MP
Martin Pabst
info
Please Note
<p>This page displays the records of the person named above and is not linked to a unique person identifier. This record may need to be merged to a profile.</p>
155 records found
1
...
Journal article
(2026)
-
Zi Xuan Hu, Jia Yu Zhang, Jitske van Ede, Yao Yao Zhang, Yu Quan Li, Mattia Ghirardello, M. Carmen Galan, Martin Pabst, Li Liu, Josef Voglmeir
Sialic acids are a diverse family of acidic sugars typically found at the terminal positions of glycan chains, mediating key physiological and pathological processes across animals - particularly vertebrates - including cell signaling and host-pathogen interactions. The distribution of sialic acids in lower animals such as mollusks, however, remains largely unresolved. Here, we report the discovery of unconjugated 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), a deaminated analogue of N-acetylneuraminic acid, in the muscle tissue of Pacific oysters (Magallana gigas). Using UPLC-ESI-MS/MS fingerprinting, we identified naturally occurring free KDN at a concentration of 1.2 ± 0.1 nmol/100 mg of oyster muscle tissue. To investigate the biosynthetic pathway, four candidate genes were identified in the M. gigas genome, and the corresponding recombinant proteins were expressed and characterized. Enzymatic assays revealed that one putative sialic acid aldolase (MgNPL) specifically catalyzes the cleavage of KDN into mannose and pyruvate. To our knowledge, this represents the first molecular evidence of KDN metabolism in mollusks and highlights both the unexpected conservation of substrate-specific aldolase activity and distinct sialic acid utilization mechanisms compared to vertebrates.
...
Sialic acids are a diverse family of acidic sugars typically found at the terminal positions of glycan chains, mediating key physiological and pathological processes across animals - particularly vertebrates - including cell signaling and host-pathogen interactions. The distribution of sialic acids in lower animals such as mollusks, however, remains largely unresolved. Here, we report the discovery of unconjugated 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), a deaminated analogue of N-acetylneuraminic acid, in the muscle tissue of Pacific oysters (Magallana gigas). Using UPLC-ESI-MS/MS fingerprinting, we identified naturally occurring free KDN at a concentration of 1.2 ± 0.1 nmol/100 mg of oyster muscle tissue. To investigate the biosynthetic pathway, four candidate genes were identified in the M. gigas genome, and the corresponding recombinant proteins were expressed and characterized. Enzymatic assays revealed that one putative sialic acid aldolase (MgNPL) specifically catalyzes the cleavage of KDN into mannose and pyruvate. To our knowledge, this represents the first molecular evidence of KDN metabolism in mollusks and highlights both the unexpected conservation of substrate-specific aldolase activity and distinct sialic acid utilization mechanisms compared to vertebrates.
Journal article
(2026)
-
Maximilienne T. Allaart, Alexander V. Tyakht, Ruth E. Ley, Martin Pabst, Gerben R. Stouten, Largus T. Angenent
D- and L-lactate are routinely produced as intermediates in fermentative ecosystems. However, the microbial fate of these stereoisomers remains poorly understood. Given that D-lactate is an unavoidable byproduct of digestion and a neurotoxin, understanding its microbial turnover not only holds ecological pertinence but also the potential to uncover new links between gut microbiota metabolism and host health. Here, we used chemostat bioreactors (pH 7.0, 37°C, and a solids retention time of 4 days) to enrich for lactate-consuming communities. DL-lactate-consuming consortia were enriched, characterized, and used as inoculum for duplicate bioreactors fed exclusively with D- or L-lactate. After steady-state was reached, the fed lactate stereoisomers were switched to assess community resilience. Regardless of the fed stereoisomer, the fermentation product spectra were consistent and dominated by acetate, propionate, and CO2. However, microbial communities and biomass yields diverged sharply, with a high relative abundance of Anaerotignum in D-lactate enrichments and Acidipropionibacterium and Propionibacterium in L-lactate enrichments. Notably, the biomass yield for D-lactate feeding was less than half that for L-lactate feeding, suggesting that the two isomers are metabolized through distinct biochemical pathways despite similar product spectra. Metagenomic and metaproteomic analyses confirmed divergence in D- and L-lactate conversion at both the phylogenetic and pathway levels. Our findings reveal how the stereoisomer identity of microbes shapes their niche specialization, with implications for understanding the ecology and clinical impact of lactate metabolism.
...
D- and L-lactate are routinely produced as intermediates in fermentative ecosystems. However, the microbial fate of these stereoisomers remains poorly understood. Given that D-lactate is an unavoidable byproduct of digestion and a neurotoxin, understanding its microbial turnover not only holds ecological pertinence but also the potential to uncover new links between gut microbiota metabolism and host health. Here, we used chemostat bioreactors (pH 7.0, 37°C, and a solids retention time of 4 days) to enrich for lactate-consuming communities. DL-lactate-consuming consortia were enriched, characterized, and used as inoculum for duplicate bioreactors fed exclusively with D- or L-lactate. After steady-state was reached, the fed lactate stereoisomers were switched to assess community resilience. Regardless of the fed stereoisomer, the fermentation product spectra were consistent and dominated by acetate, propionate, and CO2. However, microbial communities and biomass yields diverged sharply, with a high relative abundance of Anaerotignum in D-lactate enrichments and Acidipropionibacterium and Propionibacterium in L-lactate enrichments. Notably, the biomass yield for D-lactate feeding was less than half that for L-lactate feeding, suggesting that the two isomers are metabolized through distinct biochemical pathways despite similar product spectra. Metagenomic and metaproteomic analyses confirmed divergence in D- and L-lactate conversion at both the phylogenetic and pathway levels. Our findings reveal how the stereoisomer identity of microbes shapes their niche specialization, with implications for understanding the ecology and clinical impact of lactate metabolism.
