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PubMed2026

Marine Vibrio Biocatalysts as Unique Green Transformation (GX) Tools at the Time to Sustainable Development Goals (SDGs).

Vibrios have sustained various types of ocean ecosystems, being key players in marine mineral cycles and essential partners in specific groups of marine life. Observed genome plasticity and metabolic versatility are some of the unique biological features of vibrios, and these traits could contribute in expanding their ecological niche in marine environments. Vibrios are now recognized as ecophysiologically essential microbial species for our planet. At the time to "Sustainable Development Goals" (SDGs), their genome plasticity and metabolic versatility have also been studied with the aim of solving global issues such as energy production and plastic pollution by creating new microbial biocatalysts. Here, we introduce recent progress on the application of vibrios aiming towards green transformation (GX).

Vibrio↗
PubMed2026

Unlocking the molecular engineering of Geobacillus glycoside hydrolases as a source of industrial biocatalysts.

This review examines Geobacillus sensu stricto as a source of thermostable glycoside hydrolases (GH) for biomass conversion, food processing, and enzyme engineering. Recent peer-reviewed literature was assessed with emphasis on taxonomy, genome-based Carbohydrate-Active Enzymes (CAZyme) prediction, biochemical validation, structural data, and engineering case studies. Taxonomic boundaries were interpreted using current Anoxybacillaceae frameworks, with Parageobacillus treated as a related comparator rather than as Geobacillus. The strongest evidence supports GH13 alpha-amylases, xylan-active systems, beta-xylosidases, and selected accessory enzymes. Recent studies also show that genome mining must be coupled with enzymatic assays and product profiling because CAZyme annotation alone does not prove industrial function. Molecular engineering has improved relevant traits, including the longer thermal half-life of engineered G. stearothermophilus alpha-amylase variants, the increased catalytic efficiency of oligo-alpha-1,6-glucosidase variants, and improved AmyS expression in Bacillus subtilis. Geobacillus glycoside hydrolases are best interpreted as process-specific, engineerable biocatalytic templates. Their translation requires reliable taxonomy, functional validation, structural interpretation, scalable expression and testing on realistic substrates. This synthesis also recognises current limitations: many predicted CAZymes still lack biochemical validation, complete cellulolytic systems remain less mature than xylan- and starch-active systems, and scale-up data remain scarce.

Geobacillus↗
PubMed2025

Functional metaproteomics for enzyme discovery.

Discovery of microbial biocatalysts traditionally relied on activity screening of isolated bacterial strains. However, since most microorganisms cannot be cultivated in the lab, such an approach leaves the majority of the microbial enzyme diversity untapped. Metagenomic approaches, in which the DNA from a microbial community is directly isolated and then used either for the creation of an expression library or for sequencing and metagenome annotation have alleviated this shortcoming to an extent, but have their own limitations: the generation of large expression libraries is time-consuming and their screening is costly, while metagenome annotation can infer biocatalytic function only from prior knowledge. We have thus developed a functional metaproteomic approach, which combines the immediacy of traditional activity screening with the comprehensiveness of a meta-omics approach. Briefly, the whole metaproteome of an environmental sample is separated on a 2-D gel, biocatalytically active proteins are visualized in-gel through zymography, and those candidate biocatalysts are then identified through mass spectrometry, searching against a metagenome-derived database obtained from the very same environmental sample. Here we explain the process in detail, with a focus on esterases, and give guidelines on how to develop a functional metaproteomic workflow for enzyme discovery.

Proteomics↗
PubMed2025

Mining metagenomes from extremophiles as a resource for novel glycoside hydrolases for industrial applications.

