Microbial Cell Factories最新文献

{"title":"Regulatory mechanisms of acetic acid, ethanol and high temperature tolerances of acetic acid bacteria during vinegar production.","authors":"Shengkai Hua, Yuqin Wang, Leyi Wang, Qinxuan Zhou, Zhitao Li, Peng Liu, Ke Wang, Yuanyuan Zhu, Dong Han, Yongjian Yu","doi":"10.1186/s12934-024-02602-y","DOIUrl":"10.1186/s12934-024-02602-y","url":null,"abstract":"<p><p>Acetic acid bacteria (AAB) play a pivotal role in the food fermentation industry, especially in vinegar production, due to their ability to partially oxidize alcohols to acetic acid. However, economic bioproduction using AAB is challenged by harsh environments during acetic acid fermentation, among which initial ethanol pressure, subsequent acetic acid pressure, and consistently high temperatures are common experiences. Understanding the stress-responsive mechanisms is essential to developing robust AAB strains. Here, we review recent progress in mechanisms underlying AAB stress response, including changes in cell membrane composition, increased activity of membrane-bound enzymes, activation of efflux systems, and the upregulation of stress response molecular chaperones. We also discuss the potential of advanced technologies, such as global transcription machinery engineering (gTME) and Design-Build-Test-Learn (DBTL) approach, to enhance the stress tolerance of AAB, aiming to improve vinegar production.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"324"},"PeriodicalIF":4.3,"publicationDate":"2024-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11607832/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142755166","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":"An efficient and easily obtainable butelase variant for chemoenzymatic ligation and modification of peptides and proteins.","authors":"Avinash Kumar Singh, Anastasiia Antonenko, Anna Kocyła, Artur Krężel","doi":"10.1186/s12934-024-02598-5","DOIUrl":"10.1186/s12934-024-02598-5","url":null,"abstract":"<p><p>The expanding field of site-specific ligation of proteins and peptides has catalyzed the development of novel methods that enhance molecular modification. Among these methods, enzymatic strategies have emerged as dominant due to their specificity and efficiency in modifying proteins under mild conditions. Asparaginyl endopeptidase is a group of cyclotide-producing cysteine proteases from plants. These plant cysteine proteases, known for their specificity, effectively recognize the tripeptide motif (Asx-Xaa-Yaa) and cleave at the C-terminal side of Asx residues, forming acyl-enzyme intermediates that facilitate transpeptidation. Butelase 1 stands out as the most efficient AEP for protein engineering, yet challenges in its expression and purification limit its accessibility for widespread research and industrial use. To address these challenges, we engineered a new, catalytically efficient variant of Butelase 1, Butelase AY, by mutating the gatekeeping residues Val237Ala and Thr238Tyr within the LAD-1 region. These modifications significantly enhanced the stability and yield of Butelase AY, allowing for successful application in various peptide and protein engineering tasks. Butelase AY was tested on the peptide GLGKY, the globular protein GFP, and the intrinsically disordered protein α-synuclein, effectively labeling them with a fluorescent probe. Notably, Butelase AY maintained its efficiency with substrates containing unnatural amino acids, making it a promising candidate for biorthogonal applications. Importantly, the mutations did not compromise the enzyme's specificity, as it continued to process model peptides and native protein substrates with N-term NHV recognition motifs effectively. In conclusion, Butelase AY presents a novel recombinant tool for diverse protein labeling and modifications, particularly in biorthogonal strategies. This innovation has the potential to expand applications in biotechnology and therapeutic development, ultimately revolutionizing protein engineering and its utility in synthetic biology.