New biotechnology最新文献

{"title":"Characterization of an aromatic O-prenyltransferase from the edible mushroom Sparassis crispa","authors":"Pimvisuth Chunkrua,&nbsp;Martijn Hazendonk,&nbsp;Peicheng Sun,&nbsp;Janniek H. Ritsema,&nbsp;Lucia Corrà,&nbsp;Jean-Paul Vincken,&nbsp;Willem J.H. van Berkel,&nbsp;Mirjam A. Kabel,&nbsp;Wouter J.C. de Bruijn","doi":"10.1016/j.nbt.2026.01.015","DOIUrl":"10.1016/j.nbt.2026.01.015","url":null,"abstract":"<div><div>Prenylated plant phenolics are a large group of secondary metabolites known for their bioactivity that is beneficial for plant and human alike, for example as antimicrobial agents against pathogenic microbes. However, the limited availability of prenylated phenolics, especially <em>O</em>-prenylated phenolics, and the complexity of the plant metabolite mixtures hinder bioactivity studies and further application. To explore approaches for more efficient production of prenylated phenolics, we produced and characterized a novel prenyltransferase (ScPT_MY) from the edible mushroom <em>Sparassis crispa</em>. ScPT_MY belongs to the dimethylallyl tryptophan synthase (DMATS) family and was found to primarily catalyze <em>O</em>-prenylation of structurally diverse phenolics. The best substrates included <span>l</span>-tryptophan, <span>l</span>-tyrosine, stilbenes, and isoflavonoids. The ScPT_MY reactions predominantly yielded a single mono-<em>O</em>-prenylated product with an exception of the isoflavonoids, for which more products were obtained, including di-prenylated ones. Notably, we demonstrated the potential of this <em>O</em>-prenylating DMATS to enhance bioactivity by showing that enzymatic <em>O</em>4′-prenylation conferred antimicrobial activity to resveratrol, a compound with otherwise very poor antimicrobial activity. Finally, our phylogenetic analysis suggested the possibility of combining evolutionary relationships with structural insights to predict substrate scope and regioselectivity, while also revealing seven largely unexplored fungal DMATS clades that may harbor novel functions for biotechnological applications.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"93 ","pages":"Pages 21-32"},"PeriodicalIF":4.9,"publicationDate":"2026-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146119631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combining metabolic engineering and fermentation optimization to achieve cost-effective oil production by Cutaneotrichosporon oleaginosus","authors":"Zeynep Efsun Duman-Özdamar ,&nbsp;Rudolf Marcel Veloo ,&nbsp;Elena Tsepani ,&nbsp;Mattijs K. Julsing ,&nbsp;Vitor A.P. Martins dos Santos ,&nbsp;Jeroen Hugenholtz ,&nbsp;Maria Suarez-Diez","doi":"10.1016/j.nbt.2026.01.017","DOIUrl":"10.1016/j.nbt.2026.01.017","url":null,"abstract":"<div><div>Microbial oils, produced by oleaginous microorganisms, offer a sustainable alternative to plant-derived oils. Among these microorganisms, <em>Cutaneotrichosporon oleaginosus</em> is a promising microbial cell factory for sustainable oil production due to its capacity to accumulate lipids with a similar composition to palm oil. Although <em>C. oleaginosus</em> can reach higher lipid contents than other oleaginous yeasts, enhanced productivity is still necessary to have a feasible microbial oil production process via <em>C. oleaginosus</em>. In this study, we followed a combinatorial approach for strain design and bioprocess development to improve the lipid content and lipid yield. Initially, we deployed a full factorial design with genetic factors (ATP-citrate lyase (<em>ACL</em>), acetyl-CoA carboxylase (<em>ACC</em>), threonine synthase (<em>TS</em>) and carbon-to-nitrogen ratio (C/N) in the medium. The C/N ratio appeared to have the most impact on oil accumulation. Combined with genetic modifications, lipid content and lipid yield increased by 1.6-fold. In a two-stage fermentation approach at a 2 L scale, the triple transformant overexpressing <em>ACL</em>, <em>ACC</em>, and <em>TS</em> outperformed the wild-type by achieving a lipid content of 75.4 % (w/w) with lipid productivity of 0.40 g L⁻¹ h⁻¹ and around 0.30 g lipids/g glycerol. In all, we established a cultivation strategy and strain that reached almost the theoretical maximum yield, and highest lipid content reported for a medium containing glycerol as a carbon source. These results strengthen the basis of using <em>C. oleaginous</em> as a platform for microbial oil production, thereby facilitating the development of processes substituting palm oil with a sustainable alternative.