The exploration of metagenomes from extremophiles has emerged as a promising approach for discovering novel glycoside hydrolases (GHs) with potential industrial applications. Extremophiles, which thrive in harsh conditions such as high salinity, extreme temperatures, and acidic or alkaline environments, produce enzymes naturally adapted to function under these conditions. This unique adaptability makes them highly desirable for industrial processes requiring robust and efficient biocatalysts. These biocatalysts reduce reliance on harsh chemicals and energy-intensive processes, contributing to greener industrial operations. This review underscores the power of metagenomics in bypassing the need to culture large libraries of extremophiles in the lab. High-throughput sequencing and bioinformatics enable the identification of novel GH-encoding genes directly from environmental DNA. While metagenomic mining has yielded promising results, challenges such as the expression of extremophile-derived genes in mesophilic hosts, low activity yields, and scalability remain. Advances in synthetic biology and protein engineering could address these bottlenecks, enabling more efficient utilization of GHs. Additionally, integrating machine learning for predictive functional annotation may accelerate the identification of high-value candidates.

Glycoside Hydrolases↗
PubMed2026

Genomic prospecting and biochemical characterization of a novel thermostable 3-quinuclidinone reductase from hot spring metagenomes for efficient biocatalysis.

This study presents the discovery and characterization of a novel thermophilic 3-quinuclidinone reductase (ScQR) identified through metagenomic mining of hot spring environments. ScQR, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, was heterologously expressed in Escherichia coli, and its catalytic properties were systematically characterized. The enzyme demonstrates exceptional thermal stability, retaining 86% of its activity after 48 hours at 70°C. Furthermore, K+ and Mg²+ ions significantly enhanced ScQR's activity at specific concentrations. Structural analysis revealed that ScQR adopts a typical SDR fold with a conserved catalytic triad (S141-Y155-K159), and it is NAD(H) dependent. Enzyme assays indicated that ScQR is highly stereoselective for (R)-3-quinuclidinol, with no activity against its enantiomer, (S)-3-quinuclidinol. The enzyme exhibits optimal activity at pH 9 and 85°C, making it a promising candidate for industrial applications requiring high thermal stability. Molecular dynamics simulations further revealed that ScQR preserves global structural integrity up to 360 K, whereas higher temperatures induce destabilization, predominantly in the C-terminal region and residues 95-100. In addition, structure-guided computational design enabled by LigandMPNN and UniKP yielded three ScQR variants with improved substrate affinity and catalytic efficiency while maintaining the overall fold and function. This work underscores the power of metagenomics with structure-driven protein design in discovering novel enzymes with unique catalytic properties from extreme environments and establishes ScQR as a promising biocatalyst for biotechnological and pharmaceutical applications.IMPORTANCEThis study reports the discovery of ScQR, a novel thermophilic 3-quinuclidinone reductase identified via metagenomic mining. ScQR represents one of the most heat-resistant members of the SDR superfamily discovered to date, maintaining 86% activity after 48 hours at 70°C. These findings establish ScQR as a robust biocatalyst for high-temperature pharmaceutical applications and demonstrate a scalable workflow for optimizing enzymes from extreme environments, offering significant value to the fields of biocatalysis and protein engineering.

computational design↗
PubMed2026

Whole-Genome Sequence Dataset of Rhodococcus qingshengii IEGM 267-Terpenoid Biotransformer Toward Genetic Functional Annotation.

Background/Objectives: Microbial biotransformation of monoterpenoids is a promising approach for obtaining bioactive compounds. Rhodococcus species are attractive biocatalysts due to their metabolic versatility and ability to transform hydrophobic substrates. In this study, we investigated the catalytic potential of Rhodococcus qingshengii IEGM 267 toward carveol isomers and explored genomic features that may underlie this activity. Methods: The strain was cultivated in mineral medium supplemented with (-)-trans-carveol. Biotransformation products were analyzed by TLC and GC-MS. The draft genome was sequenced, assembled, taxonomically assigned, and annotated using standard bioinformatics tools. Results: Rhodococcus qingshengii IEGM 267 efficiently converted (-)-trans-carveol to carvone. Genome analysis confirmed the taxonomic assignment of the strain and revealed a large repertoire of oxidoreductases, including monooxygenases, hydroxylases, and dehydrogenases. Seven genes encoding cytochrome P450-dependent oxygenases were identified as candidate enzymes potentially involved in carveol oxidation. Conclusions: R. qingshengii IEGM 267 is an efficient and stereoselective biocatalyst for (-)-trans-carveol oxidation. The results of bioinformatics analysis suggest an alternative enzymatic basis for this transformation and provide a foundation for future functional characterization.

