Microbial Cell Factories最新文献

{"title":"Optimization of the fermentation process for fructosyltransferase production by Aspergillus niger FS054.","authors":"Yingzi Wu, Yuewen Zhang, Xiaoyu Zhong, Huiling Xia, Mingyang Zhou, Wenjin He, Yi Zheng","doi":"10.1186/s12934-025-02798-7","DOIUrl":"https://doi.org/10.1186/s12934-025-02798-7","url":null,"abstract":"<p><p>This study systematically optimized the fermentation process for fructosyltransferase (FTase) production by Aspergillus niger FS054, integrating traditional experimental designs with machine learning approaches. Single-factor experiments initially identified critical medium components (carbon source, nitrogen sources, phosphate, and metal ions) and cultivation parameters (pH, liquid volume, inoculum size, temperature, and shaking speed). Subsequent Plackett-Burman screening identified sucrose, yeast extract paste, and <math> <mrow><msub><mtext>NH</mtext> <mn>4</mn></msub> <mtext>Cl</mtext></mrow> </math> as the most influential medium factors. Through Box-Behnken response surface methodology (RSM), the optimal medium composition was determined as sucrose 156.65 g/L, yeast extract paste 42 g/L, and <math> <mrow><msub><mtext>NH</mtext> <mn>4</mn></msub> <mtext>Cl</mtext></mrow> </math> 1.68 g/L, yielding an enzyme activity of 3249.00 ± 24.39 U/L (99.16% agreement with RSM predictions). Further optimization of cultivation conditions using a hybrid backpropagation neural network-genetic algorithm (BP-GA) model identified optimal parameters as pH 5.5, a liquid volume of 96.6 mL (in a 250 mL shaker), and inoculum size of 2.4 <math><mo>×</mo></math> <math><msup><mn>10</mn> <mn>4</mn></msup> </math> spores/mL, achieving a final enzyme activity of 3422.14 ± 36.86 U/L (1.1% deviation from the predicted 3460 U/L), representing a 4.2-fold increase over initial conditions. This work demonstrates the synergistic application of classical experimental design and artificial intelligence, significantly enhancing FTase productivity and potentially offering a more economical enzyme source for industrial-scale fructooligosaccharide (FOS) biosynthesis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"173"},"PeriodicalIF":4.9,"publicationDate":"2025-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732256","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":"Enhanced production of extracellular L-asparaginase in batch culture via nitrous acid-induced mutagenesis of Aspergillus oryzae.","authors":"Sikander Ali, Sana Maqsood, Muhammad Usman Ahmad, Ifrah Shabbir, Mohammad Raish, Fozia Batool, Asad-Ur-Rehman, Iram Liaqat, Bakar Bin Khatab Abbasi, Ali Irfan, Yousef A Bin Jardan","doi":"10.1186/s12934-025-02797-8","DOIUrl":"https://doi.org/10.1186/s12934-025-02797-8","url":null,"abstract":"<p><strong>Background: </strong>L-Asparaginase (LA) is an important enzyme with therapeutic and industrial applications, particularly in the treatment of leukemia. Enhancing its production through optimization and strain improvement is crucial for commercial viability. This study aimed to increase LA production using Aspergillus oryzae by optimizing process parameters and employing chemical mutagenesis for strain improvement.</p><p><strong>Results: </strong>Among thirty-five fungal strains isolated from soil, ISL-3 A. oryzae and ISL-9 A. niger were identified as the most efficient LA producers. Using sugarcane bagasse as a substrate for solid-state fermentation, process optimization revealed that ISL-3 showed 12.15% higher yield than ISL-9 under the conditions of 5 g substrate level, 9 mL diluent MC-5, and 72 h of incubation. Chemical mutagenesis using nitrous acid resulted in the mutant NA-t3 with LA activity of 4.479 ± 0.22 U/g, significantly surpassing the parent strain. Inducible resistance was achieved on NA-cysL-C4 with 8 ppm L-cysteine HCl. Supplementation with MgSO₄·7H₂O (3 mM), ammonium nitrate (0.2%), and 2-mercaptoethanol (0.125%) further enhanced LA activity. The final mutant strain's yield increased to 16.122 ± 0.81 U/g, 2.07 times greater than the parent strain. Partial purification through ammonium sulfate precipitation (35-85%), dialysis, and chromatography achieved a 47% recovery yield, with SDS-PAGE confirming a molecular weight of 120 kDa for both strains.</p><p><strong>Conclusion: </strong>The study successfully enhanced LA production from Aspergillus oryzae through process optimization and strain improvement using chemical mutagenesis. The significantly higher yield from the mutant strain makes it a promising candidate for commercial enzyme production. ANN was also employed on results to develop a correlation between experimental and predicted results. These findings highlight the potential of optimized solid-state fermentation and genetic enhancement techniques in industrial-scale.