Applied Microbiology and Biotechnology最新文献

{"title":"Engineering natural Saccharomyces cerevisiae isolates for enhanced one-step cellulosic ethanol production.","authors":"Letitia Sabina Minnaar, Kentaro Inokuma, Tomohisa Hasunuma, Riaan den Haan","doi":"10.1007/s00253-026-13830-0","DOIUrl":"https://doi.org/10.1007/s00253-026-13830-0","url":null,"abstract":"<p><p>Engineering yeast strains for use as chassis organisms in second-generation (2G) bioethanol is a promising strategy to improve process economics. Natural isolates of Saccharomyces cerevisiae offer strain backgrounds with greater genetic diversity and enhanced robustness, with the potential for improved heterologous protein production capabilities. In this study, heterologous cellulase production using different expression strategies was evaluated in various process-relevant conditions. Enhanced cellulolytic activity was clearly demonstrated in a cell-tethered enzyme system, compared to a free enzyme system, across identical strain backgrounds. Superior secretory capacity was obtained for YI59_V2 for all individual enzymes across all process-relevant conditions tested. In addition, this strain exhibited improved hydrolysis efficiency and ethanol production from crystalline cellulose, achieving ~10 g/L after 96 h (~88% of the maximum theoretical yield) without the need for exogenous cellulase supplementation. Interestingly, enhanced strain robustness against process-relevant, secretion, and cell wall stresses was also observed in transformants with cell-tethered cellulase systems compared to those with free enzyme systems. This study highlights that the expression design strategy for cellulase-encoding genes in this natural isolate was pivotal for increasing protein titres and for influencing strain robustness. Strains exhibiting elevated cellulase activity and increased robustness represent a key step toward the industrial deployment of consolidated bioprocessing (CBP). KEY POINTS: • Cell-tethered expression greatly boosted cellulase activity and cellulose breakdown. • YI59_V2 yielded ~ 10 g/L ethanol from crystalline cellulose without added enzymes. • Tethered enzymes reshaped cell walls and altered stress tolerance.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147855893","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacillus velezensis EU07 suppresses Fusarium graminearum via transcriptomic reprogramming.","authors":"Ömür Baysal, Catherine Jimenez-Quiros, Birsen Cevher-Keskin, Mahmut Tör","doi":"10.1007/s00253-026-13855-5","DOIUrl":"https://doi.org/10.1007/s00253-026-13855-5","url":null,"abstract":"<p><p>Fusarium graminearum poses a major threat to global cereal production, necessitating sustainable control measures. This study elucidates the antagonistic mechanism of the biocontrol agent Bacillus velezensis EU07 against F. graminearum through combined transcriptomic and morphological analyses. Exposure to EU07 compromised fungal cellular integrity, causing severe hyphal distortion. RNA-seq profiling revealed that EU07 triggers a profound transcriptomic collapse rather than a standard stress response. Over 35% of the altered fungal transcriptome caused critical suppression, targeting essential metabolic hubs, secretome effectors, and master transcription factors to inhibit growth and loss of pathogenicity. In a compensatory defensive shift, the pathogen massively reallocated its transcriptional resources. This defence was characterised by a ~ 30-fold enrichment of transmembrane amino acid transporters for putative toxin efflux and an 80-fold enrichment of glutathione S-transferases to mitigate severe oxidative stress. Furthermore, EU07 suppressed core vulnerability nodes, including proline dehydrogenase (PRODH) and apolipophorin, disrupting the pathogen's osmotic resilience and membrane integrity. Molecular docking analysis indicated that the Bacillus lipopeptide iturin A directly targets apolipophorin with high affinity (- 7.2 kcal/mol). Our findings indicated that B. velezensis EU07 overwhelms F. graminearum through simultaneous metabolic starvation and loss of virulence, revealing highly vulnerable fungal targets for next-generation RNAi-based biocontrol.