Journal article
(2026)
-
Mengqing Fan, Qiang Xu, Xiaoxuan Wang, Zhiwei Fang, Mark C.M. van Loosdrecht, Martin Pabst, Yu Tao, Joan B. Rose, Walter van der Meer, Gang Liu
Fire hydrants are widely installed in drinking water distribution systems, where stagnant water forms multiple ‘high-risk zones’. The stagnant water quality at hydrant terminals has been poorly studied. Here we show that stagnant water exhibited an 18-fold increase in manganese, a 40-fold increase in total cell counts, a 13-fold increase in adenosine triphosphate and enrichment of opportunistic pathogens compared with flowing water. Notable changes were also observed in microbial communities and dissolved organic matter composition, including shifts in dominant bacterial taxa, transformation of saturated oxidized compounds and generation of unsaturated reduced compounds. This study also explored the ecological mechanisms underlying the covariation of microorganisms and dissolved organic matter after water stagnation. This finding provides an additional possibility for drinking water quality deterioration in drinking water distribution systems, highlighting the potential threat posed by stagnant water in non-consumer terminals (fire hydrants) to water safety.
...
Fire hydrants are widely installed in drinking water distribution systems, where stagnant water forms multiple ‘high-risk zones’. The stagnant water quality at hydrant terminals has been poorly studied. Here we show that stagnant water exhibited an 18-fold increase in manganese, a 40-fold increase in total cell counts, a 13-fold increase in adenosine triphosphate and enrichment of opportunistic pathogens compared with flowing water. Notable changes were also observed in microbial communities and dissolved organic matter composition, including shifts in dominant bacterial taxa, transformation of saturated oxidized compounds and generation of unsaturated reduced compounds. This study also explored the ecological mechanisms underlying the covariation of microorganisms and dissolved organic matter after water stagnation. This finding provides an additional possibility for drinking water quality deterioration in drinking water distribution systems, highlighting the potential threat posed by stagnant water in non-consumer terminals (fire hydrants) to water safety.
Journal article
(2026)
-
Yujia Zhai, Xun Wang, Xuhan Deng, Xiaoming Li, Bin Hu, Walter van der Meer, Mark C.M. van Loosdrecht, Gang Liu, Martin Pabst
Riverbank filtration is a nature-based water treatment strategy known for its effective removal of organic micropollutants. Yet, the mechanisms governing their biodegradation, especially the role of redox transitions in mediating biotransformation, remain insufficiently understood. Here, we integrate metagenomic profiling with chemical analytics in a 10 m simulated riverbank filtration system to demonstrate how sequential oxidizing–reducing degradation enhances organic micropollutant transformation. Oxygen stratification structured distinct microbial and enzymatic pathways: oxidizing zones (>+200 mV redox potential) facilitated cytochrome P450-mediated oxidation (oxidizing condition, OXD), while subsequent redox shifts to reducing conditions (←400 mV, sequential oxidizing–reducing (SOR) conditions) activated reductive transformations (e.g., via nitronate monooxygenase and aldehyde dehydrogenase) and conjugation pathways. These SOR conditions significantly enhanced the removal of recalcitrant compounds, including irbesartan (+25.3%), benzotriazole (13.4%), and gabapentin (+9.7%). Metagenomic analysis revealed redox-driven microbial specialization, with Pseudomonadota and Nitrospirota dominating in oxidizing zones and reducing microzones enriched in pathways associated with nitrotoluene and ethylbenzene degradation, providing genomic evidence for sequential organic micropollutant breakdown. These findings establish a mechanistic framework for harnessing oxidizing–reducing microbial partnerships to amplify organic micropollutant removal in nature-based water treatment systems, which can be used for riverbank filtration site selection and well field construction and optimization.
...
Riverbank filtration is a nature-based water treatment strategy known for its effective removal of organic micropollutants. Yet, the mechanisms governing their biodegradation, especially the role of redox transitions in mediating biotransformation, remain insufficiently understood. Here, we integrate metagenomic profiling with chemical analytics in a 10 m simulated riverbank filtration system to demonstrate how sequential oxidizing–reducing degradation enhances organic micropollutant transformation. Oxygen stratification structured distinct microbial and enzymatic pathways: oxidizing zones (>+200 mV redox potential) facilitated cytochrome P450-mediated oxidation (oxidizing condition, OXD), while subsequent redox shifts to reducing conditions (←400 mV, sequential oxidizing–reducing (SOR) conditions) activated reductive transformations (e.g., via nitronate monooxygenase and aldehyde dehydrogenase) and conjugation pathways. These SOR conditions significantly enhanced the removal of recalcitrant compounds, including irbesartan (+25.3%), benzotriazole (13.4%), and gabapentin (+9.7%). Metagenomic analysis revealed redox-driven microbial specialization, with Pseudomonadota and Nitrospirota dominating in oxidizing zones and reducing microzones enriched in pathways associated with nitrotoluene and ethylbenzene degradation, providing genomic evidence for sequential organic micropollutant breakdown. These findings establish a mechanistic framework for harnessing oxidizing–reducing microbial partnerships to amplify organic micropollutant removal in nature-based water treatment systems, which can be used for riverbank filtration site selection and well field construction and optimization.
