Microbial Cell Factories最新文献

{"title":"A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host.","authors":"Zhe-Chong Wang, Hayden Stegall, Takeshi Miyazawa, Adrian T Keatinge-Clay","doi":"10.1186/s12934-025-02741-w","DOIUrl":"10.1186/s12934-025-02741-w","url":null,"abstract":"<p><strong>Background: </strong>Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.</p><p><strong>Results: </strong>We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.</p><p><strong>Conclusion: </strong>Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"125"},"PeriodicalIF":4.3,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12117839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144160253","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":"Further characterization and engineering of an 11-amino acid motif for enhancing recombinant soluble protein expression.","authors":"Jiawu Bi, Elaine Tiong, Ying Sin Koo, Weibiao Zhou, Fong Tian Wong","doi":"10.1186/s12934-025-02738-5","DOIUrl":"10.1186/s12934-025-02738-5","url":null,"abstract":"<p><strong>Background: </strong>Escherichia coli (E. coli) is a popular system for recombinant protein production, owing to its low cost and availability of genetic tools. However, the expression of soluble recombinant proteins remains an issue. As such, various solubility-enhancing and yield-improving methods such as the addition of fusion tags have been developed. This study focuses on a small solubility tag (NT11), derived from the N-terminal domain of a duplicated carbonic anhydrase from Dunaliella species. The small size of NT11 (< 10 kDa) lowers the chance of protein folding interference and post-translation removal requirement, which ultimately minimizes cost of production.</p><p><strong>Results: </strong>A comprehensive analysis was performed to improve the characteristics of the 11-amino acid tag. By investigating the alanine-scan library of NT11, we achieved at least a two-fold increase in protein yield for three different proteins and identified key residues for further development. We also demonstrated that the NT11 tag is not limited to the N-terminal position and can function at either the N- or C-terminal of the protein, providing flexibility in designing constructs. With these new insights, we have successfully doubled the recombinant soluble protein yields of valuable growth factors, such as fibroblast growth factor 2 (FGF2) and human epidermal growth factor (hEGF) in E. coli.</p><p><strong>Conclusion: </strong>The further characterisation and development of the NT11 tag have provided valuable insights into the optimisation process for such small tags and expanded our understanding of its potential applications. The ability of the NT11 tag to be positioned at either the N- or C- termini within the protein construct without compromising its effectiveness to enhance soluble recombinant protein yields, makes it a valuable tool across a diverse range of proteins. Collectively, these findings demonstrate a promising approach to simplify and enhance the efficiency of soluble recombinant protein production.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"122"},"PeriodicalIF":4.3,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12103771/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144142850","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":"Engineered biosynthesis and characterization of disaccharide-pimaricin.","authors":"Xiaoshan Zuo, Liqin Qiao, Yao Dong, Xing Jin, Zhongyuan Ren, Hao Cui","doi":"10.1186/s12934-025-02742-9","DOIUrl":"10.1186/s12934-025-02742-9","url":null,"abstract":"<p><strong>Background: </strong>Disaccharide polyene macrolides exhibit superior water solubility and significantly reduced hemolytic toxicity compared to their monosaccharide counterparts, making them promising candidates for safer antifungal therapeutics. In this study, we engineered a Streptomyces gilvosporeus (pSET152-nppY) capable of producing disaccharide-pimaricin (DSP) through heterologous expression of the nppY gene, which encodes a glycosyltransferase responsible for the second sugar extension in the biosynthetic pathway.</p><p><strong>Results: </strong>The novel compound was structurally characterized and designated disaccharide-pimaricin (DSP), featuring an aglycone identical to pimaricin and a unique disaccharide moiety (mycosaminyl-α1-4-N-acetylglucosamine). A purification protocol for DSP was established. Compared to pimaricin, DSP demonstrated a 50% reduction in antifungal activity, a 12.6-fold decrease in hemolytic toxicity, and a remarkable 107.6-fold increase in water solubility. Growth analysis revealed a delayed growth cycle in the mutant strain, suggesting that nppY expression may impose additional metabolic burden. Optimization of the fermentation medium using a statistical design identified an optimal formulation, with a maximum DSP titer of 138.168 mg/L.