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147855834","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of three glycerol transporters with broad sugar specificities from Aspergillus niger.","authors":"Liinu Nummela, Christina Lyra, Leena Pitkänen, Henry N Maina, Miia R Mäkelä","doi":"10.1007/s00253-026-13854-6","DOIUrl":"https://doi.org/10.1007/s00253-026-13854-6","url":null,"abstract":"<p><p>Aspergillus niger is a versatile biotechnological workhorse whose generalist lifestyle enables the utilization of a wide range of sugar compounds, including hexoses, pentoses, and polyols. Yet most predicted A. niger sugar transporters remain uncharacterized. Here, we characterized three glycerol transporters-GlpA, GlpB, and GlpC-from A. niger. We generated single and double glp deletion strains for physiological analysis, among which the ΔglpA strain showed a clear phenotype with significantly reduced glycerol consumption, indicating that GlpA is essential for glycerol uptake. Furthermore, both growth on glycerol and its uptake were abolished in the ΔglpAΔglpB mutant, while the ΔglpAΔglpC mutant showed no growth phenotype on solid medium but lacked glycerol consumption in liquid cultures. This suggests that GlpB plays a supportive role in glycerol uptake, whereas GlpC affects it indirectly. Nevertheless, heterologous expression of the A. niger glp genes in a sugar uptake deficient Saccharomyces cerevisiae strain showed that all three transporters could transport not only glycerol but also D-mannitol, D-sorbitol, and D-xylitol. In addition, GlpC transported the hexose sugars D-fructose, D-glucose, and D-mannose. These results demonstrate the broad specificities of the A. niger Glp transporters, highlighting the need for thorough physiological and functional characterization of filamentous fungal sugar transporters. Our findings advance the understanding of polyol and sugar uptake in A. niger and provide candidate transporters for strain engineering towards biotechnological upcycling of glycerol. KEY POINTS: • GlpA is the main glycerol transporter in A. niger under the studied conditions • ΔglpAΔglpB and ΔglpAΔglpC double deletions abolished glycerol uptake in A. niger • GlpA, GlpB, and GlpC have broad polyol specificity, with GlpC also uptaking hexoses.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147855854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Understanding pellet population heterogeneity of Aspergillus niger in stirred tank and rocking motion bioreactors","authors":"Karin Engelbert,&nbsp;Tolue Kheirkhah,&nbsp;Charlotte Deffur,&nbsp;Fangxing Zhang,&nbsp;Henri Winter,&nbsp;Timothy Cairns,&nbsp;Sascha Jung,&nbsp;Heiko Briesen,&nbsp;Peter Neubauer,&nbsp;Stefan Junne,&nbsp;Vera Meyer","doi":"10.1007/s00253-026-13822-0","DOIUrl":"10.1007/s00253-026-13822-0","url":null,"abstract":"<p>The filamentous fungus <i>Aspergillus niger</i> is a well-established cell factory in biotechnology. Its productivity depends on macromorphological development which remains difficult to control, partly because the relationship between seed culture and reactor-specific shear force conditions has not been systematically investigated. This study examined how high or low shear forces affect pellet development at both micro- and macromorphological levels in stirred-tank reactors (STR, high shear regime) and rocking-motion bioreactors (RMB, low shear regime). <i>A. niger</i> seed cultures with initially either large or small pellets were used to inoculate batch STR or RMB. Comparable cultivation conditions were applied so that fermentations differed mainly in shear force regime. Growth characteristics and pellet macromorphologies were analysed using 2D and 3D image analyses, enabling us to classify pellets according to three different classes based on their inner pellet architecture. The distribution of these classes depended on both the macromorphologies of the seed culture and the reactor type. Under high shear forces in the STR, pellets underwent breakage shortly after stirrer activation, were limited in their size to an average diameter of 500–600 µm, and formed a homogeneous population. In addition, broken pellets occurred predominantly under STR conditions. In contrast, cultivations in RMB preserved the initial pellet architecture, allowed the formation of larger pellets (median diameter ~ 800 µm) and supported pellet fusion, thus resulting in a more heterogeneous macromorphological population. Notably, glucose uptake rate correlated with the surface-to-volume ratio of the pellet populations, i.e., glucose became faster consumed under STR conditions accompanied with lower biomass yields and higher protein secretion. Citric acid production, however, was detectable in both STR and RMB only when reactors were inoculated with seed cultures characterised by a loose pellet morphology. Overall, our study demonstrates how shear regime and seed culture morphology jointly shape pellet architecture, population heterogeneity and productivity in scale-up processes. Such a comprehensive understanding of morphological developments is instrumental for optimising bioprocesses and future predictive modelling approaches.