Rhodococcus↗
PubMed2026

Nitrilase-mediated degradation of insecticides flonicamid and thiacloprid by immobilized engineered Escherichia coli with a novel pathway.

The nitrile‑containing insecticides flonicamid (FLO) and thiacloprid (THI) are widely used in agriculture, posing risks to the environment and animal health. Nitrilase is a key catalyst for the degradation of nitrile compounds, and immobilized engineered bacteria are preferred in wastewater treatment. However, immobilized engineered bacteria expressing nitrilase have never been investigated for pollutant degradation. Here, engineered Escherichia coli pET28a‑VbNitA harboring the nitrilase gene VbNitA was immobilized by calcium alginate encapsulation. FLO was degraded into N-(4-trifluoromethylnicotinoyl)glycinamide and 4-(trifluoromethyl)nicotinol glycine by the immobilized cells via VbNitA. THI was converted to THI‑amide and THI‑imine using the same system. Notably, this is the first report of a nitrilase converting THI to THI‑amide and of THI‑imine as a biodegradation intermediate. Compared with free cells, the immobilized E. coli pET28a‑VbNitA showed higher tolerance to high temperature, alkaline, and acidic environments, and better long-term storage stability. The substrate inhibition model showed that the optimal initial concentrations of FLO and THI for degradation by immobilized E. coli pET28a‑VbNitA were 45.13 and 127.50 μmol/L, respectively. FLO was degraded more rapidly than THI by the immobilized cells. Molecular docking revealed that both FLO and THI formed stable interactions with VbNitA, with FLO positioned closer to Cys165 of the catalytic triad. This study presents a novel THI degradation pathway and provides a new, efficient immobilized biocatalyst for the remediation of wastewater with nitrile‑containing insecticides.

Escherichia coli↗
PubMed2026

Functional Characterization of a Novel Flavonoid O-methyltransferase From Polar Pedobacter sp. PAMC26386 and Bioactivity Assessment of Flavonoids.

Flavonoid O-methyltransferases (OMTs) catalyze the methylation of flavonoid hydroxyl groups, enhancing structural diversity and biological activity. In this study, we identified and characterized a novel Class I flavonoid OMT from the Antarctic bacterium Pedobacter sp. PAMC26386. Despite originating from a cold-adapted organism, the enzyme exhibited high catalytic activity at 55 °C and a strong preference for Co²⁺ as a cofactor. Sequence and phylogenetic analyses confirmed its classification as a flavonoid-specific OMT and revealed conserved motifs for S-adenosyl-L-methionine (SAM) binding and metal coordination. The enzyme accepted a wide range of flavonoid substrates, with quercetin and fisetin showing the highest activities. Kinetic analysis indicated greater substrate affinity for quercetin (Km = 36.55 µM) than for fisetin (Km = 49.41 µM). Whole-cell biotransformation using recombinant Escherichia coli C41 co-expressing the OMT and metK enabled efficient intracellular methylation, yielding 62.5 mg L⁻¹ of methylated quercetin and 55.5 mg L⁻¹ of 3'-O-methyl fisetin. The predicted methylation site at the 3'-hydroxyl of fisetin, determined by molecular docking, was confirmed by NMR spectroscopy. Notably, the previously reported 3'-O-methyl fisetin showed enhanced in vitro anticancer activity against mouse breast cancer cells and selectively improved antimycobacterial activity against Mycobacterium tuberculosis compared to the parent compound. To our knowledge, this study is among the first to report the enzymatic production of 3'-O-methyl fisetin using a polar microbial OMT that is optimally active at elevated temperatures in the presence of Co²⁺. The increased bioactivity compared to the parent compounds highlights the potential of this OMT as a biocatalyst for the sustainable production of pharmaceutically relevant methylated flavonoids.