</p><p><strong>Conclusions: </strong>This study underscores the potential of disaccharide polyene macrolides as safer antifungal agents and establishes a robust framework for engineering strains to produce these compounds. The findings provide critical insights into balancing biosynthetic efficiency and strain fitness, advancing the development of next-generation polyene antibiotics.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"121"},"PeriodicalIF":4.3,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12100793/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128103","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":"Functional insights from recombinant production of bacterial proteases in Saccharomyces cerevisiae.","authors":"Tova Lindh, Mattias Collin, Rolf Lood, Magnus Carlquist","doi":"10.1186/s12934-025-02732-x","DOIUrl":"10.1186/s12934-025-02732-x","url":null,"abstract":"<p><strong>Background: </strong>Proteases are important enzymes in food and pharmaceutical industries, but challenges persist in their recombinant production due to host cell proteome hydrolysis and fitness loss. The development of recombinant expression systems for directed evolution of proteolytic enzymes, and industrial production are desirable. This study evaluated Saccharomyces cerevisiae as expression host for three bacterial proteases: BdpK (from Bdellovibrio bacteriovorus), IdeS, and SpeB (both from Streptococcus pyogenes), each with distinct peptide substrate scopes.</p><p><strong>Results: </strong>We developed an experimental pipeline for analysis of protease gene expression levels and fitness effects on yeast cultures. Heterologous genes were fused with green fluorescent protein and their expression and effects on cell viability was monitored at the single-cell level by flow cytometry. IdeS-GFP fusion was produced efficiently with a gaussian distribution within the population and without compromising cell growth or viability. BdpK, on the other hand, displayed lower expression level and a more heterogenous distribution that was less stable over time. Production of SpeB was not feasible. Inserting the speB-GFP fusion gene resulted in complete growth inhibition and a significantly higher frequency of cells with compromised membrane integrity. Plasmid-based expression was compared with integrated-based expression, revealing higher total expression levels and lower degree of population heterogeneity for the latter.</p><p><strong>Conclusions: </strong>S. cerevisiae was found to be an efficient expression host for the bacterial protease IdeS. In contrast, the expression of BdpK and SpeB faced significant challenges, including lack of activity for BdpK, or imposing a substantial fitness burden on the cells for SpeB, likely due to its broad substrate scope resulting in native protein degradation. The findings of this study provide valuable insights into the limitations and possibilities of yeast as an expression host for bacterial protease production and for studying their physiological effects using yeast as a model eukaryote.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"119"},"PeriodicalIF":4.3,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12096733/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128146","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":"Region-based segmental swapping of homologous enzymes for higher cadaverine production.","authors":"Seungjin Kim, Dae-Yeol Ye, Hyun Gyu Lim, Myung Hyun Noh, Jae-Seong Yang, Gyoo Yeol Jung","doi":"10.1186/s12934-025-02739-4","DOIUrl":"10.1186/s12934-025-02739-4","url":null,"abstract":"<p><strong>Background: </strong>Cadaverine, displaying potential in medicine, agriculture and polyamide production, is biologically produced through L-lysine decarboxylation. Considering the potential of the polyamide market, its biological production has been focused on with following diverse efforts to improve the production. In Escherichia coli, lysine decarboxylase exists in two forms: CadA and LdcC, and it is known that CadA exhibits superior catalytic activity compared to LdcC. Despite its potential, cadaverine production is limited due to increased intracellular pH, which destabilizes the decameric structure of CadA and inhibits its activity.</p><p><strong>Results: </strong>In this study, based on the structural analysis, a chimeric CadA enzyme, CL2, was engineered by replacing its pH-sensitive region with a structurally stable counterpart derived from LdcC. The resulting BLCL2 strain with CL2 produced 1.12 g/L of cadaverine-1.96 times higher than BLC strain with the wild type CadA in flask culture. Compared to the wild type CadA, structural modifications enhanced pH stability and improved the affinity of CadA toward pyridoxal 5-phosphate (PLP), its cofactor.</p><p><strong>Conclusions: </strong>This study developed the improved strains for cadaverine production by creating the new enzyme, which is validated by enhanced amount of cadaverine. In addition, the segmental swapping guided by structure analysis was exhibited as the one of effective method in protein engineering strategies. These advancements offer a promising approach to optimizing cadaverine biosynthesis for industrial applications.