</p><p>• <i>2D/3D analysis of defined seed cultures in different shear-induced environments</i></p><p>• <i>High shear restricts and homogenises pellets, while low shear maintains heterogeneity</i></p><p>• <i>Highest citric acid and total protein levels were found in smaller, compact pellets. </i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"110 1","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-026-13822-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147830169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Polycationic nanopeptide-fused endolysins for the control of Mannheimia haemolytica.","authors":"Sidra Moqaddes, Hechao Du, Jieting Lin, Jae Eun Hyun, Mawra Gohar, Alexei Savchenko, Kim Stanford, Brenda Ralston, Tim A McAllister, Yan Dong Niu","doi":"10.1007/s00253-026-13835-9","DOIUrl":"https://doi.org/10.1007/s00253-026-13835-9","url":null,"abstract":"<p><p>Bovine respiratory disease (BRD) remains a major economic and animal health concern in cattle production, exacerbated by rising antimicrobial resistance and limited vaccine efficacy. Endolysins, phage-derived peptidoglycan hydrolases, have emerged as promising alternatives to antibiotics, but their activity against gram-negative bacteria is hindered by the outer membrane (OM) barrier. In this study, we identified five distinct prophage-derived endolysins from Mannheimia haemolytica and successfully expressed two (185 and 587AP2) along with their engineered polycationic nanopeptide (PCNP) fusions (PCNP-185 and PCNP-587AP2). All four recombinant proteins demonstrated muralytic activity, with original endolysins exhibiting higher enzymatic efficiency. However, PCNP-fused variants showed superior antibacterial effects as compared to those without. Especially, PCNP-185 achieved the most consistent multi-log₁₀ reductions of up to 4.5 in bacterial counts across M. haemolytica serotypes, particularly when combined with EDTA which increased permeability of the OM. These findings highlight the synergistic potential of engineered endolysins and EDTA in overcoming the gram-negative barrier. Additionally, selecting an appropriate buffer was found crucial to rule out the effect of buffers on antibacterial activity of endolysins. To our knowledge, this is the first report of engineering and evaluating prophage-derived endolysins against M. haemolytica, underscoring their promise as innovative therapeutics for BRD. KEY POINTS: • Prophage endolysins are potent antibacterials with engineering potential. • Chelator synergy boosts endolysin efficacy; buffer choice is critical. • Endolysins hold strong promise as next-generation therapeutic agents against multi-drug-resistant M. haemolytica causing bovine respiratory disease.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147855898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"One-step in vivo assembly of a 63 kb-long biosynthetic gene cluster via multiple recombination in Aspergillus oryzae.","authors":"Koichi Tamano, Haruka Takayama, Ikuko Kozone, Yukiko Abe, Akio Kanda, Kei Kudo, Hikaru Suenaga, Kazuo Shin-Ya","doi":"10.1007/s00253-026-13845-7","DOIUrl":"https://doi.org/10.1007/s00253-026-13845-7","url":null,"abstract":"<p><p>Mass production of numerous secondary plant and microbial metabolites is crucial, given their value as pharmaceutical agents, dietary supplements, and pesticides. For microbial strain, mass production generally involves improving the native producer strain to enhance overall productivity via spontaneous mutagenesis or genetic modifications. Alternatively, productivity can be enhanced through heterologous production, in which the biosynthetic genes for a secondary metabolite are expressed in a more suitable strain. However, as these biosynthetic genes commonly exist as long clusters, often exceeding several tens of kilobases (kb), their handling is labor-intensive and time-consuming, requiring multiple rounds of genetic cloning and introduction into the host. Therefore, methods enabling efficient transfer of biosynthetic genes into another microorganism in a single step of transformation without the need to clone long gene clusters have been strongly desired. Such an approach has been explored in filamentous fungi, however the maximum gene size sufficiently transferred with the approach thus