Journal article
(2026)
-
Le Min Chen, Sunanda Keisham, Hiroaki Tateno, Gijs Y. Kleine, Martin Pabst, Mario Pronk, Mark C.M. van Loosdrecht, Yuemei Lin
The authors would like to inform readers that a sentence was inadvertently omitted from the ‘Abstract’ section during the production process. The correct ‘Abstract’ section should read as follows: The long-term effects of environmental conditions, such as seawater salinity, on the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS) remain poorly understood. This study investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca. Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
...
The authors would like to inform readers that a sentence was inadvertently omitted from the ‘Abstract’ section during the production process. The correct ‘Abstract’ section should read as follows: The long-term effects of environmental conditions, such as seawater salinity, on the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS) remain poorly understood. This study investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca. Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
Journal article
(2026)
-
Carlota Vijande, Sabela Balboa, Massimo Lazzari, Juan Manuel Lema, Martin Pabst
Plastic pollution is a growing concern, especially poly(ethylene terephthalate) (PET), one of the most produced plastic polymers. Although several microorganisms capable of degrading PET have been identified, little is known about those present in wastewater treatment plants (WWTPs). This study explores their ability to degrade PET and the enzymes involved. Activated sludge from two facilities-one urban WWTP and one industrial WWTP-was cultivated with PET of different crystallinities. The inoculum source primarily determined differences in microbial community composition. Metagenomics revealed more than 300 genes homologous to PET-degrading enzymes in all biofilms; however, metaproteomics confirmed expression of only a few of these enzymes in industrial WWTP-derived biofilms. This inoculum demonstrated the ability to degrade PET breakdown products within 24 h. In addition, FTIR analysis revealed initial signs of surface alteration. In conclusion, this study reveals the presence of microorganisms in industrial wastewater treatment sludge that possess the genetic potential to degrade PET.
...
Plastic pollution is a growing concern, especially poly(ethylene terephthalate) (PET), one of the most produced plastic polymers. Although several microorganisms capable of degrading PET have been identified, little is known about those present in wastewater treatment plants (WWTPs). This study explores their ability to degrade PET and the enzymes involved. Activated sludge from two facilities-one urban WWTP and one industrial WWTP-was cultivated with PET of different crystallinities. The inoculum source primarily determined differences in microbial community composition. Metagenomics revealed more than 300 genes homologous to PET-degrading enzymes in all biofilms; however, metaproteomics confirmed expression of only a few of these enzymes in industrial WWTP-derived biofilms. This inoculum demonstrated the ability to degrade PET breakdown products within 24 h. In addition, FTIR analysis revealed initial signs of surface alteration. In conclusion, this study reveals the presence of microorganisms in industrial wastewater treatment sludge that possess the genetic potential to degrade PET.
Journal article
(2026)
-
Meryl J. Copray, Martin Pabst, Puck C. Groen, Jorke Willemse, Jeroen de Jonge, Marcel Ottens
Extended criteria donor (ECD) livers are increasingly used to mitigate organ shortage, yet they carry a higher risk of post-transplant complications. Normothermic machine perfusion (NMP) enables functional assessment, but current viability criteria cannot always distinguish transplantable from non-transplantable grafts. Here, we identify proteomic signatures and propose underlying mechanisms that determine ECD liver suitability during NMP. Using label-free shotgun proteomics, we analysed 72 perfusate samples collected during hypothermic oxygenated perfusion, controlled rewarming, and NMP of 24 human ECD livers. Non-transplanted grafts were consistently enriched in oxidative stress–related proteins, associated with disrupted glutathione metabolism and severe ischemia–reperfusion injury. In contrast, transplanted livers maintained balanced antioxidant responses and progressively upregulated functional and regenerative pathways. Oxidative stress proteins distinguished transplantable from non-transplantable grafts within 60 minutes of NMP (AUC≥0.94), while prolonged perfusion supported recovery in moderate livers. These findings propose a mechanistic basis for graft viability and demonstrate how oxidative stress and regeneration markers can refine organ assessment during machine perfusion.
...
Extended criteria donor (ECD) livers are increasingly used to mitigate organ shortage, yet they carry a higher risk of post-transplant complications. Normothermic machine perfusion (NMP) enables functional assessment, but current viability criteria cannot always distinguish transplantable from non-transplantable grafts. Here, we identify proteomic signatures and propose underlying mechanisms that determine ECD liver suitability during NMP. Using label-free shotgun proteomics, we analysed 72 perfusate samples collected during hypothermic oxygenated perfusion, controlled rewarming, and NMP of 24 human ECD livers. Non-transplanted grafts were consistently enriched in oxidative stress–related proteins, associated with disrupted glutathione metabolism and severe ischemia–reperfusion injury. In contrast, transplanted livers maintained balanced antioxidant responses and progressively upregulated functional and regenerative pathways. Oxidative stress proteins distinguished transplantable from non-transplantable grafts within 60 minutes of NMP (AUC≥0.94), while prolonged perfusion supported recovery in moderate livers. These findings propose a mechanistic basis for graft viability and demonstrate how oxidative stress and regeneration markers can refine organ assessment during machine perfusion.
