Applied Microbiology and Biotechnology最新文献

{"title":"Novel tools for genomic modification and heterologous gene expression in the phylum Planctomycetota","authors":"Tom Haufschild,&nbsp;Jonathan Hammer,&nbsp;Nico Rabold,&nbsp;Veronika Plut,&nbsp;Christian Jogler,&nbsp;Nicolai Kallscheuer","doi":"10.1007/s00253-025-13462-w","DOIUrl":"10.1007/s00253-025-13462-w","url":null,"abstract":"<p>Members of the phylum <i>Planctomycetota</i> possess a plethora of intriguing and hitherto underexplored features including an enlarged periplasmic space, asymmetric cell division (“budding”), and a mostly undiscovered small molecule portfolio. Due to the large phylogenetic distance to frequently used and easily genetically accessible model bacteria, most of the established genetic tools are not readily applicable for the here-investigated bacterial phylum. However, techniques for targeted gene inactivation and the introduction of heterologous genes are crucial to investigate the cell biology in the phylum in greater detail. In this study, the targeted genomic modification of model planctomycetes was achieved by enforcing two types of homologous recombination events: simultaneous double homologous recombination for the deletion of coding regions and insertion-duplication mutagenesis for the introduction of foreign DNA into the chromosome. Upon testing the expression of commonly used fluorescent protein-encoding genes, many of the tested native promoters could not be harnessed for variation of the expression strength. Since also four commonly used inducible gene expression systems did not work in the tested model strain <i>Planctopirus limnophila</i>, a native rhamnose-dependent transcriptional regulator/promoter pair was established as an inducible expression system. The expanded molecular toolbox will allow the future characterization of genome-encoded features in the understudied phylum.</p><p>• <i>Two recombination methods were used for the genetic modification of planctomycetes</i></p><p>• <i>Commonly used fluorescent proteins are functional in model planctomycetes</i></p><p>• <i>A rhamnose-dependent regulator was turned into an inducible expression system</i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13462-w.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143740932","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":"Enhancing production and assessing IgE reactivity of dog allergen Can f 6 in Pichia pastoris and Escherichia coli","authors":"Juta Dvareckienė,&nbsp;Gintautas Žvirblis,&nbsp;Mindaugas Zaveckas,&nbsp;Rasa Petraitytė-Burneikienė","doi":"10.1007/s00253-025-13465-7","DOIUrl":"10.1007/s00253-025-13465-7","url":null,"abstract":"<div><p>Pet allergies are increasingly prevalent in developed nations, significantly affecting humans and strongly linked with asthma and rhinitis. Allergic reactions to cats and dogs affect 15.7% of Americans and 27.2% of Europeans, with sensitization rates to dog allergens reaching 56.0% in Denmark. Despite these concerns, dog ownership remains widespread, with 25% of European and 45.5% of US households owning at least one dog. With sensitization on the rise and current diagnostic and therapeutic approaches predominantly relying on inherently inconsistent allergen extracts derived from natural sources, recombinant allergen production offers a pathway to component-resolved diagnostics, improving specificity and reliability in allergy diagnosis. The present research explored, for the first time, the production of the allergen component glycoprotein Can f 6 in the eukaryotic expression system <i>Pichia pastoris</i> and compared its IgE antigenicity to recombinant Can f 6 (rCan f 6) variants produced in <i>Escherichia coli</i>. Yields were significantly increased by fusing Can f 6 with the maltose binding protein (MBP), resulting in a 1.8-fold increase in production in <i>E. coli</i> and a threefold increase in <i>P. pastoris</i>. Antigenicity analysis showed that N-glycosylation is not critical for folding or IgE recognition of Can f 6, making both systems equally suitable for producing the allergen. Notably, <i>P. pastoris</i>-produced MBP fused protein purified through cation exchange chromatography yielded a lower protein quantity. Still, it exhibited stronger IgE reactivity than the same protein purified using anion exchange chromatography.</p><h3>Graphical abstract</h3>\u0000<div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13465-7.