Microbial Cell Factories最新文献

{"title":"Development of a novel hydroxylated recombinant human type III collagen TD-1-HrHC in Komagataella phaffii with transdermal and skin damage repair activity.","authors":"Yangyang Li, Yehai Wang, Jingjing Yue, Yan Fan, Shimei Qi, Haixia Yu, Funa Zhang, Jing Wu, Weihua Xiao","doi":"10.1186/s12934-026-03039-1","DOIUrl":"10.1186/s12934-026-03039-1","url":null,"abstract":"<p><strong>Background: </strong>Collagen has been proven to have significant potential applications value in the fields of on medical aesthetics and cosmetics. However, due to its large molecular weight, collagen is difficult to effectively penetrate the skin barrier and reach the designated location to exert the expected activities. Transdermal peptide TD-1 is a short peptide consisting of only 11 amino acids, which can assist proteins such as insulin and cytokines to penetrate the skin barrier and reach the dermis layer. To maximize the transdermal and the efficacy of Collagen III in skincare, the shortest functional fragment that containing a triple helix structure of collagen protein fused with TD-1 were designed and evaluated for its transdermal activity, safety and efficacy.</p><p><strong>Results: </strong>The recombinant protein containing transdermal TD-1 fragment and a core active fragment of human collagen III (TD-1-HrHC) was co-expressed together with specific hydroxylase using the K. phaffii expression system, the products were purified by hydrophobic interaction chromatography (HIC) and ion-exchange chromatography (IEC). The TD-1-HrHC contains about 11.89% hydroxylation modifications that are similar to the natural human collagen III. The transdermal efficiency is 2077% measured by trans-well tests compare to the nature Collagen III isolated from animals. In addition, TD-1-HrHC able to promotes skin damage repairing through regulation series of barrier factors and differentiation factors in the HaCat cell model and promotes the repair of damaged skin and induces the generation of new-born collagen in mouse model. Moreover, it also shown an excellent ability to inhibit protein carbonylation on HaCat cells.</p><p><strong>Conclusion: </strong>This study demonstrates that TD-1-HrHC can effectively cross the skin barrier and has profound ability to inhibit protein carbonylation of skin and promote skin damage repairing.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13491638/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148226254","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel utilization of the seaweed-based deoxy sugars rhamnose and fucose by engineered Corynebacterium glutamicum.","authors":"Ida Marie Stephansen, Luciana Fernandes Brito, Oliver Saur Auran, Synne Saether Lande, Fernando Pérez-García","doi":"10.1186/s12934-026-03035-5","DOIUrl":"10.1186/s12934-026-03035-5","url":null,"abstract":"<p><p>Seaweed represents a potential sustainable carbon source for industrial biotechnology, yet the workhorse bacterium Corynebacterium glutamicum cannot naturally metabolize the deoxy sugars L-rhamnose and L-fucose, abundant in green and brown seaweed, respectively. Expanding its substrate range is crucial for sustainable bioprocessing, by enabling utilization of the available biomass. In this study, we engineered C. glutamicum to utilize L-rhamnose and L-fucose by introducing the Escherichia coli operons rhaBADM and fucIKUA, enabling growth on these sugars as sole carbon sources. To enhance growth, we evaluated various transport systems and identified the non-native fucose permease (FucP) as the most efficient for L-rhamnose uptake, and the native myo-inositol transporter 2 (IolT2) as optimal for L-fucose uptake. During L-rhamnose and L-fucose utilization, L-lactaldehyde is formed as a byproduct. We demonstrate that the native acetaldehyde dehydrogenase encoded by ald also exhibits lactaldehyde dehydrogenase activity, and that its overexpression enhances L-lactaldehyde utilization. Finally, cultivation on green and brown seaweed hydrolysates enabled the engineered strains to achieve increased biomass formation through consumption of the targeted deoxy sugars. This study expands the substrate spectrum of C. glutamicum through pathway engineering, transport optimization, and functional identification of a native lactaldehyde dehydrogenase. Growth and L-lysine production on seaweed hydrolysates highlights the potential of this approach for sustainable marine biomass valorization and bioproduction of value-added compounds.