Flavonoids↗
PubMed2026

Generative artificial intelligence for enzyme design and biocatalysis.

Sparked by innovations in generative artificial intelligence (AI), the field of protein design has undergone a paradigm shift with an explosion of new models for optimizing existing enzymes or creating them from scratch. After more than one decade of low success rates for computationally designed enzymes, generative AI models are now frequently used for designing proficient enzymes. Here, we provide a comprehensive overview and classification of generative AI models for enzyme design, highlighting models with experimental validation relevant to real-world settings and outlining their respective limitations. We argue that generative AI models now have the maturity to create and optimize enzymes for industrial applications. Wider adoption of generative AI models with experimental feedback loops can speed up the development of biocatalysts and serve as a community assessment to inform the next generation of models.

Biocatalysis↗
PubMed2026

Structural Characterization and Engineering of a GH134 β-Mannanase from Aspergillus nidulans for Enhancement of Activity and Stability.

Mannans are abundant plant hemicelluloses, and endo-β-mannanases are important biocatalysts for their conversion into functional manno-oligosaccharides. Here, we report the structural and functional characterization of a glycoside hydrolase family 134 β-mannanase from Aspergillus nidulans (AnGH134) and a structure-guided engineering strategy to improve its performance on locust bean gum. The 1.75 Å crystal structure reveals the conserved lysozyme-like fold of GH134 enzymes and supports an inverting catalytic mechanism with Glu43 and Asp55 as the putative catalytic residues. Docking, mutational, and molecular dynamics analyses indicate that AnGH134 uses an extended substrate-binding groove and that groove-exit residues and the C-terminal region contribute to productive catalysis. Guided by these findings, N-terminal fusion of CBM10 enhanced catalytic efficiency and thermal stability, whereas C-terminal fusion was detrimental. These results provide a framework for engineering GH134 mannanases.

Aspergillus nidulans↗
PubMed2026

Identification of a Novel Thermal Promoter and Its Application in Glutamate Decarboxylase Protein Expression in Bacillus licheniformis.

As a thermotolerant bacterium, Bacillus licheniformis is an attractive chassis for high-temperature biomanufacturing. Here, we identified a novel temperature-responsive promoter, PycgM, which maintained strong transcriptional activity at 37-52 °C. In a promoter-mCherry reporter system, PycgM exhibited 2287.3-fold higher activity than P2 at 52 °C, demonstrating excellent compatibility with a thermotolerant host. Truncation analysis identified a 150-bp core functional region responsible for optimal activity under induction and heat stress. When applied to drive glutamate decarboxylase expression at 50 °C, PycgM enabled γ-aminobutyric acid production of 391.67 g/L with a 98.69% conversion rate, representing a 275% increase over 37 °C fermentation. The whole-cell biocatalyst retained 86% activity after five reuse cycles, and SEM analysis indicated acceptable structural stability despite moderate morphological changes. These results demonstrate that PycgM is a robust, high-temperature genetic element for efficient enzyme and metabolite production in thermotolerant hosts.

Bacillus licheniformis↗
PubMed2025

Insights into the Catalytic Activity of a Metagenome-Derived Urethanase.