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"172"},"PeriodicalIF":4.3,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718167","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":"Engineering Pichia pastoris for the efficient production of the high-value steroid intermediate 15α-OH-D-ethylgonendione.","authors":"Yu-Long Zeng, Yang-Yang Li, Bei-Feng-Chu Zheng, Dong-Qi Xie, Sheng-Qiang Tong, Yuan Yuan, Ya-Jun Wang, Bin Xue, Xiao-Guang Liu","doi":"10.1186/s12934-025-02796-9","DOIUrl":"10.1186/s12934-025-02796-9","url":null,"abstract":"<p><p>15α-OH-D-ethylgonendione (15α-OH-DE) is a key intermediate for the synthesis of steroid drug gestodene, a major component of a new generation of powerful contraceptives. Synthetic access to 15α-OH-DE by chemical means is limited by low titers and generation of toxic byproducts. To develop a sustainable process for 15α-OH-DE production, a whole-cell catalyst was constructed by engineering Pichia pastoris co-overexpressing the PRH gene from filamentous fungus Penicillium raistrickii, which encodes a steroid 15α-hydroxylase capable of selectively 15α-hydroxylating DE, and the glucose-6-phosphate dehydrogenase gene ZWF1 from the baker's yeast for enhanced NADPH production. Shake-flask cultivation was performed to optimize fermentation parameters and assess the potential of the engineered P. pastoris strains for 15α-OH-DE production. Subsequently, production was scaled up using a fed-batch strategy in a 5-L stirred-tank bioreactor, with pure methanol serving as both the carbon source and inducer. This process achieved a product titer of 5.79 g L⁻¹ with DE feeding of 10 g L^- 1 after 170 h of methanol feeding (196 h fermentation), representing the highest reported titer of 15α-OH-DE to date. The above results highlight the potential of developing P. pastoris-based biotransformation systems for the efficient production of key intermediates of steroid pharmaceuticals and other high-value fine chemicals.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"170"},"PeriodicalIF":4.3,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12278525/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675269","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":"Submicron infrared spectroscopy assessment of single-cell phenotypic diversity in microbial lipid production.","authors":"Uladzislau Blazhko, Dana Byrtusová, Volha Shapaval, Achim Kohler, Christophe Sandt, Boris Zimmermann","doi":"10.1186/s12934-025-02794-x","DOIUrl":"10.1186/s12934-025-02794-x","url":null,"abstract":"<p><strong>Background: </strong>Microbial lipid production offers a sustainable method for creating biofuels, lubricants, and high-value oils, utilizing the metabolic uniqueness of diverse organisms like bacteria, yeasts, and microalgae. However, minor physicochemical variations in bioreactors, along with subtle biochemical differences in organism's life stages, can lead to phenotypic diversity and impact the production. Therefore, monitoring, understanding and managing this diversity within bioreactors is essential in microbial biotechnology. Optical photothermal infrared (O-PTIR) spectroscopy can provide label-free chemical characterization of individual cells at sub-micron level. Here, we demonstrate the use of O-PTIR to evaluate metabolic heterogeneity within a population of oleaginous yeast Rhodotorula graminis in the production of free fatty acids (FFAs) and triacylglycerols (TAGs).</p><p><strong>Results: </strong>Forty yeast cells were measured by acquiring six single-point O-PTIR spectra per cell. Cell sizes were estimated from the corresponding microscopy images, while reference bulk infrared measurements of yeast biomass and pure compounds were obtained by Fourier transform infrared spectroscopies. Within the population, most of the cells have similar chemical composition, though several cells have quite different composition from the population average. Moreover, a number of cells have relatively large intra-cell chemical variability. The main chemical differences between the cells are correlated with cell sizes, and there are statistically significant size-dependent differences in cellular chemistry. Specifically, small cells have higher content of proteins than mid-size and large cells, and large cells have higher TAG-to-FFA ratio compared to mid-size cells. Characteristic wavenumbers for TAGs, FFAs and proteins can be used to estimate content of these compounds, namely 1748, 1714 and 1659 cm^- 1 respectively.