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13479540/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148226359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering Komagataella phaffii for secreted and surface-displayed production of SARS-CoV-2 receptor-binding domain and its immunogenicity evaluation.","authors":"Kostyantyn Dmytruk, Olena Dmytruk, Marta Semkiv, Roksolana Vasylyshyn, Lyubov Fayura, Lidia Gaffke, Magdalena Podlacha, Zuzanna Cyske, Karolina Pierzynowska, Patrick Budylowski, Mario Ostrowski, Grzegorz Wegrzyn, Andriy Sibirny","doi":"10.1186/s12934-026-03043-5","DOIUrl":"10.1186/s12934-026-03043-5","url":null,"abstract":"<p><strong>Background: </strong>The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein represents a key antigen for vaccine development due to its critical role in the ACE2 receptor recognition. Yeast-based expression systems, particularly Komagataella phaffii, offer scalable and cost-effective platforms for recombinant protein production. In addition to secretion, cell surface display provides an alternative strategy enabling direct antigen delivery, including applications via mucosal routes.</p><p><strong>Results: </strong>In this study, we engineered K. phaffii strains for the production of SARS-CoV-2 RBD in two formats: as a secreted recombinant protein and as a cell surface-displayed antigen using a Sag1 anchoring system. Incorporation of glycine-serine linkers enhanced secretion efficiency, yielding up to 50 mg/L of RBD, with minimal intracellular retention. Western blot analysis indicated the presence of glycosylated forms of RBD, and the recombinant protein was subsequently purified for further characterization. Surface localization of RBD was validated by immunofluorescence microscopy and quantitative fluorescence measurements. Immunogenicity studies in mice demonstrated that intraperitoneal administration of purified RBD elicited a strong humoral immune response. Importantly, sera from immunized animals efficiently inhibited spike protein binding to the Ace2 receptor, indicating potent neutralizing activity. Comparable results were obtained following oral administration of K. phaffii cells displaying RBD on their surface, demonstrating the feasibility of a whole-cell yeast-based vaccine approach.</p><p><strong>Conclusions: </strong>A yeast-based system enabling both secretion and surface display of SARS-CoV-2 RBD has been developed. Both delivery strategies, intraperitoneal administration of the purified protein and oral administration of recombinant yeast cells, induced robust and functionally relevant immune responses in mice. These findings highlight the potential of K. phaffii as a versatile system for the development of cost-effective subunit and oral vaccine candidates.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13491813/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148226204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering primary metabolism for sustainable production of isoprene in Synechocystis sp. PCC 6803.","authors":"Paul Bolay, Kim N Janssen, Stefan Timm, Martin Hagemann, Pia Lindberg","doi":"10.1186/s12934-026-03040-8","DOIUrl":"10.1186/s12934-026-03040-8","url":null,"abstract":"<p><p>The imminent climate crisis calls for the development of truly sustainable alternatives in virtually every aspect of human life. The photosynthetic production of chemicals like isoprene, a critical precursor for wide ranging applications, presents a promising alternative to conventional petroleum-based processes. While cyanobacteria can offer the ability for direct CO₂ to isoprene conversion, highly efficient strains are required to reduce costs and resource usage. In this study, we enhanced isoprene productivity in the model cyanobacterium Synechocystis sp. PCC 6803 by engineering the primary metabolism of an isoprene producing strain. We inactivated synthesis of the carbon and energy storage polymer polyhydroxybutyrate (PHB) and increased isoprene synthase copy number in a previously published deletion mutant of PirC (sll0994), a recently discovered regulator involved in the fate of newly fixed carbon during limited nutrient supply. The engineered strains achieved an isoprene production of 278 µg*L^- 1*OD^- 1*h^- 1 and showed elevated productivity under fluctuating nitrogen supply, ultimately resulting in significantly enhanced nitrogen use efficiency for isoprene production. As nitrate represents the ingredient with the highest cost contribution and climate impact in conventionally used cyanobacterial growth media, the presented strategy could reduce cost and environmental footprint without impeding performance for a wide array of compounds sustainably produced with cyanobacteria.