Aerobic Granular Sludge (AGS) is an innovative and efficient biotechnology for wastewater treatment that has been successfully applied on full-scale worldwide. Full-scale municipal AGS systems typically contain both granular sludge (granules) and flocculent sludge (flocs). Studies on the different roles of granules and flocs remain limited. In this study, a laboratory-scale AGS reactor fed with complex synthetic wastewater was operated to simulate full-scale AGS systems and to study the different functional roles of granules and flocs. The laboratory reactor achieved a coexistence of granules and flocs with a floc mass fraction of 17 %. The activities of different size fractions were evaluated using batch experiments and compared for carbon, nitrogen, and phosphorus removal: flocs (FL; <0.2 mm), small granules (SG; 0.2∼1.0 mm), medium granules (MG; 1.0∼2.0 mm), and large granules (LG; >2.0 mm). During feeding, large granules and medium granules exhibited more substrate uptake than small granules and flocs due to preferential substrate access. For aerobic conversion, flocs and small granules showed higher biomass-specific nitrification rates, while medium granules and large granules showed higher phosphorus uptake and denitrification capacity. Furthermore, large granules and medium granules showed stronger mass transfer limitation of oxygen, which limits their nitrification capability. Microbial community analysis using metagenomics and metaproteomics was performed across size fractions, and distinct communities in granules and flocs were shown. Granules showed a high abundance of Candidatus Accumulibacter (polyphosphate-accumulating organisms, PAOs) and Candidatus Competibacter (glycogen-accumulating organisms, GAOs). Flocs showed a high abundance of Nitrosomonas (ammonium-oxidizing bacteria, AOB) and Tetrasphaera (fermentative PAOs) and a low abundance of Ca. Accumulibacter. The distribution of microbial activities and microbial community over sludge size fractions in the laboratory reactor is similar to full-scale AGS systems, indicating that this laboratory setup can simulate full-scale systems and can be used for future research. Overall, this study highlights the importance of maintaining a good balance between different granule sizes and flocs to optimize nutrient removal.
...
Aerobic Granular Sludge (AGS) is an innovative and efficient biotechnology for wastewater treatment that has been successfully applied on full-scale worldwide. Full-scale municipal AGS systems typically contain both granular sludge (granules) and flocculent sludge (flocs). Studies on the different roles of granules and flocs remain limited. In this study, a laboratory-scale AGS reactor fed with complex synthetic wastewater was operated to simulate full-scale AGS systems and to study the different functional roles of granules and flocs. The laboratory reactor achieved a coexistence of granules and flocs with a floc mass fraction of 17 %. The activities of different size fractions were evaluated using batch experiments and compared for carbon, nitrogen, and phosphorus removal: flocs (FL; <0.2 mm), small granules (SG; 0.2∼1.0 mm), medium granules (MG; 1.0∼2.0 mm), and large granules (LG; >2.0 mm). During feeding, large granules and medium granules exhibited more substrate uptake than small granules and flocs due to preferential substrate access. For aerobic conversion, flocs and small granules showed higher biomass-specific nitrification rates, while medium granules and large granules showed higher phosphorus uptake and denitrification capacity. Furthermore, large granules and medium granules showed stronger mass transfer limitation of oxygen, which limits their nitrification capability. Microbial community analysis using metagenomics and metaproteomics was performed across size fractions, and distinct communities in granules and flocs were shown. Granules showed a high abundance of Candidatus Accumulibacter (polyphosphate-accumulating organisms, PAOs) and Candidatus Competibacter (glycogen-accumulating organisms, GAOs). Flocs showed a high abundance of Nitrosomonas (ammonium-oxidizing bacteria, AOB) and Tetrasphaera (fermentative PAOs) and a low abundance of Ca. Accumulibacter. The distribution of microbial activities and microbial community over sludge size fractions in the laboratory reactor is similar to full-scale AGS systems, indicating that this laboratory setup can simulate full-scale systems and can be used for future research. Overall, this study highlights the importance of maintaining a good balance between different granule sizes and flocs to optimize nutrient removal.
Journal article
(2026)
-
Yujia Zhai, Xun Wang, Xuhan Deng, Xiaoming Li, Bin Hu, Walter van der Meer, Mark C.M. van Loosdrecht, Gang Liu, Martin Pabst
Organic micropollutants (OMPs) in wastewater treatment plant (WWTP) effluent pose persistent risks to aquatic ecosystems and drinking water sources. Riverbank filtration (RBF) is a nature-based treatment process, yet the compartment-specific roles of riverbed sediment and downstream soil in OMP attenuation remain poorly resolved under wastewater-impacted conditions. Here, we combined targeted chemical analysis, OMP property compilation, shotgun metagenomics, EnviPath-based biotransformation annotation, and exploratory network analysis to investigate OMP attenuation in a laboratory-scale RBF system treating real WWTP effluent for 10 months. Nineteen OMPs were monitored along a sequential sediment–soil filtration pathway. Sediment preferentially attenuated hydrophilic or charged compounds, including lidocaine, amantadine, and sotalol, whereas soil contributed more strongly to the attenuation of naproxen, atenolol, and losartan. Metagenomic profiling revealed distinct microbial communities and functional gene repertoires between sediment and soil after long-term operation. Sediment harbored higher relative abundances of genes associated with oxidative xenobiotic transformation, including cytochrome P450-related enzymes, demethylases, dehydrogenases, oxidases, and aromatic compound degradation pathways. An exploratory Spearman network further identified associations among microbial genera, EnviPath-annotated candidate biotransformation genes, and OMP removal rates, including 17 KO–OMP links supported by both correlation and pathway annotation. These findings indicate that sediment and soil develop complementary microbial functional potentials that may support compound-specific OMP attenuation. This study provides a mechanistic basis for optimizing sediment–soil configurations in wastewater-impacted RBF systems and for improving nature-based barriers against diverse OMP mixtures.