</p><p><strong>Conclusions: </strong>The O-PTIR method allows estimation of chemical composition of individual yeast cells and differentiation of two types of lipids (TAGs and FFAs). We have demonstrated that measurement at only four wavenumbers (the aforementioned wavenumbers for TAGs, FFAs and proteins plus one reference wavenumber at 1800 cm^- 1) provides the assessment of major chemical constituents of high importance for optimization of SCO production. We foresee that rapid data acquisition through O-PTIR imaging will significantly aid in understanding and managing phenotypic diversity in microbial cells by providing a detailed representation of individual cells for population statistics.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"171"},"PeriodicalIF":4.3,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12278496/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675270","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":"Biogenic Zinc nanoparticles: green approach to synthesis, characterization, and antimicrobial applications.","authors":"Myada S M Ouf, Mahmoud E A Duab, Dina I Abdel-Meguid, Ebaa E El-Sharouny, Nadia A Soliman","doi":"10.1186/s12934-025-02788-9","DOIUrl":"10.1186/s12934-025-02788-9","url":null,"abstract":"<p><strong>Background: </strong>Biogenic synthesis of zinc nanoparticles (ZnNPs) has attracted significant interest due to their unique properties and potential biological applications. Unlike chemical and physical methods, biogenic synthesis offers a greener and more eco-friendly alternative. This study explores the synthesis of zinc-based nanoparticles using two distinct bacterial strains.</p><p><strong>Results: </strong>In this study, zinc nanoparticles were synthesized in two forms: single-phase zinc sulfide nanoparticles (ZnS NPs) and mixed-phase zinc sulfide-oxide nanoparticles (ZnS-ZnO NPs), using Achromobacter sp. S4 and Pseudomonas sp. S6. The synthesis conditions were optimized for each strain, with pH playing a crucial role: Achromobacter sp. S4 favored basic conditions (pH 8.0) for zinc nanoparticles production, while Pseudomonas sp. S6 preferred acidic conditions (pH 4.7). TEM analysis revealed that Zn NPs from Pseudomonas sp. S6 were rod-shaped, whereas those from Achromobacter sp. S4 were spherical. Further characterization using EDX, XRD, and FTIR confirmed the successful synthesis of single phase ZnS NPs and hybride phase ZnS-ZnO NPs. Antimicrobial dose-response testing showed that single-phase ZnS NPs inhibited Klebsiella pneumoniae, while mixed-phase ZnS-ZnO NPs were effective against Staphylococcus epidermidis at 100 µg/ml based on inhibition zone measurements.Furthermore, the mixed-phase ZnS-ZnO NPs at 25 µg/ml demonstrated superior inhibition of microbial growth in sludge samples, likely due to a synergistic effect.</p><p><strong>Conclusion: </strong>The study demonstrates the successful biogenic synthesis of ZnS NPs, and ZnS-ZnO NPs using two bacterial strains, with distinct morphological and functional properties. The use of two bacterial species was to assess strain-specific differences in nanoparticle synthesis and performance. The synthesized nanoparticles exhibited promising antimicrobial and environmental remediation potential, highlighting their applicability in both biomedical and environmental fields.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"168"},"PeriodicalIF":4.3,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12273035/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144659604","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":"Multiple factors based adaptive laboratory evolution strategy enhances DHA production in Aurantiochytrium sp. PKU#Mn16 by rewiring key metabolic pathways.","authors":"Xueyan Ding, Xingyu Zhu, Yuetong Zhao, Ningdong Xie, Guangyi Wang","doi":"10.1186/s12934-025-02792-z","DOIUrl":"10.1186/s12934-025-02792-z","url":null,"abstract":"","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"169"},"PeriodicalIF":4.3,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12272968/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667979","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":"Review of research progress in immobilization and chemical modification of microbial enzymes and their application.","authors":"Walaa A Abdel Wahab","doi":"10.1186/s12934-025-02791-0","DOIUrl":"10.1186/s12934-025-02791-0","url":null,"abstract":"<p><p>Enzyme stabilization is one of the most critical steps help in applying the enzymes on industrial scale efficiently. Enzymes have use in a variety of areas, including medical, industry, food, and even beauty and cosmetics. The industrial application of enzymes is constantly limited by stability and cost. Enzyme synthesis on a large scale involves multiple steps, therefore stability and repeatability are critical. These two goals are crucial on an industrial scale because they translate to reduced time, effort, and cost. Enzyme stabilization provides the stability and reusability required for successful application. Immobilization with appropriate carriers and conjugation with chemically modified polysaccharides are the most common and low-cost strategies used for enzyme stabilization. These tactics enhance the enzyme's physicochemical characteristics, making it better suited for industrial applications that benefit our daily lives. This review is an attempt to provide a spot on each method, their progress, benefits and draw backs, and their application.