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"325"},"PeriodicalIF":4.3,"publicationDate":"2024-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11607802/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142755138","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 the secretome of Aspergillus niger for cellooligosaccharides production from plant biomass.","authors":"Fernanda Lopes de Figueiredo, Fabiano Jares Contesini, César Rafael Fanchini Terrasan, Jaqueline Aline Gerhardt, Ana Beatriz Corrêa, Everton Paschoal Antoniel, Natália Sayuri Wassano, Lucas Levassor, Sarita Cândida Rabelo, Telma Teixeira Franco, Uffe Hasbro Mortensen, André Damasio","doi":"10.1186/s12934-024-02578-9","DOIUrl":"10.1186/s12934-024-02578-9","url":null,"abstract":"<p><strong>Background: </strong>Fermentation of sugars derived from plant biomass feedstock is crucial for sustainability. Hence, utilizing customized enzymatic cocktails to obtain oligosaccharides instead of monomers is an alternative fermentation strategy to produce prebiotics, cosmetics, and biofuels. This study developed an engineered strain of Aspergillus niger producing a tailored cellulolytic cocktail capable of partially degrading sugarcane straw to yield cellooligosaccharides.</p><p><strong>Results: </strong>The A. niger prtT∆ strain created resulted in a reduced extracellular protease production. The prtT∆ background was then used to create strains by deleting exoenzyme encoding genes involved in mono- or disaccharide formation. Consequently, we successfully generated a tailored prtT∆bglA∆ strain by eliminating a beta-glucosidase (bglA) gene and subsequently deleted two cellobiohydrolases and one beta-xylosidase encoding genes using a multiplex strategy, resulting in the Quintuple∆ strain (prtT∆; bglA∆; cbhA∆; cbhB∆; xlnD∆). When applied for sugarcane biomass degradation, the tailored secretomes produced by A. niger resulted in a higher ratio of cellobiose and cellotriose compared with glucose relative to the reference strain. Mass spectrometry revealed that the Quintuple∆ strain secreted alternative cellobiohydrolases and beta-glucosidases to compensate for the absence of major cellulases. Enzymes targeting minor polysaccharides in plant biomass were also upregulated in this tailored strain.</p><p><strong>Conclusion: </strong>Tailored secretome use increased COS/glucose ratio during sugarcane biomass degradation showing that deleting some enzymatic components is an effective approach for producing customized enzymatic cocktails. Our findings highlight the plasticity of fungal genomes as enzymes that target minor components of plant cell walls, and alternative cellulases were produced by the mutant strain. Despite deletion of important secretome components, fungal growth was maintained in plant biomass.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"323"},"PeriodicalIF":4.3,"publicationDate":"2024-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11606201/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142755164","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":"Genetic engineering of Nannochloropsis oceanica to produce canthaxanthin and ketocarotenoids.","authors":"Davide Canini, Flavio Martini, Stefano Cazzaniga, Tea Miotti, Beatrice Pacenza, Sarah D'Adamo, Matteo Ballottari","doi":"10.1186/s12934-024-02599-4","DOIUrl":"10.1186/s12934-024-02599-4","url":null,"abstract":"<p><strong>Background: </strong>Canthaxanthin is a ketocarotenoid with high antioxidant activity, and it is primarily produced by microalgae, among which Nannochloropsis oceanica, a marine alga widely used for aquaculture. In the last decade, N. oceanica has become a model organism for oleaginous microalgae to develop sustainable processes to produce biomolecules of interest by exploiting its photosynthetic activity and carbon assimilation properties. N. oceanica can accumulate lipids up to 70% of total dry weight and contains the omega-3 fatty acid eicosapentaenoic acid (EPA) required for both food and feed applications. The genome sequence, other omics data, and synthetic biology tools are available for this species, including an engineered strain called LP-tdTomato, which allows homologous recombination to insert the heterologous genes in a highly transcribed locus in the nucleolus region. Here, N. oceanica was engineered to induce high ketocarotenoid and canthaxanthin production.</p><p><strong>Results: </strong>We used N. oceanica LP-tdTomato strain as a background to express the key enzyme for ketocarotenoid production, a β-carotene ketolase (CrBKT) from Chlamydomonas reinhardtii. Through the LP-tdTomato strain, the transgene insertion by homologous recombination in a highly transcribed genomic locus can be screened by negative fluorescence. The overexpression of CrBKT in bkt transformants increased the content of carotenoids and ketocarotenoids per cell, respectively, 1.5 and 10-fold, inducing an orange/red color in the bkt cell cultures. Background (LP) and bkt lines productivity were compared at different light intensities from 150 to 1200 µmol m^-2 s^-1: at lower irradiances, the growth kinetics of bkt lines were slower compared to LP, while higher productivity was measured for bkt lines at 1200 µmol m^-2 s^-1. Despite these results, the highest canthaxanthin and ketocarotenoids productivity were obtained upon cultivation at 150 µmol m^-2 s^-1.