...
Organic micropollutants (OMPs) in wastewater treatment plant (WWTP) effluent pose persistent risks to aquatic ecosystems and drinking water sources. Riverbank filtration (RBF) is a nature-based treatment process, yet the compartment-specific roles of riverbed sediment and downstream soil in OMP attenuation remain poorly resolved under wastewater-impacted conditions. Here, we combined targeted chemical analysis, OMP property compilation, shotgun metagenomics, EnviPath-based biotransformation annotation, and exploratory network analysis to investigate OMP attenuation in a laboratory-scale RBF system treating real WWTP effluent for 10 months. Nineteen OMPs were monitored along a sequential sediment–soil filtration pathway. Sediment preferentially attenuated hydrophilic or charged compounds, including lidocaine, amantadine, and sotalol, whereas soil contributed more strongly to the attenuation of naproxen, atenolol, and losartan. Metagenomic profiling revealed distinct microbial communities and functional gene repertoires between sediment and soil after long-term operation. Sediment harbored higher relative abundances of genes associated with oxidative xenobiotic transformation, including cytochrome P450-related enzymes, demethylases, dehydrogenases, oxidases, and aromatic compound degradation pathways. An exploratory Spearman network further identified associations among microbial genera, EnviPath-annotated candidate biotransformation genes, and OMP removal rates, including 17 KO–OMP links supported by both correlation and pathway annotation. These findings indicate that sediment and soil develop complementary microbial functional potentials that may support compound-specific OMP attenuation. This study provides a mechanistic basis for optimizing sediment–soil configurations in wastewater-impacted RBF systems and for improving nature-based barriers against diverse OMP mixtures.
Journal article
(2026)
-
Le Min Chen, Sunanda Keisham, Hiroaki Tateno, Gijs Y. Kleine, Martin Pabst, Mario Pronk, Mark C.M. van Loosdrecht, Yuemei Lin
The long-term effects of environmental conditions, such as seawater salinity, on the extracellular investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca.Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
...
The long-term effects of environmental conditions, such as seawater salinity, on the extracellular investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca.Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.
Wastewater metaproteomics
Tracking microbial and human protein biomarkers
Journal article
(2026)
-
Claudia G. Tugui, Filine Cordesius, Willem van Holthe, Mark C.M. Van Loosdrecht, Martin Pabst
Wastewater-based surveillance has become a powerful tool for monitoring the spread of pathogens, antibiotic resistance genes, and measuring population-level exposure to pharmaceuticals and chemicals. While surveillance methods commonly target small molecules, DNA, or RNA, wastewater also contains a vast spectrum of proteins. However, despite recent advances in environmental proteomics, large-scale monitoring of protein biomarkers in wastewater is still far from routine. Analyzing raw wastewater presents a challenge due to its heterogeneous mixture of organic and inorganic substances, microorganisms, cellular debris, and various chemical pollutants. To overcome these obstacles, we developed a wastewater metaproteomics approach including efficient protein extraction and an optimized data-processing pipeline. The pipeline utilizes de novo sequencing to customize large public sequence databases to enable comprehensive metaproteomic coverage. Using this approach, we analyzed wastewater samples collected over approximately three months from two urban locations. This revealed a core microbiome comprising a broad spectrum of microbes, gut bacteria and potential opportunistic pathogens. Additionally, we identified nearly 200 human proteins, including promising population-level health indicators, such as immunoglobulins, uromodulin, and cancer-associated proteins.
...
Wastewater-based surveillance has become a powerful tool for monitoring the spread of pathogens, antibiotic resistance genes, and measuring population-level exposure to pharmaceuticals and chemicals. While surveillance methods commonly target small molecules, DNA, or RNA, wastewater also contains a vast spectrum of proteins. However, despite recent advances in environmental proteomics, large-scale monitoring of protein biomarkers in wastewater is still far from routine. Analyzing raw wastewater presents a challenge due to its heterogeneous mixture of organic and inorganic substances, microorganisms, cellular debris, and various chemical pollutants. To overcome these obstacles, we developed a wastewater metaproteomics approach including efficient protein extraction and an optimized data-processing pipeline. The pipeline utilizes de novo sequencing to customize large public sequence databases to enable comprehensive metaproteomic coverage. Using this approach, we analyzed wastewater samples collected over approximately three months from two urban locations. This revealed a core microbiome comprising a broad spectrum of microbes, gut bacteria and potential opportunistic pathogens. Additionally, we identified nearly 200 human proteins, including promising population-level health indicators, such as immunoglobulins, uromodulin, and cancer-associated proteins.