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"167"},"PeriodicalIF":4.3,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12273361/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144659605","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":"Enhanced anaerobic synthesis of rhamnolipids and emulsification of crude oil by genetically engineered Pseudomonas aeruginosa strains.","authors":"Feng Zhao, Yuting Wu, Lu Liu","doi":"10.1186/s12934-025-02793-y","DOIUrl":"10.1186/s12934-025-02793-y","url":null,"abstract":"<p><strong>Background: </strong>Pseudomonas aeruginosa is a facultative anaerobic bacterium widely distributed in oil reservoirs. Efficient production of rhamnolipids by P. aeruginosa in anoxic environments of oil reservoirs contributes to oil displacement. How to enhance the synthesis of rhamnolipids by P. aeruginosa under anaerobic conditions has become a new scientific question.</p><p><strong>Results: </strong>Pseudomonas aeruginosa SG anaerobically synthesized much lower yield of rhamnolipids than that under aerobic conditions. The key genes rmlBDAC (m), rhlABRI (h) and rhlC (c) and their combination genes were overexpressed in P. aeruginosa. Among seven genetically engineered strains, P. aeruginosa SGhm anaerobically synthesized the highest yield of rhamnolipids, 1.34 g/L, which was about 4.5-fold higher than that of the wild-type strain SG (0.24 g/L). The results of Plackett-Burman (PB) design indicated that glycerol and nitrate were the nutrient factors that significantly influenced the anaerobic production of rhamnolipids by strain SGhm. Optimization of culture medium by response surface method improved the rhamnolipids production of strain SGhm to 1.54 g/L under anaerobic conditions. Pseudomonas aeruginosa SGhm emulsified oil in anaerobic tubes through anaerobic synthesis of rhamnolipids. The percentage of oil droplets formed with a diameter of 0-5 μm was 89.4%.</p><p><strong>Conclusions: </strong>Genes rmlBDAC and rhlABRI were the best overexpression combination for improvement of rhamnolipids production in P. aeruginosa under anaerobic conditions. Overexpression of key genes and optimization of medium synergistically increase rhamnolipids production of P. aeruginosa under anaerobic conditions. Pseudomonas aeruginosa SGhm is promising for emulsification of crude oil through anaerobic synthesis of rhamnolipids. This study provided an excellent microbial strain resource and nutrients for in-situ production of rhamnolipids in anoxic environments of oil reservoirs.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"166"},"PeriodicalIF":4.3,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12265193/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144642934","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":"Response surface optimization of sedimentation efficiency for sustainable green microalgae harvesting using automated non-invasive methods.","authors":"Amr M Ayyad, Eladl G Eltanahy, Mervat H Hussien, Dina A Refaay","doi":"10.1186/s12934-025-02765-2","DOIUrl":"10.1186/s12934-025-02765-2","url":null,"abstract":"<p><strong>Background: </strong>Microalgae such as Chlorella sorokiniana and Monoraphidium convolutum are promising sources for biofuels, pharmaceuticals, nutraceuticals, and wastewater treatment. However, biomass harvesting remains a cost-intensive bottleneck. Conventional methods like centrifugation and flocculation pose challenges due to energy demands and contamination risks. Sedimentation offers a passive, eco-friendly alternative but is highly sensitive to environmental and physiological variables. This study integrates response surface methodology with a novel, non-invasive photographic imaging technique to optimize sedimentation efficiency.