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"93 ","pages":"Pages 1-8"},"PeriodicalIF":4.9,"publicationDate":"2026-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146135690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterising complex metabolic responses in an engineered, cross-feeding microbial co-culture using quantitative proteomics","authors":"Mengxun Shi ,&nbsp;Josie McQuillan ,&nbsp;Caroline Evans ,&nbsp;Yanmeng Liu ,&nbsp;Xiaoxia Nina Lin ,&nbsp;Brett Barney ,&nbsp;Jagroop Pandhal","doi":"10.1016/j.nbt.2026.02.001","DOIUrl":"10.1016/j.nbt.2026.02.001","url":null,"abstract":"<div><div>Microbial communities play a key role in biogeochemical transformations in a wide range of ecosystems, but they also hold significant potential to enhance the bioproduction of desired chemicals. Although designing synthetic microbial consortia has generated a lot of interest, a more in-depth understanding of the interactions between strains is required, particularly when strains are engineered to cross-feed, but are not isolated from related environments. Challenges include enhancing stability, productivity and controllability. Here, we used a synthetic microbial co-culture consisting of engineered strains of the photosynthetic cyanobacterium <em>Synechococcus elongatus</em> PCC 7942 cscB/SPS and nitrogen-fixing bacterium <em>Azotobacter vinelandii</em> AV3. Each relies on the other for conversion of atmospheric carbon (CO₂) and nitrogen (N₂) into organic forms, <em>i.e.</em> sucrose and ammonia, respectively, resources which can be shared. As both strains have such contrasting growth dynamics in co-culture compared to monoculture, we applied a label-free quantitative proteomics approach to characterise metabolism in both strains. The proteomes of both shifted when in co-culture to reflect adaptive restructuring of carbon and nitrogen metabolism, although <em>A. vinelandii</em> appeared to transition to a more stressed state, inducing proteins linked to polymer biosynthesis. An analysis of the co-culture over 16 days led to phenotypic changes, including cell structure alterations in <em>A. vinelandii</em> AV3 over time, with the proteome suggesting cell envelope remodelling and potentially encystment. These findings suggest that physiological control of parameters, such as oxygen and nutrient availability, may enable cultivation of more stable co-cultures.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"93 ","pages":"Pages 9-20"},"PeriodicalIF":4.9,"publicationDate":"2026-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146132493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in microbial strain selection and carbon source valorization for Polyhydroxyalkanoates (PHA) production","authors":"William Mawuko Siegu ,&nbsp;Piotr Drożdżyński ,&nbsp;Vignesh Kumaravel ,&nbsp;Olga Marchut-Mikołajczyk","doi":"10.1016/j.nbt.2026.01.016","DOIUrl":"10.1016/j.nbt.2026.01.016","url":null,"abstract":"<div><div>Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by various microorganisms as intracellular carbon and energy reserves. Their potential to replace petroleum-based plastics has made them central to sustainable materials research. However, their large-scale commercialization is hindered by high production costs, prompting efforts to improve yield and identify low-cost, non-food carbon sources. This review examines PHA biosynthesis optimization via advances in microbial strain-substrate selection ensuring economic feasibility. Focus is placed on three key bacterial genera, <em>Cupriavidus</em>, <em>Pseudomonas</em>, and <em>Escherichia coli</em> analyzing their metabolic flexibility, suitable substrate range and PHA content (g product/ g substrate). <em>Escherichia coli</em> species and <em>Cupriavidus necator</em> demonstrate high polymer contents from largely simple sugars and fatty-acid substrates respectively, while <em>Pseudomonas</em> species offer a broad substrate adaptability, particularly waste streams. The environmental impact assessment of some <em>Cupriavidus necator</em> and <em>Pseudomonas</em> strains highlight low carbon footprint from waste lipids compared to PHA production from glucose or bottle-grade PET, however these results are contingent to specific scenarios without a reproducible and quantifiable GWP values across these genera. Integrating microbial engineering of agro-industrial and organic wastes in adherence to regulatory frameworks around waste valorization and optimization, genus-specific LCA bioprocessing approach offers a path toward economically viable and environmentally sustainable PHA production.