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143726747","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":"The role of ATP citrate lyase, phosphoketolase, and malic enzyme in oleaginous Rhodotorula toruloides","authors":"Alīna Reķēna,&nbsp;Kristjan Pals,&nbsp;Srðan Gavrilović,&nbsp;Petri-Jaan Lahtvee","doi":"10.1007/s00253-025-13454-w","DOIUrl":"10.1007/s00253-025-13454-w","url":null,"abstract":"<p><i>Rhodotorula toruloides</i> is an oleaginous yeast recognized for its robustness and the production of high content of neutral lipids. Early biochemical studies have linked ATP citrate lyase (ACL), phosphoketolase (PK), and cytosolic malic enzyme (cMAE) with de novo lipid synthesis. In this study, we discovered that upon a CRISPR/Cas9-mediated knockout of the ACL gene, lipid content in <i>R. toruloides</i> IFO0880 decreased from 50 to 9% of its dry cell weight (DCW) in glucose medium and caused severe growth defects (reduced specific growth rate, changes in cell morphology). In xylose medium, the lipid content decreased from 43 to 38% of DCW. However, when grown on acetate as the sole carbon source, the lipid content decreased from 45 to 20% of DCW. Significant growth defects as a result of ACL knockout were observed on all substrates. In contrast, PK knockout resulted in no change in growth or lipid synthesis. Knocking out cMAE gene resulted in lipid increase of 2.9% of DCW and 23% increase in specific growth rate on glucose. In xylose or acetate medium, no change in lipid production as a result of cMAE gene knockout was observed. These results demonstrated that ACL plays a crucial role in lipid synthesis in <i>R. toruloides</i> IFO0880, as opposed to PK pathway or cMAE, whose presence in some conditions even disfavors lipid production. These results provided valuable information for future metabolic engineering of <i>R. toruloides</i>.</p><p>• <i>ACL is crucial for the fatty acid synthesis and growth in R. toruloides IFO0880.</i></p><p>• <i>Lipid production and cell growth is are unchanged as a result of PK knockout.</i></p><p>• <i>Cytosolic malic enzyme does not play a significant role in lipogenesis.</i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13454-w.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143726748","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":"An update on thermostable keratinases for protein engineering against feather pollutants","authors":"Bhagya Jyothi J L,&nbsp;Immanuel Dhanasingh","doi":"10.1007/s00253-025-13459-5","DOIUrl":"10.1007/s00253-025-13459-5","url":null,"abstract":"<p>Every year, the poultry business worldwide produces at least 8.5 billion tonnes of chicken feathers, making it one of the major landfill pollutants in the world. Biodegradation and recycling of native feathers is difficult due to the presence of numerous disulfide linkages in the feather’s major constituent, keratin. Denaturation of such recalcitrant protein is thermodynamically favored at high temperatures. Therefore, the lookout for the enzymes that degrade keratin (keratinases) from thermophilic bacteria resulted in the identification of thermostable enzymes favoring feather degradation at high temperatures. This review presents a comprehensive analysis of the biochemical properties and structural attributes of thermostable keratinases, emphasizing their catalytic mechanisms, stability at high temperatures, and substrate specificity. Our exploration of structural features enables us to understand the molecular architecture of these enzymes for protein engineering that might enhance the keratinolytic activity and thermostability further. As the field of protein engineering advances, there exists a pressing requirement for integration of structural data with pragmatic engineering applications. Our review addresses for the first time the detailed structural aspects of thermostable bacterial keratinolytic enzymes that will facilitate the development of modified keratinases through protein engineering for a broad range of industrial applications, such as in the production of biofuels, leather processing, and waste management.</p><p><i>• Efficient eco-friendly bioremediation of feather landfill pollutant using thermophilic keratinases.</i></p><p><i>• Detailed structural and biochemical aspects of different thermophilic bacterial keratinases.</i></p><p><i>• Combinations of thermostable keratinases for the enhanced feather degradation process</i></p><p>Feather waste degradation using bacterial keratinases: an eco-friendly bioprocess for degradation of keratin-rich feather wastes into nutrient-rich byproducts, biofertilizers, and animal feed, using bacterial keratinases. A recycling strategy, contributing to pollutant degradation and waste management.