Journal article
(2026)
-
Agustina Ziliani, Patricia Bovio-Winkler, Martin Pabst, Angela Cabezas, Claudia Etchebehere, Hector A. Garcia, Carlos M. López-Vázquez, Damir Brdjanovic, Mark C.M. van Loosdrecht, Francisco J. Rubio-Rincón
Amino acids are less studied substrates in enhanced biological phosphorus removal (EBPR) systems. Glycine, a prevalent amino acid in wastewater, was used in this study to evaluate its role in EBPR processes. We operated a sequencing batch reactor (SBR) for over three months with glycine as the sole carbon source to investigate phosphorus removal performance and microbial dynamics using chemical and molecular analyses. The reactor supported EBPR activity, with glycine enabling anaerobic phosphorus release followed by aerobic uptake. The dissolved organic carbon to phosphorus (DOC:P) removal ratio of 100:9.9 closely matched values reported for systems dominated by polyphosphate-accumulating organisms (PAOs), and net phosphorus removal (20 mg PO₄-P L−1) fell within the range reported for laboratory-scale EBPR systems fed with mixed carbon sources. Community analyses showed enrichment of Saccharimonadales alongside putative PAOs, including Ca. Phosphoribacter and Ca. Propionivibrio. Genome-resolved analyses indicate distinct but complementary metabolic potentials, including glycine transformation and lactate-related pathways, suggesting distributed carbon processing within the community. Together, these findings expand the understanding of amino acid utilization in EBPR systems and identify potential metabolic linkages that influence phosphorus removal under glycine-fed conditions.
...
Amino acids are less studied substrates in enhanced biological phosphorus removal (EBPR) systems. Glycine, a prevalent amino acid in wastewater, was used in this study to evaluate its role in EBPR processes. We operated a sequencing batch reactor (SBR) for over three months with glycine as the sole carbon source to investigate phosphorus removal performance and microbial dynamics using chemical and molecular analyses. The reactor supported EBPR activity, with glycine enabling anaerobic phosphorus release followed by aerobic uptake. The dissolved organic carbon to phosphorus (DOC:P) removal ratio of 100:9.9 closely matched values reported for systems dominated by polyphosphate-accumulating organisms (PAOs), and net phosphorus removal (20 mg PO₄-P L−1) fell within the range reported for laboratory-scale EBPR systems fed with mixed carbon sources. Community analyses showed enrichment of Saccharimonadales alongside putative PAOs, including Ca. Phosphoribacter and Ca. Propionivibrio. Genome-resolved analyses indicate distinct but complementary metabolic potentials, including glycine transformation and lactate-related pathways, suggesting distributed carbon processing within the community. Together, these findings expand the understanding of amino acid utilization in EBPR systems and identify potential metabolic linkages that influence phosphorus removal under glycine-fed conditions.
Journal article
(2026)
-
Aswin Muralidharan, Ana Rita Costa, Desi Fierlier, Daan Frits van den Berg, Halewijn van den Bossche, Adja Damba Zoumaro-Djayoon, Martin Pabst, Martin Pacesa, Stan J. J. Brouns, More authors...
Jumbo phages protect their genomes from DNA-sensing bacterial defense systems by enclosing them within vesicles and nucleus-like compartments. Very little is known about defense systems specialized to counter these phages. Here, we show that AVAST type 5 (Avs5) systems, part of the signal transduction ATPases of numerous domains (STAND) superfamily, confer conserved immunity against jumbo phages. Using fluorescence microscopy and biotin proximity labeling, we demonstrate that Avs5 localizes to early infection vesicles, where it senses an essential, early-expressed phage protein named JADA (Jumbo phage Avs5 Defense Activator). Recognition of phage infection triggers the Sir2-like effector domain of Avs5 across three Avs5 clades, resulting in rapid NAD + hydrolysis, disruption of phage nucleus formation, and arrest of infection. These findings reveal a spatially coordinated bacterial immune strategy that targets an early vulnerability in jumbo phage infection.
...
Jumbo phages protect their genomes from DNA-sensing bacterial defense systems by enclosing them within vesicles and nucleus-like compartments. Very little is known about defense systems specialized to counter these phages. Here, we show that AVAST type 5 (Avs5) systems, part of the signal transduction ATPases of numerous domains (STAND) superfamily, confer conserved immunity against jumbo phages. Using fluorescence microscopy and biotin proximity labeling, we demonstrate that Avs5 localizes to early infection vesicles, where it senses an essential, early-expressed phage protein named JADA (Jumbo phage Avs5 Defense Activator). Recognition of phage infection triggers the Sir2-like effector domain of Avs5 across three Avs5 clades, resulting in rapid NAD + hydrolysis, disruption of phage nucleus formation, and arrest of infection. These findings reveal a spatially coordinated bacterial immune strategy that targets an early vulnerability in jumbo phage infection.