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"93 ","pages":"Pages 44-53"},"PeriodicalIF":4.9,"publicationDate":"2026-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146132415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Beyond CAT, BSD, and URA: SUL, A novel selectable marker for Cyanidioschyzon merolae","authors":"Yissell Borges-Rodríguez ,&nbsp;Martha R. Stark ,&nbsp;Elise Kerckhofs ,&nbsp;Julie Mueller ,&nbsp;Kyle J. Lauersen ,&nbsp;Daniel Schubert ,&nbsp;Stephen D. Rader","doi":"10.1016/j.nbt.2026.01.012","DOIUrl":"10.1016/j.nbt.2026.01.012","url":null,"abstract":"<div><div>The unicellular red alga <em>Cyanidioschyzon merolae</em> is a valuable model organism for studying pre-mRNA splicing, stress adaptation, and biotechnological applications. However, the limited availability of selectable markers has constrained its potential in genetic engineering. In this study, we evaluated the <em>sul1</em> gene, which encodes a sulfadiazine-resistant variant of dihydropteroate synthase, as a new selectable marker (SUL) for <em>C. merolae</em> transformation. SUL has previously been used for this purpose in plants and green algae. We analyzed the sensitivity of <em>C. merolae</em> to sulfadiazine and determined the concentration that effectively inhibited cell growth. To test the effectiveness of SUL as a selectable marker<strong>,</strong> we designed a transformation construct containing SUL directed to the algal mitochondria through a native targeting peptide, along with mVenus to visualize transformation. We integrated the construct into a neutral genomic locus via homologous recombination. Fluorescence microscopy confirmed stable mVenus expression, and sulfadiazine selection successfully enriched transformed cells. As a demonstration of the utility of this marker, we rescued the large-cell phenotype of a cell division cycle-like kinase 2 (CmClk2) mutant by replacing the CAT-marked kinase domain deletion with the SUL-marked native kinase domain, thereby restoring CmClk2 function and recycling the CAT marker. The deletion phenotype provides evidence for a conserved cell-cycle regulatory role for CmClk2 in <em>C. merolae</em>. Beyond establishing SUL as an effective selectable marker, this highlights how SUL facilitates functional genetic studies of essential cellular regulators.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"93 ","pages":"Pages 33-43"},"PeriodicalIF":4.9,"publicationDate":"2026-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146125760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The GigaAssay: A combined high throughput and highly accurate platform for variant classification","authors":"Christopher J. Giacoletto ,&nbsp;Liz J. Valente ,&nbsp;Lancer Brown ,&nbsp;Sara Patterson ,&nbsp;Rewatee Gokhale ,&nbsp;Susan M. Mockus ,&nbsp;Wayne W. Grody ,&nbsp;Hong-Wen Deng ,&nbsp;Jerome I. Rotter ,&nbsp;Martin R. Schiller","doi":"10.1016/j.nbt.2025.12.001","DOIUrl":"10.1016/j.nbt.2025.12.001","url":null,"abstract":"<div><div><em>ERBB2</em> (HER2) is a critical oncogene, yet the comprehensive and standardized assessment of its variant effects, particularly concerning receptor tyrosine kinase function, remains a major challenge. To compare the oncogenic potential of benchmark variants, we utilized the GigaAssay, a modular high-throughput one-pot assay system capable of simultaneously measuring their molecular function. We designed the GigaAssay to evaluate the relative oncogenic potential of HER2 variants by measuring HER2 phosphorylation at Y1248 (pHER2) in a ligand-free system. Using a benchmark dataset, the HER2 GigaAssay demonstrated robust performance, achieving 100 % accuracy, sensitivity, and F1 score in classifying gain-of-function (GOF) variants linked to hyperphosphorylation and wild type (WT) variants. Furthermore, the normalization of GOF HER2 variants revealed a gradient of elevated phosphorylation activity. Three previously reported oncogenic mutants (Q679L, L726F, and H878Y) were identified as non-pY1248 GOF mutations, aligning with literature and independent flow cytometry measurements, and highlighting the complexity of HER2 signaling. This study establishes the GigaAssay as a highly accurate and standardized platform for the precision classification of HER2 variant function.