</p><p><strong>Conclusions: </strong>Through targeted gene redesign and heterologous transformation, ketocarotenoids and canthaxanthin content were significantly increased, achieving 0.3% and 0.2% dry weight. Canthaxanthin could be produced using CO₂ as the only carbon source at 1.5 mg/L titer. These bkt-engineered lines hold potential for industrial applications in fish or poultry feed sectors, where canthaxanthin and ketocarotenoids are required as pigmentation agents.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"322"},"PeriodicalIF":4.3,"publicationDate":"2024-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11606307/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751338","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":"Editorial expression of concern:Characterization and high-efciency secreted expression in bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic bacillus clausii strain S10.","authors":"Cheng Zhou, Yanfen Xue, Yanhe Ma","doi":"10.1186/s12934-024-02580-1","DOIUrl":"10.1186/s12934-024-02580-1","url":null,"abstract":"","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"321"},"PeriodicalIF":4.3,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11600909/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142730277","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":"Increased cytoplasmic expression of PETase enzymes in E. coli.","authors":"Luke M Carter, Chris E MacFarlane, Samuel P Karlock, Tridwip Sen, Joel L Kaar, Jason A Berberich, Jason T Boock","doi":"10.1186/s12934-024-02585-w","DOIUrl":"10.1186/s12934-024-02585-w","url":null,"abstract":"<p><strong>Background: </strong>Depolymerizing polyethylene terephthalate (PET) plastics using enzymes, such as PETase, offers a sustainable chemical recycling route. To enhance degradation, many groups have sought to engineer PETase for faster catalysis on PET and elevated stability. Considerably less effort has been focused toward expressing large quantities of the enzyme, which is necessary for large-scale application and widespread use. In this work, we evaluated several E. coli strains for their potential to produce soluble, folded, and active IsPETase, and moved the production to a benchtop bioreactor. As PETase is known to require disulfide bonds to be functional, we screened several disulfide-bond promoting strains of E. coli to produce IsPETase, FAST-PETase and Hot-PETase.</p><p><strong>Results: </strong>We found expression in SHuffle T7 Express results in higher active expression of IsPETase compared to standard E. coli production strains such as BL21(DE3), reaching a purified titer of 20 mg enzyme per L of culture from shake flasks using 2xLB medium. We characterized purified IsPETase on 4-nitrophenyl acetate and PET microplastics, showing the enzyme produced in the disulfide-bond promoting host has high activity. Using a complex medium with glycerol and a controlled bioreactor, IsPETase titer reached 104 mg per L for a 46-h culture. FAST-PETase was found to be produced at similar levels in BL21(DE3) or SHuffle T7 Express, with purified production reaching 65 mg per L culture when made in BL21(DE3). Hot-PETase titers were greatest in BL21(DE3) reaching 77 mg per L culture.</p><p><strong>Conclusions: </strong>We provide protein expression methods to produce three important PETase variants. Importantly, for IsPETase, changing expression host, medium optimization and movement to a bioreactor resulted in a 50-fold improvement in production amount with a per cell dry weight productivity of 0.45 mg_PETase g_CDW^-1 h^-1, which is tenfold greater than that for K. pastoris. We show that the benefit of using SHuffle T7 Express for expression only extends to IsPETase, with FAST-PETase and Hot-PETase better produced and purified from BL21(DE3), which is unexpected given the number of cysteines present. This work represents a systematic evaluation of protein expression and purification conditions for PETase variants to permit further study of these important enzymes.