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13459-5.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143688498","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":"Effect of zirconia nanoparticles in heat-cured acrylic resins on bacterial adhesion in vegan beverages","authors":"Hayriye Yasemin Yay Kuscu,&nbsp;Ilhan Gun,&nbsp;Kemal Dogan","doi":"10.1007/s00253-025-13457-7","DOIUrl":"10.1007/s00253-025-13457-7","url":null,"abstract":"<div><p>This study aims to investigate the bacterial adhesion effect of heat-curing acrylic resins modified with zirconia nanoparticles when interacting with vegan beverages using SEM and FTIR analyses. The bacterial adhesion of heat-cured acrylic resins from Imicryl and Procryla was investigated. The focus was on modified versions of Procryla containing 1 wt% and 3 wt% zirconia nanoparticles. A total of 192 specimens (<i>n</i> = 48 per group) were soaked in four different solutions: distilled water, mineral water, almond milk, and water kefir. Repeated measures ANOVA revealed statistically significant differences (<i>P</i> &lt; .05) in bacterial adhesion among resin groups, beverage types, and immersion times. Specifically, the addition of 1% by weight zirconia to the specimens reduced bacterial adhesion in groups immersed in distilled water (<i>P</i> &lt; .001). Similarly, 3 wt% zirconia reduced adhesion in mineral water (<i>P</i> &lt; .001). However, in groups exposed to other beverages, including mineral water, almond milk, and water kefir, the incorporation of either 1 wt% or 3 wt% zirconia increased bacterial adhesion significantly (<i>P</i> &lt; .001). SEM images corroborated these findings, showing varying patterns of bacterial adhesion on the surfaces of different resin groups under different environmental conditions. The findings indicate the importance of resin composition and beverage type in dictating bacterial interactions on acrylic surfaces. Notably, the addition of zirconia nanoparticles, particularly in Procryla 1Z, demonstrated a significant reduction in bacterial adhesion, highlighting its potential to enhance the antimicrobial properties of acrylic materials.\u0000</p><p>Key points</p><p>• <i>Zirconia reduces bacterial adhesion in water.</i></p><p>• <i>Higher zirconia increases adhesion in vegan beverages.</i></p><h3>Graphical Abstract</h3>\u0000<div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13457-7.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143698447","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":"Enhancement of exogenous protein stability in AcMNPV by overexpressing lef5 gene during passaging","authors":"Jie Pei,&nbsp;Ruilun Liu,&nbsp;Wei Li,&nbsp;Shasha Qian,&nbsp;Jie Yang,&nbsp;Shengli Meng,&nbsp;Shuo Shen,&nbsp;Jing Guo","doi":"10.1007/s00253-025-13451-z","DOIUrl":"10.1007/s00253-025-13451-z","url":null,"abstract":"<p>The baculovirus insect cell expression system is pivotal for exogenous protein expression. However, serial passages of baculovirus in insect cells often result in defective virus generation and a rapid decline in exogenous protein expression, limiting its wider application. Previous studies have shown that the expression of the late expression factor 5 (<i>lef5</i>) from other baculoviruses can enhance the stability of exogenous genes in <i>Autographa californica multiple nucleopolyhedrovirus</i> (AcMNPV). In this study, we engineered diverse recombinant strains of AcMNPV to express the enhanced green fluorescent protein (eGFP) and enterovirus 71 virus-like particles (EV71-VLPs), co-expressed with the P1 and 3CD proteins of EV71. We investigated the influence of <i>lef5</i> overexpression, regulated by various promoters, on the stability of exogenous genes and their protein expressions. Notably, <i>lef5</i> overexpression significantly improved the stability of <i>eGFP</i> and <i>EV71-P1</i> gene and protein expressions during serial passages in Sf9 cells. Specifically, the <i>lef5</i> overexpression driven by the op166 promoter was more beneficial for enhancing the expression stability of complex exogenous proteins, such as EV71-VLPs. This study underscores the importance of <i>lef5</i> overexpression in maintaining the stability of exogenous gene expression in baculovirus systems, particularly for producing complex proteins such as VLPs of enterovirus.