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"92 ","pages":"Pages 79-88"},"PeriodicalIF":4.9,"publicationDate":"2026-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145834328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Locus-specific quantification reveals dynamics of ribosomal ac4C modifications","authors":"Ningning Qin,&nbsp;Yin Wang,&nbsp;Qiaoling Yuan,&nbsp;Li Chen,&nbsp;Guo-Sheng Qu","doi":"10.1016/j.nbt.2026.01.003","DOIUrl":"10.1016/j.nbt.2026.01.003","url":null,"abstract":"<div><div>Eukaryotic ribosomes carry two evolutionarily conserved N4-acetylcytidine (ac4C) modifications within critical functional regions. However, fundamental questions remain regarding their deposition kinetics, potential dynamics, and functional interplay with other rRNA modifications. Here, using ac4C-Sanger, we quantitatively profiled ribosomal ac4C modifications across various rRNA precursors and in mature rRNA under varied conditions in <em>Saccharomyces cerevisiae</em>. We show that ac4C accumulates progressively during 18S rRNA maturation, with distinct kinetics at each site. Moreover, ribosomal ac4C levels are dynamic and respond to growth phase and thermal stress in a site-specific manner. Changes in modifications under heat stress are accompanied by alterations in translation fidelity and efficiency, although evidence is still lacking to establish a causal relationship. Genetic perturbation of the box C/D snoRNA snR40, which guides 2′-O-methylation Gm1271, revealed a differential effect on both ac4C sites, despite the spatial proximity of Gm1271 to only one of them. Beyond ac4C-Sanger, we also demonstrate that High-Resolution Melting (HRM) and SELECT (a single-base elongation- and ligation-based qPCR amplification method) can serve as orthogonal approaches to effectively detect and quantitatively measure ac4C at known sites. Collectively, our work establishes ribosomal ac4C as a dynamic epitranscriptomic mark, suggests intricate interdependencies governing its biogenesis, and provides accessible tools for its investigation, thereby underscoring the integrated and adaptive nature of the ribosomal modification landscape.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"92 ","pages":"Pages 62-73"},"PeriodicalIF":4.9,"publicationDate":"2026-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145934410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microalgal cultivation on ricotta cheese whey: Modulating nutrient supply during cultivation to improve culture growth","authors":"Paolo Capano,&nbsp;Gaia Santini,&nbsp;Natascia Biondi,&nbsp;Giacomo Sampietro,&nbsp;Liliana Rodolfi","doi":"10.1016/j.nbt.2026.01.007","DOIUrl":"10.1016/j.nbt.2026.01.007","url":null,"abstract":"<div><div>Dairy industry generates significant effluent streams, comprising wastewaters and nutrient-rich by-products such as cheese whey, which pose environmental challenges for disposal, but also offer valorisation opportunities. Microalgae cultivation on whey represents a sustainable biotechnological approach that couples biomass production with effluent treatment. This study aimed at optimizing microalgal cultivation using ricotta cheese whey as substrate. Eighteen microalgal strains, mainly belonging to <em>Scenedesmus</em> and <em>Chlorella</em>, were initially screened for their ability to grow on whey. Selected strains were further tested in 300-mL photobioreactors under different management options: Low Whey Load (LWL), High Whey Load (HWL), Salinity and Micronutrient (S&amp;M), and Moderate Whey Load (MWL) trials. Results showed that most of the tested strains grew better when cultivated on diluted whey than on standard synthetic media, with increases from 56 to 590 % during the first days, and with high removal efficiencies. Several issues were identified during the trials, including nutrient shortage, salinity increase, micronutrients deficiency and potential inhibitory effects of whey compounds, which have hindered long term cultivation of microalgae on whey. In MWL trial, optimization of whey concentration (12 % v/v), replenishment strategy (30 % of the culture volume every 3-days), and micronutrient supplementation overcame these issues extending the cultivation period and improving biomass yield (203.6 billion cells L⁻¹ whey⁻¹ with the best strain). Overall, this work underscores the importance of carefully optimize microalgal cultivation management to maximise exploitation of dairy by-products and minimize growth-limiting effects of whey accumulation, thus offering a promising starting point for large-scale applications of whey in microalgae cultivation.