The discovery of urethanases shows an opportunity to access the biotechnological recycling of polyurethane-based plastics (PURs), widely used in the manufacture of everyday materials. However, the mechanistic understanding of these enzymes remains under debate. In this work, we report a QM/MM-based mechanistic study of the metagenome-derived urethanase UMG-SP2 catalyzing the degradation of a urethane-like model compound, 4-nitrophenyl benzylcarbamate (pNC). A high-quality structural model generated with AlphaFold2, prior to the availability of the crystal structure, accurately captured the Ser-Ser-Lys catalytic triad characteristic of amidase signature enzymes. Highly accurate constant-pH nonequilibrium molecular dynamics and Monte Carlo (neMD/MC) simulations provided the full titration curve of active site Lys, explaining the need for alkaline media for the enzyme to be active. The generation of the free energy landscape, obtained by means of free energy perturbation methods with the M06-2X DFT functional describing the QM region of the full system, reveals an esterase-like three-step mechanism of UMG-SP2, i.e., acylation, hydrolysis, and decarboxylation, with all steps being kinetically feasible. Our computational results show very good agreement with experimental kinetic data, with a calculated free energy barrier of 21.2 kcal·mol-1 for the rate-determining step compared to 22.9 kcal·mol-1 derived from the experimentally measured turnover frequency (TOF). The present results also open the door for the final decarboxylation occurring in the solution after the release of the product of the hydrolysis step or within the active site. These findings provide an atomistic insight into the urethanase function and establish a robust framework for the future design of biocatalysts targeting polyurethane degradation.

Metagenome↗
PubMed2026

Exploring biohydrogen producing potential of Arctic ice and water through metagenomics and dark fermentation kinetics.

Cryospheric ecosystems in the high Arctic harbor largely unexplored microbiomes with significant biotechnological potential. The present study evaluates the biohydrogen production capabilities of the indigenous microbiome of Ny-Ålesund, Svalbard, using glacial ice and surface water samples. Dark fermentation batch assays were performed at 4 °C and 20 °C with 2-bromoethanesulfonate (BES), a methanogenic inhibitor, to track the succession of metabolic and taxonomic diversity. Metagenomic and functional analyses revealed that under 20 °C and BES conditions, psychrotolerant microbial communities maximize biohydrogen production to 85% of the total biogas produced, with an acetate-dominant fermentation pathway, as inferred from volatile fatty acid (VFA) analysis. This evolves into a highly coordinated system utilizing a coupled Rnf-nitrogenase route alongside Formate Hydrogenlyase and [FeFe]-hydrogenase pathways. Kinetic modelling using the Modified Gompertz equation, along with Q10 temperature-sensitivity indices, demonstrated a very high latent catalytic potential in these cold-adapted microbiomes. This study indicates that Arctic microbiomes are highly elastic thermodynamically and could serve as highly efficient, manipulatable biocatalysts for the environmental recovery of bioenergy through engineered low-temperature systems.

Fermentation↗
PubMed2025

Physiological and metabolic responses of Zymomonas mobilis to lignocellulosic hydrolysate.

Zymomonas mobilis is a promising biocatalyst for the sustainable conversion of lignocellulosic sugars into biofuels and bioproducts, yet its response to lignocellulosic hydrolysates remains poorly understood. Here, we investigate the physiological response of Z. mobilis to ammonia fiber expansion (AFEX)-pretreated switchgrass hydrolysate using a systems-level approach integrating LC-MS/MS-based lipidomics and shotgun proteomics. Growth on hydrolysate induced substantial shifts in fatty acid and membrane phospholipid composition, alongside broad proteomic remodeling. Notably, Z. mobilis exhibited a stress response characterized by the upregulation of heat shock proteins and efflux transporters and the downregulation of cell motility proteins. Unexpectedly, hydrolysate exposure also led to a robust upregulation of the Entner-Doudoroff pathway, the ethanol fermentation pathway, and other central carbon metabolism enzymes, indicating a substantial cellular investment potentially driven by additional nutrient availability in hydrolysate. These findings provide new insights into the metabolic adaptations of Z. mobilis to lignocellulosic hydrolysates, informing strategies to enhance its biofuel production capabilities.IMPORTANCEBiomass pretreatment processes release fermentable sugars from lignocellulosic biomass, but they also generate inhibitors that can impact microbial metabolism. This study provides a systems-level evaluation of how Zymomonas mobilis responds to hydrolysate stress, revealing distinct physiological and lipid membrane remodeling responses. While some stress responses overlap with those induced by ethanol and isobutanol toxicity, both valuable biofuels, hydrolysate exposure elicits unique metabolic shifts. These findings offer valuable insights for engineering Z. mobilis strains with improved tolerance and performance for efficient bioconversion of lignocellulosic hydrolysates into biofuels and bioproducts.