far is only approximately 20 kb. In this study, we transferred 63 kb pairs of DNA encoding a secondary metabolite-biosynthetic genes into the chromosome of Aspergillus oryzae, a filamentous fungus, using a single-step transformation approach based on multiple homologous recombination events. This study expands the potential of using A. oryzae as a host for efficient heterologous metabolite production. The results serve as a useful reference, providing insights, such as the DNA fragment number and assembled cluster length in host cells, for the cases where heterologous production of a secondary metabolite proves desirable in filamentous fungi. KEY POINTS: • One-step transfer of 24 DNA fragments to A. oryzae and the in vivo ordered assembly. • The assembled 63 kb DNA region enabled heterologous secondary metabolite production. • The transfer method adopted in this study may be applied to other filamentous fungi.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147833089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of a novel bifunctional endo-β-1,4-glucanase/mannanase from Aspergillus oryzae.","authors":"Akira Watanabe, Tomohiko Matsuzawa, Seiryu Ujiie, Takahiro Shintani, Katsuya Gomi","doi":"10.1007/s00253-026-13838-6","DOIUrl":"https://doi.org/10.1007/s00253-026-13838-6","url":null,"abstract":"<p><p>The present study involved a global search of the Aspergillus oryzae genome database. As a result, the gene (gene ID: AO090011000715) encoding a putative endo-β-1,4-glucanase of the glycoside hydrolase family 5 possessing a carbohydrate-binding module was identified. Overexpression of this gene in A. oryzae, followed by the purification of the gene product, revealed that the protein exhibits endoglucanase activity with carboxymethyl cellulose as a substrate. Notably, further enzymatic characterization showed that this enzyme could also hydrolyze glucomannan and galactomannan with relatively lower activity than that of endoglucanase, indicating that the enzyme is likely a bifunctional endoglucanase/mannanase. Detailed analyses of the substrate specificity revealed that the enzyme could hydrolyze cello-oligosaccharides larger than cellotetraose, whereas manno-oligosaccharides could not be hydrolyzed unless they were larger than mannopentaose. Therefore, this enzyme has different modes of action toward cello- and manno-oligosaccharides. To the best of our knowledge, this is the first study to purify and characterize a bifunctional endo-β-1,4-glucanase/mannanase from the Aspergillus genus. The ability of the enzyme to degrade both mannan and cellulose could be used for more efficient degradation of lignocellulosic biomass, indicating that the enzyme shows promise for applications in biomass utilization and the food industry. KEY POINTS: • A novel bifunctional endoglucanase/mannanase, EgmA, is identified from Aspergillus oryzae. • EgmA is the second bifunctional endo-β-1,4-glucanase/mannanase of fungal origin. • EgmA has different mechanisms of action toward cello- and manno-oligosaccharides.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147833072","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Motility and growth Burkholderia two-component: a new regulatory system of Burkholderia seminalis.","authors":"Juliana Cardinali-Rezende, Carolina A P T Silva, Alessandra Costa, Leandro M Garrido, Milene Ferro, Mauricio Bacci, Ana Olivia De Souza, Welington Luiz Araújo","doi":"10.1007/s00253-026-13775-4","DOIUrl":"https://doi.org/10.1007/s00253-026-13775-4","url":null,"abstract":"<p><p>The two-component regulatory system (TCS) is a highly conserved global signal transduction mechanism that enables microorganisms to adapt to changing environments in response to signals. Burkholderia seminalis TC3.4.2R3 has attracted attention as a potential phytopathogenic biological control agent. In this study, a TCS mutant was obtained and evaluated in comparison to wild type using different approaches. The transcriptome analysis revealed 374 differentially expressed genes, some of which are involved in motility and chemotaxis (downregulated) and related to iron uptake and pyochelin production (upregulated). Swarming and swimming motility were impaired in the mutant, while biofilm production increased, leading to differences in growth profiles. However, the biocontrol efficacy was similar. These findings suggest that the new TCS, named motility and growth Burkholderia two-component regulator (MgbTC), positively regulates motility and chemotaxis genes while negatively controlling genes related to biofilm production, iron uptake and pyochelin siderophore production in the Burkholderia seminalis TC3.4.2R3. KEY POINTS: • The new motility and growth of Burkholderia two-component regulator (MgbTC) system • MgbTC positively regulates motility and chemotaxis genes in B. seminalis TC3.4.2R3 • MgbTC negatively controls iron uptake and pyochelin and biofilm production genes.