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"92 ","pages":"Pages 25-39"},"PeriodicalIF":4.9,"publicationDate":"2026-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145959356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Zero-shot deep learning with multi-objective optimization improves thermostability of zearalenone hydrolase and xylanase","authors":"Fan Wu ,&nbsp;Rui Wu ,&nbsp;Linghui Chen ,&nbsp;Quan Chen ,&nbsp;Haiyan Liu","doi":"10.1016/j.nbt.2026.01.009","DOIUrl":"10.1016/j.nbt.2026.01.009","url":null,"abstract":"<div><div>Enhancing enzyme thermostability is crucial for industrial applications requiring robust performance under extreme conditions. Structure-based protein design models excel at improving thermal stability but often compromise enzymatic activity, while sequence-based models better preserve activity but struggle to enhance thermostability. It is challenging to efficiently generate multi-site mutants with both improved thermostability and intact activity using minimal experimental effort. Here, we used zearalenone hydrolase (RmZHD) and xylanase as model systems to evaluate different strategies for multi-site mutation design: (i) structure-based design with the ABACUS-R model, (ii) sequence-based design with the ProGen2 or MSA Transformer, (iii) integrated approaches combining either ProGen2 or MSA Transformer with ABACUS-R via Markov Chain Monte Carlo sampling with multi-objective scoring. Results showed that designing with ABACUS-R increased thermal stability by ∼15°C but caused 80 to 100 % activity loss. Sequence-based designs retained ∼19 % wild-type activity but failed to improve thermostability. Notably, zero-shot designs from integrating ABACUS-R with MSA Transformer achieved significant thermostability gains (<em>ΔT</em>_<em>m</em> ∼8°C) while preserving &gt; 95 % wild-type activity. This highlights the potential of combining sequence-and structure-based deep learning models for developing industrially relevant thermostable enzymes.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"92 ","pages":"Pages 51-61"},"PeriodicalIF":4.9,"publicationDate":"2026-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146019193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ability of a microfluidic droplets system to assess the dynamics of Green Fluorescence Protein (GFP) production by Escherichia coli for modelling and bioprocess purposes","authors":"Hassan El Itawi ,&nbsp;Aya Zoghlami,&nbsp;Madeleine Charbonnier,&nbsp;Yusmel González-Hernández,&nbsp;Firas Ghali,&nbsp;Rafik Balti,&nbsp;Patrick Perré","doi":"10.1016/j.nbt.2026.01.006","DOIUrl":"10.1016/j.nbt.2026.01.006","url":null,"abstract":"<div><div>Understanding microbial growth and metabolism under controlled environments is critical for both fundamental research and bioprocess development. In this study, we present a cost-effective droplet-based microfluidic device enabling high-throughput screening of GFP expression in <em>Escherichia coli</em> (<em>E. coli</em>) under varying glucose concentrations. Fluorinated Ethylene Propylene (FEP) tubing was selected for its low cost and compatibility with stable water-in-oil emulsification, facilitating robust droplet generation. The system achieved a 33333-fold reduction in media consumption compared to traditional Erlenmeyer flask cultures. Growth kinetics and GFP expression were assessed in both Erlenmeyer flasks and microdroplets, showing high qualitative correlation between platforms. Low glucose levels (5–10 g/L) supported rapid initial growth and early GFP production, followed by a fluorescence decline due to nutrient depletion. In contrast, higher glucose concentrations (25–50 g/L) prolonged the exponential phase and enhanced GFP production per unit biomass, though growth was slowed by overflow metabolism. In microdroplets, delayed GFP expression at 25 and 50 g/L were observed, and parallel bioreactor experiments confirmed that this delay is caused by oxygen limitation at high glucose concentrations. Importantly, the microfluidic device enables controlled variation of oxygen availability simply by adjusting droplet size or generation frequency, providing a powerful means to probe oxygen-sensitive metabolic behaviors. These results validate the microfluidic platform’s ability to mimic Erlenmeyer flask-scale dynamics, while uniquely allowing precise modulation of oxygen transfer conditions at the microscale<strong>.</strong> The system offers a reliable, miniaturized alternative for optimizing microbial bioprocesses with drastically reduced reagent use and increased experimental throughput.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"92 ","pages":"Pages 12-24"},"PeriodicalIF":4.9,"publicationDate":"2026-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145985253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}