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13262354/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148176251","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Untapped microbial factories for next-generation therapeutics: Endophytic fungi of medicinal plants.","authors":"Abdullah M Nagib, Mahmoud H Sultan, Hussein H El-Sheikh, Amr H Hashem","doi":"10.1186/s12934-026-03036-4","DOIUrl":"10.1186/s12934-026-03036-4","url":null,"abstract":"<p><p>Endophytic fungi residing within medicinal plants represent a rich and sustainable source of bioactive secondary metabolites with diverse pharmacological applications. These symbiotic microorganisms establish mutualistic relationships with their host plants, contributing to enhanced stress tolerance, growth promotion, and defense against pathogens. In recent years, endophytic fungi have gained considerable attention as alternative biofactories for the production of valuable compounds such as alkaloids, terpenoids, phenolics, lignans, and polysaccharides. These metabolites exhibit a wide range of biological activities, including antimicrobial, antiviral, anticancer, antioxidant, anti-inflammatory, and antidiabetic effects. Advances in cultivation strategies, such as OSMAC, co-culture, and epigenetic modification, have significantly improved metabolite yield and diversity. Moreover, the integration of genomics, transcriptomics, and metabolomics has revolutionized the understanding of biosynthetic gene clusters and metabolic pathways, enabling the discovery of novel compounds and optimization of production processes. Despite these advances, challenges such as low yield, silent gene clusters, and difficulties in large-scale production remain significant barriers. Nevertheless, continued progress in multi-omics technologies, synthetic biology, and biotechnological tools holds great promise for unlocking the full potential of endophytic fungi. Herein, endophytic fungi represent a powerful and eco-friendly platform for the development of new therapeutic agents and sustainable pharmaceutical applications.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13262479/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148163826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acyl CoA reductases useful for bioproduction of hydrocarbons.","authors":"Masakazu Ito, Shigenobu Kishino, Masayoshi Muramatsu, Jun Ogawa","doi":"10.1186/s12934-026-03038-2","DOIUrl":"10.1186/s12934-026-03038-2","url":null,"abstract":"&lt;p&gt;&lt;strong&gt;Background: &lt;/strong&gt;Hydrocarbon-based biofuels-so-called drop-in fuels-have gained attention as sustainable alternatives to petroleum-derived fuels, yet their biological production remains limited by the availability of efficient enzymatic pathways for generating hydrocarbon precursors. Medium-chain alkanes produced by microorganisms represent a promising target, but the aldehyde-producing capabilities of acyl-CoA reductases (ACRs) from bacteria, plants, and animals have not been systematically compared. Because ACRs generate fatty aldehydes-key intermediates in hydrocarbon biosynthesis-understanding their diversity is essential for expanding biological fuel production strategies. In this study, we established a whole‑cell proxy screening framework in E. coli, a widely used microbial production host, to identify promising ACR candidates in this chassis and to provide new insights into ACR diversity and performance in a microbial production host that can help inform future development of microbial hydrocarbon bioproduction pathways.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;Sixteen acyl-CoA reductases (ACRs) from microorganisms, plants, and animals were cloned and expressed in Escherichia coli and screened using hydrocarbon formation as a practical proxy readout by coexpressing each enzyme with a cyanobacterial aldehyde decarbonylase. Several Arabidopsis thaliana ACRs produced higher alkane levels than microbial and animal enzymes. To further examine plant-derived enzymes, ACR homologs with high amino acid similarity to A. thaliana ACR1 and ACR2 were cloned from multiple plant species and tested. In the E. coli proxy‑screening context used here, some plant ACR homologs yield detectable medium‑chain hydrocarbon proxy readouts. Among these, introduction of ACR2 from Glycine max resulted in the highest alkane and alkene formation levels. Phylogenetic analysis of fourteen plant ACRs with considerable medium chain hydrocarbon proxy-readout showed that ACRs similar to GmACR2 generated higher levels of C13 alkanes, although no clear trend was observed for C15 alkane or C17 alkene. In an E. coli strain coexpressing GmACR2 and SpALDH, we detected a small C17 alkene (1‑heptadecene) signal above the empty‑vector background under the conditions tested, although enzyme‑specific attribution remains unresolved in the absence of single‑expression controls. This preliminary observation suggests that a C17 alkene signal can be detected in an ACR-ALDH coexpression background, suggesting a potentially broader space of ACR-ALDH combinations for future exploration in microbial hydrocarbon production.