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"319"},"PeriodicalIF":4.3,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11587651/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142710605","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":"Retraction Note: Potential use of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition and prevention method in viral infection.","authors":"Khursheed Muzammil, Mohammad Hosseini Hooshiar, Shirin Varmazyar, Thabit Moath Omar, Manal Morad Karim, Sadeq Aadi, Shaylan Kalavi, Saman Yasamineh","doi":"10.1186/s12934-024-02594-9","DOIUrl":"10.1186/s12934-024-02594-9","url":null,"abstract":"","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"316"},"PeriodicalIF":4.3,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11587653/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142710609","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":"Combined metabolic engineering and lipid droplets degradation to increase vitamin A production in Saccharomyces cerevisiae.","authors":"Jing-Yuan Lin, Xiao Bu, Yi-Bin Lan, Chang-Qing Duan, Guo-Liang Yan","doi":"10.1186/s12934-024-02596-7","DOIUrl":"10.1186/s12934-024-02596-7","url":null,"abstract":"<p><strong>Background: </strong>In microbial cell factories, substrate accessibility to enzyme is a key factor affecting the biosynthesis of natural products. As a robust chassis cells for biofuels and bioproducts, Saccharomyces cerevisiae also encounters the challenge since different enzymes and precursors are typically compartmentalized in different organelles. Such spatial separation could largely limit the efficiency of enzymatic reactions. In this study, the production of the hydrophobic product (vitamin A) was highly improved by metabolic engineering combined with degrading lipid droplets (the primary organelle storing β-carotene) to achieve efficient contact between β-carotene and 15, 15'-β-carotene monooxygenases in Saccharomyces cerevisiae.</p><p><strong>Results: </strong>To efficiently produce vitamin A in Saccharomyces cerevisiae, ten 15, 15'-β-carotene monooxygenases (BCMOs) were firstly evaluated. The strain carrying marine bacterium 66A03 (Mb. BCMO) achieved the highest vitamin A titer. Co-adding 10% dodecane and 1% dibutylhydroxytoluene increased vitamin A titer to 19.03 mg/L in two-phase fermentation. Since most β-carotene is stored in LDs while BCMO is located in the cytosol, we developed a strategy to release β-carotene from LDs to better contact with BCMO. By overexpressing TGL3 and TGL4 using an ion-responsive promoter after high accumulation of β-carotene in LDs, LDs were sequentially degraded, which dramatically improved vitamin A production. Finally, by overexpressing tHMG1, ERG20, and CrtI and introducing Vitreoscilla hemoglobin, vitamin A titer reached 219.27 mg/L, which was a 10.52-folds increase over the original strain in shake flasks, and finally reached 1100.83 mg/L in fed-batch fermentation. The effectiveness of LDs degradation on promoting the formation of β-carotene cleaved product has also been verified in β-ionone synthesis with 44.07% increased yield.</p><p><strong>Conclusions: </strong>Overall, our results highlighted the significance of sequential degrading LDs on vitamin A overproduction in recombinant yeast, and verified that combining metabolic and LDs engineering is an efficient strategy to improve vitamin A production. This integrated strategy can be applied to the overproduction of other hydrophobic compounds with similar characteristics.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"317"},"PeriodicalIF":4.3,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11587636/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142710603","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":"High cell density cultivation by anaerobic respiration.","authors":"Marte Mølsæter Maråk, Ricarda Kellermann, Linda Liberg Bergaust, Lars Reier Bakken","doi":"10.1186/s12934-024-02595-8","DOIUrl":"10.1186/s12934-024-02595-8","url":null,"abstract":"<p><strong>Background: </strong>Oxygen provision is a bottleneck in conventional aerobic high cell density culturing (HCDC) of bacteria due to the low O₂ solubility in water. An alternative could be denitrification: anaerobic respiration using nitrogen oxides as terminal electron acceptors. Denitrification is attractive because NO₃^- is soluble in water, the end-product (N₂) is harmless, and denitrification is widespread among bacteria, hence suitable organisms for most purposes can be found. The pH must be controlled by injection of an inorganic acid to compensate for the pH increase by NO₃^--consumption, resulting in salt accumulation if feeding the bioreactor with NO₃^- salt. We avoid this with our novel pH-stat approach, where the reactor is supplied with 5 M HNO₃ to compensate for the alkalization, thus sustaining NO₃^--concentration at a level determined by the pH setpoint. Here we present the first feasibility study of this method, growing the model strain Paracoccus denitrificans anaerobically to high densities with glucose as the sole C-source and NO₃^- as the N-source and electron acceptor.