue injury.

Animals↗
PubMed2024

Investigating the degradation potential of microbial consortia for perfluorooctane sulfonate through a functional "top-down" screening approach.

Perfluorooctane sulfonate (PFOS) is a prominent perfluorinated compound commonly found in the environment, known to pose various risks to human health. However, the removal of PFOS presents significant challenges, primarily due to the limited discovery of bacteria capable of effectively degrading PFOS. Moreover, single degradation bacteria often encounter obstacles in individual cultivation and the breakdown of complex pollutants. In contrast, microbial consortia have shown promise in pollutant degradation. This study employed a continuous enrichment method, combined with multiple co-metabolic substrates, to investigate a microbial consortium with the potential for PFOS degradation. By employing this methodology, we effectively identified a microbial consortium that demonstrated the capacity to reduce PFOS when exposed to an optimal concentration of methanol. The consortium predominantly comprised of Hyphomicrobium species (46.7%) along with unclassified microorganisms (53.0%). Over a duration of 20 days, the PFOS concentration exhibited a notable decrease of 56.7% in comparison to the initial level, while considering the exclusion of adsorption effects. Furthermore, by comparing the predicted metabolic pathways of the microbial consortium with the genome of a known chloromethane-degrading bacterium, Hyphomicrobium sp. MC1, using the KEGG database, we observed distinct variations in the metabolic pathways, suggesting the potential role of the unclassified microorganisms. These findings underscore the potential effectiveness of a "top-down" functional microbial screening approach in the degradation of stubborn pollutants.

Fluorocarbons↗
PubMed2026

Simultaneous determination of imiquimod and terbinafine in skin permeation studies: Validation of a liquid chromatography method with fluorescence detection.

Chromoblastomycosis is a chronic, neglected subcutaneous mycosis posing significant therapeutic challenges. A topical strategy combining terbinafine (TBF), an antifungal, with imiquimod (IMQ), a TLR-7/8 agonist immunomodulator, has emerged a promising alternative. However, no validated analytical method is currently available to simultaneously quantify both drugs in skin, which is crucial for novel formulation development. This study reports the development and validation of a simple HPLC method with fluorescence detection (excitation 236 nm, emission 340 nm) for the simultaneous determination of TBF and IMQ extracted from porcine skin. Separation was achieved on a C8 reversed-phase column (125 × 4.0 mm, 5 μm) using a mobile phase of methanol and water (60,40, v/v), both containing 0.1% formic acid at a flow rate of 0.8 mL/min. The method showed excellent linearity (r > 0.999) over 0.01-1.0 μg/mL for IMQ and 0.1-2.0 μg/mL for TBF. Intra- and inter-day precision demonstrated coefficients of variation below 5%, and recovery rates from skin (79-105%) confirmed accuracy. Limits of detection were 0.001 μg/mL for IMQ and 0.004 μg/mL for TBF, with quantification limits of 0.02 μg/mL and 0.16 μg/mL, respectively. This selective, sensitive, and reproducible method represents a valuable analytical tool for supporting the development and quality control of topical formulations for chromoblastomycosis and other fungal skin diseases.

Animals↗
PubMed2024

ZASP: A Highly Compatible and Sensitive ZnCl2 Precipitation-Assisted Sample Preparation Method for Proteomic Analysis.

Universal sample preparation for proteomic analysis that enables unbiased protein manipulation, flexible reagent use, and low protein loss is required to ensure the highest sensitivity of downstream liquid chromatography-mass spectrometry (LC-MS) analysis. To address these needs, we developed a ZnCl2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl2 and methanol-induced protein precipitation at room temperature (RT). ZASP can remove trypsin digestion and LC-MS incompatible detergents such as SDS, Triton X-100, and urea at high concentrations in solution and unbiasedly recover proteins independent of the amount of protein input. We demonstrated the sensitivity and reproducibility of ZASP in an analysis of samples with 1 μg to 1000 μg of proteins. Compared to commonly used sample preparation methods such as SDC-based in-solution digestion, acetone precipitation, FASP, and SP3, ZASP has proven to be an efficient approach. Here, we present ZASP, a practical, robust, and cost-effective proteomic sample preparation method that can be applied to profile different types of samples.

Proteomics↗
PubMed2026

Evidence of direct methane production from long-chain fatty acids by thermophilic Archaeoglobi.

Methanogenic degradation of long-chain fatty acids has traditionally been thought to occur through syntrophic partnerships between fatty acid-degrading bacteria and methanogenic archaea. However, recent genomic evidence suggests that certain archaea may independently carry out the entire process. Here we report the enrichment of an archaeon from the class Archaeoglobi, Candidatus Methanoglobus sphaerolipidus DLY3, from hot spring sediments in Tengchong, China. The results of selective enrichment, growth experiments, microscopy, stable isotope tracing, metagenomics and metatranscriptomics suggest that Ca. M. sphaerolipidus directly converts long-chain fatty acids to methane. This transformation involves the beta-oxidation pathway, the Wood-Ljungdahl pathway and methanogenic methyl-coenzyme M reductase and methyltransferase complexes-a process we term liparotrophy. In addition to oleic acid, Ca. M. sphaerolipidus is also capable of utilizing methanol as a substrate for methanogenesis. Our findings expand the known substrate range for methanogenic archaea beyond carbon dioxide reduction, acetoclastic methanogenesis, methylotrophy, methyl reduction, methoxydotrophy and the recently reported alkylotrophy.

Journal Article↗
PubMed2026

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae↗