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion: &lt;/strong&gt;This study identifies multiple microbial and plant-derived ACRs, particularly GmACR2, as a strong candidate in this E. coli whole‑cell proxy screening assay for medium-chain hydrocarbon biosynthesis. Coexpression of GmACR2 with S. pombe aldehyde dehydrogenase provides an initial proof-of-concept indication that hydrocarbon formation can","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13483699/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148150627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in the heterologous biosynthesis of QS saponins: a review.","authors":"Yanjie Tian, Wanze Zhang, Min Tang, Mingzhang Wen, Jianjun Qiao, Hongji Zhu, Weiguo Li, Qinggele Caiyin","doi":"10.1186/s12934-026-03034-6","DOIUrl":"10.1186/s12934-026-03034-6","url":null,"abstract":"<p><p>QS saponins are a type of triterpenoid glycosides derived from the bark of Quillaja saponaria (Chilean soapbark tree). They are widely used in detergents, food and cosmetics, especially as vaccine adjuvants, due to their cleansing function and immunostimulatory activities. Among these compounds, QS-21 stands out as one of the most potent adjuvants in modern vaccinology due to its remarkable capacity to enhance antigen-specific immunogenicity and elicit robust immune responses. It shows potential for development in both human and veterinary vaccine research. With the rise of synthetic biology technology, the production of QS saponins using microbial cell factories has become a research hotspot. The review systematically summarizes the mechanism of action and practical applications of the most promising QS saponin QS-21, and outlines the biosynthesis pathways of QS saponins, with a focus on exploring the synthetic biology strategies employed for the microbial synthesis of QS saponins. In addition, targeted response strategies have been proposed for the current research challenges. This review aims to lay a theoretical foundation for promoting the medical utilization and sustainable production of QS saponins.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13393811/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148032618","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fungal exopolysaccharides as next-generation microbial biomaterials: molecular biosynthesis, structural architecture, and translational biomanufacturing strategies.","authors":"Hassan A H Ibrahim, Yasser H El-Halmouch, Dalia El Badan, Rana Diab, Sameh S Ali","doi":"10.1186/s12934-026-03002-0","DOIUrl":"10.1186/s12934-026-03002-0","url":null,"abstract":"<p><p>Fungal exopolysaccharides (EPSs) are increasingly recognized as structurally programmable microbial polymers with applications spanning biomedicine, materials engineering, food systems, and environmental technologies. While previous reviews have often addressed fungal EPS diversity, production variables, or application domains separately, an integrated framework linking biosynthesis, molecular architecture, process control, and translational manufacturing remains underdeveloped. This review positions fungal EPSs as next-generation biomaterials by integrating (i) biochemical and genetic regulation of EPS biosynthesis, (ii) structure-function mapping across major polymer classes, (iii) cultivation and downstream processing workflows that enable reproducible product specifications, and (iv) industrial translation pathways within scalable and sustainability-aligned biomanufacturing systems. Gene-cluster-resolved case studies and process-to-product design principles illustrate how metabolic flux, fermentation parameters, and polymer modification shape functional performance. Current bottlenecks-including strain-dependent variability, purification complexity, quality harmonization, and techno-economic constraints-are critically evaluated to distinguish laboratory potential from scalable feasibility. By shifting from descriptive cataloging toward platform-based engineering logic, this review provides a translational roadmap for rational fungal EPS design within standardized and application-driven manufacturing frameworks.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13202872/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148016473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Harnessing the thermotolerant methylotroph Bacillus methanolicus for methanol-based synthetic L-proline production.","authors":"Christine Frank, David Virant, Gregor Kosec, Tamara Hoffmann, Oskar Zelder, Max F Felle, Erhard Bremer","doi":"10.1186/s12934-026-03032-8","DOIUrl":"10.1186/s12934-026-03032-8","url":null,"abstract":"<p><strong>Background: </strong>Bacillus methanolicus MGA3 is a thermotolerant methylotroph that utilizes methanol, a renewable C₁ substrate, as its sole carbon and energy source. The strain naturally overproduces and secretes L-glutamate, making it a promising platform for engineering pathways toward L-glutamate-derived amino acids such as L-proline, which has applications in nutrition, stress protection, and industry.