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"120"},"PeriodicalIF":4.3,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12096693/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128147","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":"Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization.","authors":"Jeffrey J Czajka, Ziyu Dai, Tijana Radivojević, Joonhoon Kim, Shuang Deng, Teresa Lemmon, Marie Swita, Meagan C Burnet, Nathalie Munoz, Yuqian Gao, Young-Mo Kim, Beth Hofstad, Jon K Magnuson, Hector Garcia Martin, Kristin E Burnum-Johnson, Kyle R Pomraning","doi":"10.1186/s12934-025-02705-0","DOIUrl":"10.1186/s12934-025-02705-0","url":null,"abstract":"<p><strong>Background: </strong>Lipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial chassis for biofuel and chemical production. However, the feasibility of redirecting L. starkeyi lipid flux away from lipids and towards other products remains relatively unexplored. Here, we engineer the native metabolism to produce malic acid by introducing the reductive TCA pathway and a C4-dicarboxylic acid transporter to the yeast.</p><p><strong>Results: </strong>Heterogeneous expression of two genes, the Aspergillus oryzae malate transporter and malate dehydrogenase, enabled L. starkeyi malic acid production. Overexpression of a third gene, the native pyruvate carboxylase, allowed titers to reach approximately 10 g/L during shaking flasks cultivations, with production of malic acid inhibited at pH values less than 4. Corn-stover hydrolysates were found to be well-tolerated, and controlled bioreactor fermentations on the real hydrolysate produced 26.5 g/L of malic acid. Proteomic, transcriptomic and metabolomic data from real and mock hydrolysate fermentations indicated increased levels of a S. cerevisiae hsp9/hsp12 homolog (proteinID: 101453), glutathione dependent formaldehyde dehydrogenases (proteinIDs: 2047, 278215), oxidoreductases, and expression of efflux pumps and permeases during growth on the real hydrolysate. Simultaneously, machine learning based medium optimization improved production dynamics by 18% on mock hydrolysate and revealed lower tolerance to boron (a trace element included in the standard cultivation medium) than other yeasts.</p><p><strong>Conclusions: </strong>Together, this work demonstrated the ability to produce organic acids in L. starkeyi with minimal byproducts. The fermentation characterization and omic analyses provide a rich dataset for understanding L. starkeyi physiology and metabolic response to growth in hydrolysates. Identified upregulated genes and proteins provide potential targets for overexpression for improving growth and tolerance to concentrated hydrolysates, as well as valuable information for future L. starkeyi engineering work.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"117"},"PeriodicalIF":4.3,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12093598/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111389","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":"Comparative analysis of pre-treatment strategies and bacterial strain efficiency for improvement of feather hydrolysis.","authors":"Clémentine Isembart, Boris Zimmermann, Josipa Matić, Cristian Bolaño Losada, Nils K Afseth, Achim Kohler, Svein J Horn, Vincent Eijsink, Piotr Chylenski, Volha Shapaval","doi":"10.1186/s12934-025-02743-8","DOIUrl":"10.1186/s12934-025-02743-8","url":null,"abstract":"<p><strong>Background: </strong>Feathers are a major by-product of the poultry industry, which poses an environmental challenge due to the recalcitrant structure of keratin, making them resistant to degradation. Traditional methods of feather handling, like conversion to feather meal, are energy-intensive and have limited efficiency. Biotechnological approaches, particularly microbial hydrolysis, offer a novel and more sustainable alternative for keratin degradation. This study evaluated feather hydrolysis by two bacterial strains, newly characterized cold-adapted Arthrobacter oryzae (BIM B-1663) and Bacillus licheniformis (CCM 2145^T), known as a keratin degrader, under various feather pre-treatment conditions, including washing, autoclaving, drying, and grinding.</p><p><strong>Results: </strong>Both bacterial strains were able to degrade pretreated feathers with a degradation efficiency of 75 to 90%, resulting in high ratios of nitrogen to carbon in the hydrolysates. B. licheniformis confirmed its enzymatic capabilities with high levels of general and specific protease activity and furthermore presented enriched amounts of amino acids of industrial interest. A. oryzae showed a much higher keratinase/protease activity ratio, demonstrating high specificity and efficiency of its enzymes. Autoclaving emerged as the most important determinant of microbial degradation efficiency and influenced the composition (peptide pattern, amino acid content, and chemical composition assessed through FTIR) of the resulting hydrolysates. Feather drying, although not improving microbial degradation efficiencies, had a considerable impact on hydrolysate composition.