</p><p>• <i>The overexpression of lef5 stabilizes exogenous gene expression in the baculovirus system.</i></p><p>• <i>Promoter choice affects lef5-mediated gene expression stability.</i></p><p>• <i>The overexpression of lef5 is crucial for producing complex proteins like EV71-VLPs.</i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13451-z.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143676400","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":"Quantification of intracellular influenza A virus protein dynamics in different host cells after seed virus adaptation","authors":"Jan Küchler,&nbsp;Patricia Opitz,&nbsp;Ingo Jordan,&nbsp;Yvonne Genzel,&nbsp;Dirk Benndorf,&nbsp;Udo Reichl","doi":"10.1007/s00253-025-13423-3","DOIUrl":"10.1007/s00253-025-13423-3","url":null,"abstract":"<p>Influenza A virus is a major human pathogen, and its replication is widely studied. One important aspect for effective virus propagation is the host cell, since cellular properties can limit or favor virus entry, viral genome and viral protein synthesis and virus release. To establish detailed mathematical models for these processes, quantitative experimental data on the intracellular dynamics of viral compounds together with the number of infectious and non-infectious virus particles released are required. In this study, we report results obtained from an optimized mass spectrometry assay for the quantification of viral proteins that was applied to compare the production of influenza A virus HA, NP, NA, M1, and NS1 proteins for different seed viruses and host cells of batch cultures. With canine MDCK cell-adapted seed virus, a maximum of about 1.0E+08 copies/cell were found for all five viral proteins after infection of avian AGE1.CR and human HEK293 cells. These intracellular levels are about fivefold lower than in MDCK cells. However, after five passages of seed virus adaptation, intracellular protein copy numbers comparable to those in MDCK cells were achieved. Highest levels were found for the NS1 protein with about 1.0E+09 copies/cell. Furthermore, the onset of virus particle release started earlier for both cell lines (about 3–6 h). In contrast, the maximum virus titers did not change for AGE1.CR cells but increased for HEK293 cells. Nevertheless, the highest HA titers were always obtained for MDCK cells. Overall, the experimental data indicate that influenza A virus replication is different due to specifics of innate host cell immune response, viral protein production, precursor consumption, and degradation rates.</p><p>•<i> Application of absolute quantification for five major proteins of influenza A virus.</i></p><p>•<i> NS1 protein most abundant protein with 1.0E+09 copies/cell at the end of infection.</i></p><p>•<i> Virus adaptation leads to earlier release and higher virus titers in HEK293 cell.</i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13423-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143676378","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":"A novel two-step purification process for highly stable C-phycocyanin of analytical grade purity and its properties","authors":"Anna Antecka,&nbsp;Rafał Szeląg,&nbsp;Stanisław Ledakowicz","doi":"10.1007/s00253-025-13458-6","DOIUrl":"10.1007/s00253-025-13458-6","url":null,"abstract":"<p>Efficient and economic purification of phycobiliproteins can be achieved by a novel relatively simple two-step process involving foam fractionation and ion exchange chromatography. Foam fractionation, which has not previously been used to concentrate phycobiliproteins, is a low-cost and environmentally friendly method that provides a significant volume reduction prior to the chromatography step. Two C-phycocyanin fractions with purities of 4.66 and 4.25 with slightly different characteristics and an allophycocyanin fraction with a purity of 3.23 were obtained. Both C-phycocyanins contain α-subunits of 15.0 kDa and β-subunits of 16.4 kDa, whereas the molecular weight of allophycocyanin is 15.5 kDa. The resulting C-phycocyanin retains its properties at pH in the range of 3–10, whereas strong alkaline pH leads to its rapid degradation. The purified protein is completely resistant to temperature changes in the range of 4 to 50 °C and loses only about 13% of its initial concentration during a 5 h incubation at 60 °C. Interestingly, purified C-phycocyanin is relatively resistant to photochemical degradation, as the loss in concentration after 10 h exposure to light is only about 14%. The most suitable storage conditions are temperature of 4 °C and pH in the range 4–5. The final product with an analytical purity greater than 4 is suitable for use in food, biomedicine and as a therapeutic agent.