The Metaproteomics Initiative
Five years of community-driven progress
Journal article
(2026)
-
Tim Van Den Bossche, Lucia Grenga, Gelio Alves, Magnus Arntzen, Dirk Benndorf, Madita Brauer, Daniel Figeys, Martin Pabst, Benoit J. Kunath, More Authors
The Metaproteomics Initiative was officially launched in 2021 to strengthen collaboration, promote knowledge exchange, and support and lead standardization efforts within the growing metaproteomics community. Over the past 5 years, the Initiative has developed into a structured, global network of researchers. It has launched community-driven benchmark studies, helped shape emerging metadata and reporting standards, developed practical guidance and training materials, organized international symposia, and fostered connections across the microbiome research landscape (https://metaproteomics.org/). We outline the Initiative’s organization, activities, achievements, and ongoing efforts, and reflect on how sustained, community-led coordination has shaped the development of metaproteomics as a field. We further position the Grand Metaproteome Challenges as a next step toward coordinated, community-scale biological research, aimed at advancing functional microbiome studies across clinical, industrial, and environmental application domains, and invite engagement from the wider microbiome and omics communities.
...
The Metaproteomics Initiative was officially launched in 2021 to strengthen collaboration, promote knowledge exchange, and support and lead standardization efforts within the growing metaproteomics community. Over the past 5 years, the Initiative has developed into a structured, global network of researchers. It has launched community-driven benchmark studies, helped shape emerging metadata and reporting standards, developed practical guidance and training materials, organized international symposia, and fostered connections across the microbiome research landscape (https://metaproteomics.org/). We outline the Initiative’s organization, activities, achievements, and ongoing efforts, and reflect on how sustained, community-led coordination has shaped the development of metaproteomics as a field. We further position the Grand Metaproteome Challenges as a next step toward coordinated, community-scale biological research, aimed at advancing functional microbiome studies across clinical, industrial, and environmental application domains, and invite engagement from the wider microbiome and omics communities.
A Special Issue of Mass Spectrometry Reviews to Honor Professor Renato Zenobi
A Lifetime of Mentorship and Innovation in Mass Spectrometry
Decoding Sugars
Mass Spectrometric Advances in the Analysis of the Sugar Alphabet
Monosaccharides play a central role in metabolic networks and in the biosynthesis of glycomolecules, which perform essential functions across all domains of life. Thus, identifying and quantifying these building blocks is crucial in both research and industry. Routine methods have been established to facilitate the analysis of common monosaccharides. However, despite the presence of common metabolites, most organisms utilize distinct sets of monosaccharides and derivatives. These molecules therefore display a large diversity, potentially numbering in the hundreds or thousands, with many still unknown. This complexity presents significant challenges in the study of glycomolecules, particularly in microbes, including pathogens and those with the potential to serve as novel model organisms. This review discusses mass spectrometric techniques for the isomer-sensitive analysis of monosaccharides, their derivatives, and activated forms. Although mass spectrometry allows for untargeted analysis and sensitive detection in complex matrices, the presence of stereoisomers and extensive modifications necessitates the integration of advanced chromatographic, electrophoretic, ion mobility, or ion spectroscopic methods. Furthermore, stable-isotope incorporation studies are critical in elucidating biosynthetic routes in novel organisms.
...
Monosaccharides play a central role in metabolic networks and in the biosynthesis of glycomolecules, which perform essential functions across all domains of life. Thus, identifying and quantifying these building blocks is crucial in both research and industry. Routine methods have been established to facilitate the analysis of common monosaccharides. However, despite the presence of common metabolites, most organisms utilize distinct sets of monosaccharides and derivatives. These molecules therefore display a large diversity, potentially numbering in the hundreds or thousands, with many still unknown. This complexity presents significant challenges in the study of glycomolecules, particularly in microbes, including pathogens and those with the potential to serve as novel model organisms. This review discusses mass spectrometric techniques for the isomer-sensitive analysis of monosaccharides, their derivatives, and activated forms. Although mass spectrometry allows for untargeted analysis and sensitive detection in complex matrices, the presence of stereoisomers and extensive modifications necessitates the integration of advanced chromatographic, electrophoretic, ion mobility, or ion spectroscopic methods. Furthermore, stable-isotope incorporation studies are critical in elucidating biosynthetic routes in novel organisms.
Journal article
(2025)
-
Robert Heyer, Maximilian Wolf, Dirk Benndorf, Sergio Uzzau, Jana Seifert, Lucia Grenga, Martin Pabst, Heike Schmitt, Bart Mesuere, More authors...
One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions.
...
One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions.
Journal article
(2025)
-
Francesc Corbera-Rubio, Alje S. Boersma, Weren de Vet, Martin Pabst, Paul W.J.J. van der Wielen, Maartje A.H.J. van Kessel, Mark C.M. van Loosdrecht, Doris van Halem, Sebastian Lücker, Michele Laureni
Methane removal is an essential step in drinking water production from methane-rich groundwaters. Conventional aeration-based stripping results in significant direct methane emissions, contributing up to one-third of a treatment plant's total carbon footprint. To address this, a full-scale trickling filter was operated for biological methane oxidation upstream of a submerged sand filter, and its performance was compared to a conventional aeration–submerged sand filtration set-up. Full-scale data were combined with ex-situ batch assays and metagenome-resolved metaproteomics to quantify the individual contribution of the main (a)biotic processes and characterize the enriched microbial communities. Both treatment setups fully removed methane, iron, ammonium, and manganese, yet the underlying mechanisms differed significantly. Methane was completely removed from the effluent after trickling filtration, with stripping and biological oxidation each accounting for half of the removal, thereby halving overall methane emissions. Methane-oxidizing bacteria not only outcompeted nitrifiers in the trickling filter, but also likely contributed directly to ammonia oxidation. In contrast to the submerged filter preceded by methane stripping, signatures of biological iron oxidation were almost completely absent in the trickling filter, suggesting that the presence of methane directly or indirectly promotes chemical iron oxidation. All systems had similar ex-situ manganese oxidation capacities, yet removal occurred only in the submerged filters but not the trickling filter. Ultimately, our results demonstrate that trickling filtration is effective in promoting biological methane oxidation at comparable produced drinking water quality, highlighting its potential for advancing sustainable drinking water production.