</p><p><strong>Conclusions: </strong>The results show that both tested bacterial strains can efficiently degrade autoclaved feathers but use distinct enzymatic strategies to do so. Enriched profiles in amino acids and high nitrogen content in the hydrolysates also advocate for the benefits of microbial feather hydrolysis over an enzymatic one. To the authors' knowledge this study is the first to report a comprehensive evaluation of the impact of various feather pre-treatment methods on the efficiency of subsequent microbial feather hydrolysis and is the first one to report enrichment in phenylalanine, lysine, and tyrosine secreted by B. licheniformis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"118"},"PeriodicalIF":4.3,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12093666/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111375","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":"Optimized expression of oxazolomycins in engineered Streptomyces longshengensis and their activity evaluation.","authors":"Huiying Sun, Xiang Liu, Junyue Li, Yang Xu, Yue Li, Yuqing Tian, Huarong Tan, Jihui Zhang","doi":"10.1186/s12934-025-02726-9","DOIUrl":"10.1186/s12934-025-02726-9","url":null,"abstract":"<p><strong>Background: </strong>To cope with the growing number of severe diseases and intractable pathogens, drug innovation in both chemical structures and pharmacological efficiency has become an imperative global mission. Oxazolomycins are a unique family of polyketide-polypeptide antibiotics from Streptomyces with diverse functional groups in their structures, conferring them multifarious activities. But further development into clinical applications has been hindered for decades for many reasons. Among them, the yield improvement is a critical basis for activity evaluation and drug-like property optimization. This study aims to enhance the production of oxazolomycins in Streptomyces longshengensis through metabolic engineering and evaluate their bioactivity against clinically relevant pathogens.</p><p><strong>Results: </strong>Co-transcriptional analyses suggested that two operons (the transcriptional unit from gene oxaG to oxaB, and that from gene oxaH to oxaQ) could be included in the oxazolomycin biosynthetic gene cluster (oxa BGC) of S. longshengensis. So a strategy was designed to replace the native promoter regions between oxaG and oxaH with constitutive promoters P_neo and P_kasO* following functional module evaluation. In the resultant strain (SL_OE), the production of oxazolomycin component Toxa5 was increased to 4-fold of that in the wild-type strain. Accordingly, the transcription of all related genes in oxa was clearly promoted. SL_OE was then subjected to sublethal dose of gentamicin to induce mutagenesis for optimizing the genetic background, generating a resistant mutant SL_ROE. With the introduction of transporter genes (ozmS and oxaA) into SL_ROE, 175 mg/L of Toxa5 was achieved, representing the highest yield in shake-flask fermentation to the best of our knowledge. Finally, the purified Toxa5 showed significant inhibition on the growth of clinically important Gram-negative pathogenic bacterium, Pseudomonas aeruginosa, and the biofilm formation of Bacillus subtilis. Intriguingly, an unprecedented antioxidant activity was also demonstrated.</p><p><strong>Conclusions: </strong>An oxazolomycin high-producing system of S. longshengensis was established by employing genetic engineering strategies to facilitate the bioactivity exploitation. Oxazolomycin Toxa5 showed interesting inhibitory effects against multiple Gram-negative and -positive pathogens as well as antioxidant capacity, indicating its great potential in clinical applications. The findings provide an efficient strategy for the overproduction and activity evaluation of oxazolomycins.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"114"},"PeriodicalIF":4.3,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12090451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111391","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":"Development of a set of bacterial engineered glycoconjugates as novel serogroup-specific antigens for the serodiagnosis of Escherichia coli O26, O111, O103 and O45 infections associated to hemolytic uremic syndrome.","authors":"Ana J Caillava, Luciano J Melli, Malena Landoni, Stella Maris Landivar, Isabel Chinen, Alicia S Couto, Marta Rivas, Juan E Ugalde, Diego J Comerci, Andrés E Ciocchini","doi":"10.1186/s12934-025-02694-0","DOIUrl":"10.1186/s12934-025-02694-0","url":null,"abstract":"<p><p>Hemolytic uremic syndrome associated to Shiga toxin-producing Escherichia coli infection (STEC-HUS) is a life-threatening condition characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. Among