</p><p><strong>Results: </strong>Both species exhibited optimal growth in Bold Basal Medium, achieving cell densities of 29.59 and 9.5 million cells per mL, respectively. Automated cell counting strongly correlated with manual methods (R² = 98.99%). Biochemical analysis revealed a higher protein content in C. sorokiniana (61.6%) and greater lipid content in M. convolutum (39.31%). Sedimentation efficiency was highest at acidic pH and low salinity, reaching 96.14% for C. sorokiniana and 88.7% for M. convolutum. Sealed vessels and smaller culture volumes further enhanced sedimentation efficiency. RSM predictive models achieved high accuracy (adjusted R² > 99%). A novel, real-time photographic method for sedimentation assessment was introduced, offering a non-invasive, sampling-free alternative to conventional techniques. This method strongly correlated with OD-based measurements (R² = 94.89%) and presents a scalable solution for continuous biomass monitoring. Compared to conventional centrifugation, the optimized sedimentation approach is estimated to reduce harvesting costs by 77-79%.</p><p><strong>Conclusions: </strong>This study advances sedimentation-based harvesting of C. sorokiniana and M. convolutum by integrating RSM with a novel, automated, non-invasive imaging technique for sedimentation monitoring. This approach, rarely applied in microalgae harvesting, enables real-time assessment without disturbing the culture, enhancing process control and scalability. Sedimentation efficiency was influenced by cell morphology, biochemical composition, and environmental factors such as pH, salinity, gas exchange, and culture volume. The optimized conditions not only improved harvesting precision and reproducibility but also reduced harvesting costs, highlighting the method's potential for economic and environmentally sustainable deployment in large-scale microalgae-based production systems for biofuels, bioplastics, and high-value compounds.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"165"},"PeriodicalIF":4.3,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12255085/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619054","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":"Construction of primary chassis cells with efficient protein expression in Thermus thermophilus.","authors":"Yuqian Liang, Mohamed Motawaa, Xuying Bu, Junwei Wei, Yuan Shao, Yingjun Li","doi":"10.1186/s12934-025-02785-y","DOIUrl":"10.1186/s12934-025-02785-y","url":null,"abstract":"<p><strong>Background: </strong>Thermus thermophilus HB27 is a promising thermophilic chassis for recombinant thermostable protein production, owing to its high optimal growth temperature, which can simplify downstream processing and reduce contamination risks. However, maximizing its potential requires optimized genetic tools and host strains. Key limitations include a shortage of well-characterized strong constitutive promoters and potential degradation of recombinant proteins by proteases. To address these, we established a β-galactosidase reporter system (endogenous TTP0042) to screen for strong constitutive promoters and investigated the impact of deleting specific protease genes on protein expression.</p><p><strong>Results: </strong>Screening of 13 endogenous promoter regions identified P0984 as exhibiting significantly 13-fold higher activity than the control promoter driving the reporter gene. Constructing a plasmid-free strain (HB27ΔpTT27) successfully minimized 270 kb of the genome; it exhibited auxotrophy for cobalamin (requiring 0.1 µg/ml AdoCbl for growth) and a slightly reduced growth rate compared to the wild-type, while its transformation efficiency remained comparable. Notably, a CRISPR-deficient precursor strain (HB27ΔIII-ABΔI-CΔCRF3) showed a significant (~ 100-fold) increase in transformation efficiency compared to the wild-type, facilitating subsequent genetic manipulations. Systematic knockout of 16 predicted non-essential protease loci was performed. Characterization revealed that deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) significantly reduced extracellular proteolytic activity. Iterative deletion based on phenotypic analysis led to strain DSP9 (10 protease loci deletions), which maintained robust growth and exhibited enhanced accumulation of the β-galactosidase reporter protein compared to the parental strains.</p><p><strong>Conclusions: </strong>This study provides foundational advancements for T. thermophilus HB27 chassis development, and genetic tools represent valuable resources for optimizing T. thermophilus as a platform for heterologous thermostable protein production and ideas for antibiotic-free systems.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"163"},"PeriodicalIF":4.3,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12243296/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144608747","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}