...
Methane removal is an essential step in drinking water production from methane-rich groundwaters. Conventional aeration-based stripping results in significant direct methane emissions, contributing up to one-third of a treatment plant's total carbon footprint. To address this, a full-scale trickling filter was operated for biological methane oxidation upstream of a submerged sand filter, and its performance was compared to a conventional aeration–submerged sand filtration set-up. Full-scale data were combined with ex-situ batch assays and metagenome-resolved metaproteomics to quantify the individual contribution of the main (a)biotic processes and characterize the enriched microbial communities. Both treatment setups fully removed methane, iron, ammonium, and manganese, yet the underlying mechanisms differed significantly. Methane was completely removed from the effluent after trickling filtration, with stripping and biological oxidation each accounting for half of the removal, thereby halving overall methane emissions. Methane-oxidizing bacteria not only outcompeted nitrifiers in the trickling filter, but also likely contributed directly to ammonia oxidation. In contrast to the submerged filter preceded by methane stripping, signatures of biological iron oxidation were almost completely absent in the trickling filter, suggesting that the presence of methane directly or indirectly promotes chemical iron oxidation. All systems had similar ex-situ manganese oxidation capacities, yet removal occurred only in the submerged filters but not the trickling filter. Ultimately, our results demonstrate that trickling filtration is effective in promoting biological methane oxidation at comparable produced drinking water quality, highlighting its potential for advancing sustainable drinking water production.
Journal article
(2025)
-
Jitske M. van Ede, Suzanne van der Steen, Geert M. van der Kraan, Mark C.M. van Loosdrecht, Martin Pabst
The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.
...
The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.
Journal article
(2025)
-
Christian M. Heckmann, Derren J. Heyes, Martin Pabst, Edwin Otten, Nigel S. Scrutton, Caroline E. Paul
Enzymes are attractive catalysts due to their high chemo-, regio-, and enantioselectivity. In recent years, the application of enzymes in organic synthesis has expanded dramatically, especially for the synthesis of chiral alcohols and amines, two very important functional groups found in many active pharmaceutical ingredients (APIs). Indeed, many elegant routes employing such compounds have been described by industry. Yet, for the synthesis of chiral thiols and thioethers, likewise found in APIs albeit less ubiquitous, only very few biocatalytic syntheses have been reported, and stereocontrol has proved challenging. Here, we apply ene-reductases (EREDs), whose ability to initiate and control chemically challenging radical chemistries has recently emerged, to the synthesis of chiral thioethers from α-bromoacetophenones and pro-chiral vinyl sulfides, without requiring light. Depending on the choice of ERED either enantiomer of the product could be accessed. The highest conversion and selectivity were achieved with GluER T36A using fluorinated substrates, reaching up to 82% conversion and >99.5% ee. With α-bromoacetophenone and α-(methylthio)styrene, the reaction could be performed on a 100 mg scale, affording the product in a 46% isolated yield with a 93% ee. Finally, mechanistic studies were carried out using stopped-flow spectroscopy and protein mass spectrometry, providing insight into the preference of the enzyme for the intermolecular reaction. This work paves the way for new routes for the synthesis of thioether-containing compounds.
...
Enzymes are attractive catalysts due to their high chemo-, regio-, and enantioselectivity. In recent years, the application of enzymes in organic synthesis has expanded dramatically, especially for the synthesis of chiral alcohols and amines, two very important functional groups found in many active pharmaceutical ingredients (APIs). Indeed, many elegant routes employing such compounds have been described by industry. Yet, for the synthesis of chiral thiols and thioethers, likewise found in APIs albeit less ubiquitous, only very few biocatalytic syntheses have been reported, and stereocontrol has proved challenging. Here, we apply ene-reductases (EREDs), whose ability to initiate and control chemically challenging radical chemistries has recently emerged, to the synthesis of chiral thioethers from α-bromoacetophenones and pro-chiral vinyl sulfides, without requiring light. Depending on the choice of ERED either enantiomer of the product could be accessed. The highest conversion and selectivity were achieved with GluER T36A using fluorinated substrates, reaching up to 82% conversion and >99.5% ee. With α-bromoacetophenone and α-(methylthio)styrene, the reaction could be performed on a 100 mg scale, affording the product in a 46% isolated yield with a 93% ee. Finally, mechanistic studies were carried out using stopped-flow spectroscopy and protein mass spectrometry, providing insight into the preference of the enzyme for the intermolecular reaction. This work paves the way for new routes for the synthesis of thioether-containing compounds.