</p><p>• <i>Foam fractionation and ion chromatography for the purification of phycobiliproteins</i>.</p><p>• <i>C-phycocyanin stable over a wide temperature and pH range without a stabilizing agent</i>.</p><p>• <i>C-phycocyanin of analytical purity for food, medical and pharmaceutical applications</i>.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13458-6.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143676401","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":"CUsKit: a test to revolutionise the evaluation of the disinfection process","authors":"Débora Castro,&nbsp;Isabel Ferreri,&nbsp;Mariana Henriques,&nbsp;Isabel Carvalho","doi":"10.1007/s00253-025-13456-8","DOIUrl":"10.1007/s00253-025-13456-8","url":null,"abstract":"<p>The overuse of surface disinfectants has been shown to lead to increased microbial resistance, often due to their incorrect application. The aim of this work was to develop and investigate the use of an indicative detection kit for hydrogen peroxide-based disinfectants on porous and non-porous surfaces. The detection kit allows for the detection of the disinfectant up to 7 days after its application, resulting in a colour change from transparent to yellow-orange on both on porous and non-porous surfaces. Concurrent antimicrobial tests were performed to establish the correlation between the colour change and the retention of the disinfectant on the surfaces, thereby confirming the maintenance of its antimicrobial efficacy.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13456-8.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143668033","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":"Dual one-step recombinase-aided PCR for rapid detection of Candida in blood","authors":"Xiaona Lyu,&nbsp;Kenan Peng,&nbsp;Zhiqiang Han,&nbsp;Hongyi Li,&nbsp;Xiaoping Chen,&nbsp;Shijue Gao,&nbsp;Yanqing Tie,&nbsp;Yuan Gao,&nbsp;Yuxin Wang,&nbsp;Jie Wang,&nbsp;Xinxin Shen,&nbsp;Xuejun Ma,&nbsp;Zhishan Feng","doi":"10.1007/s00253-025-13452-y","DOIUrl":"10.1007/s00253-025-13452-y","url":null,"abstract":"<p>This study presents a novel dual one-step recombinase-aided PCR (DO-RAP) method, combined with recombinant human mannan-binding lectin protein (rhMBL; i.e., M1 protein)-conjugated magnetic bead (M1 bead) enrichment, for the early detection of <i>Candida krusei</i> and <i>Candida parapsilosis</i> bloodstream infections. Unlike previous studies that utilized the characteristic of docosane being solid at room temperature and melting above 44 °C as an impermeable barrier to separate two reaction steps, DO-RAP simplifies the process by eliminating this step. Specificity tests with the genomic DNAs from 11 bacterial strains and 3 fungi related to bloodstream infections (BSIs) confirmed no cross-reactivity, while sensitivity analysis demonstrated detection limits of 1 copy/μL for recombinant plasmids containing 26S ribosomal RNA gene fragment from <i>C. krusei</i> and NADH5 mitochondrial gene fragment from <i>C. parapsilosis</i> and 10⁻⁷ ng/μL for DNAs from standard strains of <i>C. krusei</i> and <i>C. parapsilosis</i>. In simulated infection samples enriched with M1 beads, DO-RAP achieved detection thresholds of 1 CFU/mL (colony-forming unit per milliliter) in simulated samples within 3.5 h, surpassing quantitative PCR (qPCR) performance, which has detection limits of 3–5 CFU/mL. Clinical validation showed strong agreement between DO-RAP and qPCR, with Kappa values of 0.936 for <i>C. krusei</i> and 0.904 for <i>C. parapsilosis</i> (<i>P</i> &lt; 0.05). This integrated approach improves detection speed and sensitivity, eliminates the need for culturing, and offers a more efficient alternative to qPCR for diagnosing invasive <i>Candida</i> infections.</p><p>• <i>The DO-RAP method achieves a detection sensitivity of 1 CFU/mL, surpassing conventional qPCR.</i></p><p>• <i>This approach eliminates the need for docosane, streamlining operations, and accelerating detection.</i></p><p>• <i>M1 magnetic bead enrichment enhances pathogen capture, facilitating rapid Candida diagnosis.</i></p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"109 1","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00253-025-13452-y.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143667976","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}