STEC, E. coli O157:H7 is the dominant serotype related with human disease worldwide; however, a subset of STEC non-O157 serotypes -named the \"Big-Six\"- that include the E. coli serogroups O145, O121, O26, O111, O103 and O45 became of a great concern for their potential to cause HUS. Previously, we have demonstrated that serological tests based on bacterial engineered glycoconjugates developed by exploiting the Campylobacter jejuni N-glycosylation machinery, notably increases the association rate of HUS to O157, O145 and O121 STEC infections. In this work, we developed the recombinant glycoproteins O26-AcrA, O111-AcrA, O103-AcrA and O45-AcrA by co-expressing in E. coli the gene cluster required for the synthesis of the O polysaccharide corresponding to each serogroup, the C. jejuni oligosaccharyltransferase (OTase) PglB, and the carrier protein AcrA. The glycans attached to AcrA in the produced and purified glycoconjugates were characterized by mass spectrometry. The glycoconjugates were evaluated as antigens for detection of IgM antibodies against the O polysaccharide of the lipopolysaccharide of O26, O111 and O103 STEC strains in human serum samples. Our results demonstrate that O26-AcrA, O111-AcrA and O103-AcrA allow a clear discrimination between negative and positive samples obtained from patients with HUS associated to O26, O111 and O103 STEC infections. Additionally, these novel antigens are serospecific allowing E. coli serogroup identification which may contribute to the epidemiological surveillance of STEC-HUS patients and their contacts.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"116"},"PeriodicalIF":4.3,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12093740/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111376","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":"pTripleTREP - A vector for tightly controlled expression and purification of virulence factors in Staphylococcus aureus.","authors":"Hannes Wolfgramm, Christopher Saade, Marco Harms, Larissa M Busch, Josephine Lange, Maximilian Schedlowski, Kristin Surmann, Manuela Gesell Salazar, Christian Hentschker, Leif Steil, Stephan Michalik, Uwe Völker, Alexander Reder","doi":"10.1186/s12934-025-02736-7","DOIUrl":"10.1186/s12934-025-02736-7","url":null,"abstract":"<p><strong>Background: </strong>Recombinant proteins facilitate and contribute to detailed studies of the virulence mechanisms and pathophysiology of the major human pathogen Staphylococcus aureus. Of particular interest are secreted virulence factors. However, due to their potential toxicity and specific post-translational processing, virulence factors are difficult targets for heterologous protein production. Purified proteins with native conformation and adequate purity can therefore often only be achieved by elaborate multi-step purification workflows. While homologous expression in S. aureus theoretically offers a promising alternative in this regard, its application remains limited due to the lack of systems that ensure both tightly controlled expression and subsequent efficient purification.</p><p><strong>Results: </strong>To bridge this gap, we present pTripleTREP as a versatile expression vector for S. aureus, which enables the homologous expression and purification of staphylococcal virulence factors. It features a strong SigA-dependent staphylococcal promoter overlapped by three tetracycline responsive elements (TRE), which ensures tight repression under control conditions and high expression levels upon induction of the target gene. This allowed very precise controlled production of the exemplary targets, serine protease-like protein A (SplA) and B (SplB). A simple single-step protein purification workflow using a Twin-Strep-tag and Strep-Tactin^®XT coated magnetic beads yielded endotoxin-free Spl samples with purities above 99%. Thereby, the homologous production host facilitates native secretion and maturation without the need to engineer the target gene sequence. Proper signal peptide cleavage and the corresponding enzymatic activity of the generated protein products were confirmed for SplA and B.</p><p><strong>Conclusion: </strong>The expression vector pTripleTREP adds an important element to the staphylococcal molecular toolbox, facilitating the tightly controlled homologous expression and rapid native purification of secreted staphylococcal virulence factors. The optimised architecture and genetic features of the vector additionally provide a solid background for further applications such as plasmid-based complementation or interaction studies. Thus, pTripleTREP will support research on the role of staphylococcal virulence factors, paving the way for future therapeutic strategies to combat this pathogen.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"115"},"PeriodicalIF":4.3,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12090601/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111393","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}