</p><p><strong>Results: </strong>Heterologous expression of an osmotic stress-responsive L-proline biosynthetic operon from the mesophile Bacillus licheniformis in the thermotolerant B. methanolicus strain MGA3 did not increase L-proline levels but instead led to accumulation of L-citrulline. This was likely due to heat sensitivity of pyrroline-5-carboxylate reductase (ProH), the last enzyme of the osmoregulatory L-proline biosynthetic route, and metabolic crosstalk between L-proline and L-arginine pathways operating in Bacilli. To overcome these limitations, a synthetic operon containing the native anabolic proBA and proI L-proline biosynthetic genes from B. methanolicus MGA3 was engineered to remove transcriptional T-box regulation and biochemical feedback inhibition of ProB enzyme activity. Expression of this engineered operon enhanced L-proline synthesis and triggered its secretion during methanol-based growth of B. methanolicus MGA3 at 50° C. In fed-batch fermentation with methanol as carbon and energy source, extracellular L-proline levels reached 262 ± 20 mg L⁻¹ after 40 h. During the fermentation process, a stepwise increase in medium osmolarity was observed, likely due to large-scale L-glutamate excretion, which impaired cellular growth.</p><p><strong>Conclusions: </strong>This study links osmolarity dynamics to methanol-based fermentation in B. methanolicus MGA3 and demonstrates its potential as a cell factory for L-proline and L-citrulline production. These findings support further strain optimization for producing value-added amino acids and highlight the relevance of methylotrophic thermophiles in sustainable biotechnology.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"25 1","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13198045/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148005990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of an improved chemically defined medium for high-efficiency expression of glucokinase in Escherichia coli.","authors":"Yuhan Ma, Jiahao Kong, Zongjin Wang, Sitong Chen, Xu Yang, Min Chen, Zhenguo Wen, JianYe Xia","doi":"10.1186/s12934-026-03027-5","DOIUrl":"10.1186/s12934-026-03027-5","url":null,"abstract":"<p><strong>Background: </strong>Recombinant glucokinase (GLK), an important enzyme for blood glucose diagnostics, is commonly prepared in Escherichia coli (E.coli )for laboratory research and related biosynthetic applications. In small-scale laboratory cultivation, complex media are often used because of their convenience and strong growth-supporting capacity, but their ill-defined composition and batch-to-batch variability can compromise reproducibility and increase medium-related costs. Although minimal defined medium (e.g., M9 medium) are generally preferred in scalable E. coli bioprocesses because of their compositional consistency and process controllability, their relatively simple nutrient composition may limit recombinant protein expression performance under laboratory-scale conditions. Therefore, developing an improved chemically defined medium represents a practical strategy to balance expression efficiency, compositional control, and medium cost for recombinant GLK production.</p><p><strong>Results: </strong>To address this, we developed a chemically defined medium including amino acids using high-throughput respiration activity monitoring system (RAMOS) and a high-throughput growth curve analysis system (Growth Profiler 960, GP960), alongside a machine learning-driven iterative optimization system (Machine-learning guided Experimental Trials for Improvement of Systems, METIS). The optimized medium enhanced soluble GLK expression by 33 fold compared to the pre-optimization medium, reaching 74% of the titer obtained in complex media, while reducing medium costs by 74%. Moreover, soluble protein expression per cell increased by 80% compared to the complex medium, lowering the medium usage cost per unit of bacterial protein by 85%. Validation in a 7.5 L bioreactor demonstrated that the optimized medium supported continuous growth to an optical density at 600 nm (OD₆₀₀) of 103, achieving 4-fold and 5.4-fold higher biomass than Luria-Bertina (LB) and M9 media, respectively, with soluble GLK titer reaching 920 mg L^- 1, which exceeded conventional media by more than 10-fold.</p><p><strong>Conclusion: </strong>This study tackles the challenge of suboptimal media in GLK fermentation, offering a cost-effective alternative and a transferable strategy for optimizing fermentation processes for other high-value enzymes.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13491839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148005908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}