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147833111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacterial lipoxygenases for fatty acid oxyfunctionalization: advances and future directions.","authors":"Ruth Chrisnasari, Tom A Ewing, Willem J H van Berkel, Jean-Paul Vincken, Marie Hennebelle","doi":"10.1007/s00253-026-13837-7","DOIUrl":"https://doi.org/10.1007/s00253-026-13837-7","url":null,"abstract":"<p><p>The shift to a biobased economy aims to reduce dependence on fossil fuels by converting biological materials, such as plant-derived and algae-derived fatty acids, into valuable compounds. The enzymatic oxyfunctionalization of fatty acids is of special interest because it enables the eco-friendly synthesis of a wide range of products with unique properties. Lipoxygenases (LOXs) are key oxyfunctionalization enzymes featuring broad substrate specificities and distinct regioselectivities. Among them, bacterial LOXs remain underexplored compared to their eukaryotic counterparts, representing an untapped resource with unique structural and functional traits. This review highlights recent advances in bacterial LOXs, emphasizing their versatile oxidative capabilities via dioxygenase and hydroperoxide isomerase activities. Key strategies, including protein engineering and oxygen level modulation, are discussed for modifying substrate specificity, regioselectivity, and preferred catalytic pathway. Multi-enzyme cascade reactions further expand the scope of LOX-derived products, unlocking new applications in biotechnological contexts. We address challenges in harnessing bacterial LOXs' biocatalytic potential and propose solutions to optimize their performance. This work enhances understanding of bacterial LOXs and highlights their potential for transformative biotechnological applications in fatty acid derivatization.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147833117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Real-time NMR monitoring of HMF biotransformation by Ectopseudomonas oleovorans CECT 5344 R1D.","authors":"Mar Gómez-Ortega, Sara Diego, Felipe Morales-Durán, Faustino Merchán, Rafael Blasco, Ana G Neo, Carlos F Marcos","doi":"10.1007/s00253-026-13853-7","DOIUrl":"https://doi.org/10.1007/s00253-026-13853-7","url":null,"abstract":"<p><p>This research identifies Ectopseudomonas oleovorans CECT 5344 R1D as a novel and highly efficient biocatalyst for the sustainable production of 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). By employing benchtop Nuclear Magnetic Resonance (NMR) spectroscopy with a recirculating flow system, we achieved real-time monitoring and a detailed stoichiometric characterisation of the 5-hydroxymethylfurfural (HMF) detoxification pathway. This work contributes to the specialised field of real-time metabolic tracking of living biocatalysts, a niche where in situ NMR data is still scarce compared to end-point analytical methods. Our findings demonstrate that this strain, while not utilising HMF for biomass growth, maintains exceptional metabolic stability and quantitative molar yields over 125 h of operation, requiring significantly lower cell densities than other reported Pseudomonas strains. The in-line NMR setup enabled the definitive identification of 2,5-bis(hydroxymethyl)furan (BHMF) as a transient intermediate, clarifying previous metabolic misidentifications and revealing a highly specific, non-consuming \"cell factory\" behaviour. Furthermore, the system allowed for the assessment of bacterial tolerance thresholds and the exploration of the enzymatic substrate scope against other environmentally relevant aldehydes. This work underscores the synergy between a robust, living biocatalyst and a precise monitoring platform, providing an optimised workflow for bioprocess development in sustainable aldehyde valorisation. KEY POINTS: • Real-time monitoring of a microbial process via in-line benchtop NMR. • Versatile biodetoxification of various natural and synthetic aldehydes. • Green chemistry alternative to petrochemicals using renewable feedstocks.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147833065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}