</p><p><strong>Results: </strong>Our fed-batch culture reached 20 g cell dry weight L^-1, albeit with slower growth rates than observed in low cell density batch cultures. We explored reasons for slow growth, and the measured trace element uptake indicates it is not a limiting factor. Bioassays with spent medium excluded accumulation of inhibitory compounds at high cell density as the reason for the slow growth. The most plausible reason is that high metabolic activity led to CO₂/H₂CO₃ accumulation, thus suppressing pH, leading to a paucity in HNO₃-feeding until N₂-sparging had removed sufficient CO₂. The three free intermediates in the denitrification pathway (NO₃^- → NO₂^- → NO → N₂O → N₂) can all reach toxic concentrations if the electron flow is unbalanced, and this did occur if cells were glucose-limited. On the other hand, accumulation of polyhydroxyalkanoates occurred if the cells were NO₃^--limited. Carefully balancing glucose provision according to the HNO₃ injected is thus crucial.</p><p><strong>Conclusions: </strong>This work provides a proof of concept, while also identifying CO₂/H₂CO₃ accumulation as a hurdle that must be overcome for further development and optimization of the method.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"320"},"PeriodicalIF":4.3,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11590539/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142716776","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 production of a truncated form of the recombinant neuraminidase of influenza virus in Escherichia coli as host with suitable functional activity.","authors":"Fatemeh Sadat Shariati, Fatemeh Fotouhi, Behrokh Farahmand, Zahra Barghi, Kayhan Azadmanesh","doi":"10.1186/s12934-024-02587-8","DOIUrl":"10.1186/s12934-024-02587-8","url":null,"abstract":"<p><strong>Background: </strong>To discover effective drugs for treating Influenza (a disease with high annual mortality), large amounts of recombinant neuraminidase (NA) with suitable catalytic activity are needed. However, the functional activity of the full-length form of this enzyme in the bacterial host (as producing cells with a low cost) in a soluble form is limited. Thus, in the present study, a truncated form of the neuraminidase (derived from California H1N1 influenza strain) was designed, then biosynthesized in Escherichia coli BL21 (DE3), Shuffle T7, and SILEX systems. E. coli BL21 (DE3) was selected as a best host for statistical optimization. Using central composite design methodology, neuraminidase expression level was measured at 20 different runs considering most effective factors including; concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), temperature, and induction time.</p><p><strong>Result: </strong>The recombinant neuraminidase was purified using Ni-affinity chromatography in soluble form. The neuraminidase expression was confirmed by western blot technique with a molecular mass of 48 kDa. The optimum expression condition was at temperature (30°C), induction time (3 h), and concentration of IPTG (0.6 mM) resulting in maximum neuraminidase expression (7.6 µg/mL) with P < 0.05. The analysis of variance with the significant value of R² (0.97) indicated that the quadratic model utilized for this prediction was highly significant (p < 0.0001). Applying the optimized condition led to a ~ 2.2-fold increase in NA expression level (from 3.4 to 7.6 µg/ml). The kinetic parameters were also confirmed by fluorescent signals (by 2'-(4-Methylumbelliferyl)-α-D-N acetyl neuraminic acid substrate) with specific activity; ~3.5 IU/mg and Km: 86.49 ± 0.1 µ, close to the Vibrio Cholera neuraminidase with specific activity; 4 IU/mg. The neuraminidase inhibition test confirmed the inhibition of the neuraminidase activity by the drug inhibitor (Oseltamivir) compared to the control sample.</p><p><strong>Conclusion: </strong>The high quality and proper functional activity of the truncated neuraminidase described in this research show that E. coli can be a suitable host for a wide range of applications with less cost and risk compared to eukaryotic expression systems.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"318"},"PeriodicalIF":4.3,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11587648/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142710608","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}