Zymomonas↗
PubMed2026

Broad-spectrum biodegradation of aliphatic and aliphatic-aromatic polyesters by Papiliotrema laurentii isolated from locust frass.

Biodegradable aliphatic and aliphatic-aromatic polyesters, such as poly(butylene adipate-co-terephthalate) (PBAT), polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), and polyhydroxyalkanoates (PHA), are increasingly used as sustainable alternatives to petrochemical plastics. However, their depolymerization outside industrial composting facilities is often incomplete. This study characterized Papiliotrema laurentii strain 62UF-13, isolated from migratory locust frass, for broad-spectrum polyester hydrolysis. Emulsion assays demonstrated hydrolytic activity across all five polymers, with PCL and PBS showing the highest clearance rates. Solid-film assays revealed substantial gravimetric mass loss of PCL, PLA, and PHA cast films, whereas a commercial PBAT-PLA mulch film in minimal medium, underwent progressive fragmentation/disintegration, as assessed by the remaining film area. Incubation with the PBAT-PLA film was accompanied by the release of adipic acid (49.60 mg/L, week 1) and terephthalic acid (maximum 21.62 mg/L, week 4), followed by a decrease to 0.26 mg/L by week 8, coinciding with the emergence of putative 3,4-dihydroxymandelic acid and a putative acetylated derivative. Scanning electron microscopy (SEM) revealed pronounced pitting and erosion, while Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) indicated ester-bond scission and changes in crystallinity/melting behavior. Whole-genome sequencing identified eight candidate polyesterases, including cutinases and esterases, with ≥ 60% amino acid identity to known hydrolases active on PCL, PBS, PHA, and PLA. This study is the first report of P. laurentii degrading a broad range of aliphatic and aliphatic-aromatic polyesters, including partial biotransformation of terephthalate moieties from PBAT. Integration of phenotypic assays and genomic evidence positions P. laurentii 62UF-13 as a viable biocatalyst for decentralized management of biodegradable plastic waste under mild environmental conditions.

Papiliotrema laurentii↗
PubMed2026

Comparative Characterization of σ32-Dependent Promoters for the Heat-Inducible Expression of FAST-PETase in Escherichia coli.

Efficient regulation of recombinant enzyme expression is an important consideration for the development of microbial biocatalysts. Heat-inducible promoters regulated by the alternative sigma factor σ32 provide an inducer-free strategy for controlling gene expression in Escherichia coli. In this study, four σ32-dependent promoters (PdnaK, PgrpE, PibpA, and PclpB) were comparatively characterized using the PET-degrading enzyme FAST-PETase fused to superfolder green fluorescent protein as a model recombinant protein. Promoter performance was evaluated based on basal leakage, induction kinetics, and expression strength following heat induction. Among the promoters examined, PdnaK exhibited the strongest heat-inducible expression and was dissected to examine the autonomous and combinatorial behavior of its promoter-derived elements. Molecular docking analysis further supported the experimental observations by showing qualitative agreement between predicted σ32-DNA interactions and promoter performance. Together, these findings provide a comparative characterization of σ32-dependent promoters and identify promoter architectures that may facilitate the development of heat-inducible recombinant enzyme expression systems in E. coli.

Escherichia coli↗
PubMedDate not supplied

[Synthesis of organic acids by immobilized propionic bacteria in the flow system and stabilization of the process].

The capacity of immobilized cells of propionic bacteria to synthesize organic acids was examined. Propionibacterium shermanii cells incorporated into polyacrylamide gel were capable to synthesize propionic, acetic and pyruvic acids in the flow system. As a carbon source glucose, lactate-Na or whey lactose was used. The greatest amount of the acids was synthesized with the use of lactate-Na. The life-time of the biocatalyst (immobilized cells) can be increased by its reactivation with a nutrient medium required for optimal cell proliferation.

Acrylamides↗