Microbial Cell Factories最新文献

{"title":"Management of a ciprofloxacin as a contaminant of emerging concern in water using microalgaebioremediation: mechanism, modeling, and kinetic studies.","authors":"Heba Salah, Nabila Shehata, Noha Khedr, Khaled N M Elsayed","doi":"10.1186/s12934-024-02591-y","DOIUrl":"10.1186/s12934-024-02591-y","url":null,"abstract":"<p><p>Pharmaceutical residues, now recognized as a new category of environmental pollutants, have potentially risks to both ecosystems and human health effects. Recently, biosorption has emerged as one of the most promising strategies for managing these pharmaceutical wastes in water. Nevertheless, the environmental impact of the adsorbents presents a challenge to the advancement of this process. Therefore, the present study proposed two biosorbent: Chlorella vulgaris and Synechocystis sp. microalgae to manage Ciprofloxacin (CIP) in water. The experimental findings revealed that the optimal conditions for adsorption conditions are CIP initial concentration 4.0 mg/L and pH 5 and 3 for Synechocystissp. and C. vulgaris, respectively. The adsorption process followed the Pseudo-second-order kinetic model. The main mechanism of biosorption is the complexation of CIP with carboxyl, hydroxyl, carbonyl, and amido groups which was confirmed by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDX) analyses. These analyses confirmed the presence of CIP on the surface of tested microalgal cells. These results indicated that the adsorption mechanism of CIP by Synechocystis sp. PCC6803 and C. vulgaris offers theoretical insights into the biosorption mechanisms of pharmaceutical residues.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"329"},"PeriodicalIF":4.3,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11650846/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142837737","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":"Tailored culture strategies to promote antimicrobial secondary metabolite production in Diaporthe caliensis: a metabolomic approach.","authors":"Laura V Hoyos, Luis E Vasquez-Muñoz, Yuliana Osorio, Daniela Valencia-Revelo, Daiana Devia-Cometa, Miriam Große, Esteban Charria-Girón, Nelson H Caicedo-Ortega","doi":"10.1186/s12934-024-02567-y","DOIUrl":"10.1186/s12934-024-02567-y","url":null,"abstract":"<p><strong>Background: </strong>In the search for new antimicrobial secondary metabolites of fungi, optimizing culture conditions remains a critical challenge, as standard laboratory approaches often result in low yields. While non-selective methods, such as modifying culture media, have been effective in expanding the chemical diversity of fungal metabolites, they have not yet established a direct link to key process parameters crucial for further optimization. This study investigates the capacity of Diaporthe caliensis as a biofactory for biologically active secondary metabolites, employing tailored culture media to explore the relationship between chemical diversity and critical process variables.</p><p><strong>Results: </strong>The metabolomic profiles, antibacterial activities, and production yields of the extracts were analyzed to progressively adjust the culture conditions. This study was conducted in five steps, evaluating carbon and nitrogen source concentration, nitrogen source type, salt supplementation, and pH adjustment. Altering the rice starch concentration affected biomass yield per unit of oxygen consumed, while modifications to the nitrogen source concentration influenced both the bioactivity and chemical space by Diaporthe caliensis. Despite changes at the metabolome level, the extracts consistently exhibited potent antibacterial activities, influenced by the nitrogen source, added salts and pH adjustments. For instance, when using corn steep liquor and rice starch, supplemented with micronutrients, different metabolites were produced depending on whether buffer or water was used, though both conditions showed similar antibacterial activities (IC₅₀ ≈ 0.10 mg mL^- 1 against Staphylococcus aureus and ≈ 0.14 mg mL^- 1 against Escherichia coli). In the treatment where buffer was used to stabilize pH change, there was an increase in the production of phomol-like compounds which are associated with known antibiotic properties. In contrast, in the treatments using water, the drop in pH stimulated the production of previously unidentified metabolites with potential antimicrobial activity.</p><p><strong>Conclusions: </strong>This study proposes a strategic methodology for the tailored formulation of culture media aiming to promote the biosynthesis of diverse secondary metabolites. This approach revealed the critical role of nutrient limitation and pH regulation in stimulating the production of polyketide-lactone derivatives, including the antibiotic phomol. Ultimately, the systematic, custom-designed culture conditions developed in this work offer a promising strategy for expanding the chemical diversity of Diaporthe caliensis, while providing valuable insights into the key parameters needed for optimizing this fungal biofactory.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"328"},"PeriodicalIF":4.3,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11619134/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142786165","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":"Efficient calcium fumarate overproduction from xylose and corncob-derived xylose by engineered strains of Aureobasidium pullulans var. Aubasidani DH177.","authors":"Peng Wang, Hao Chen, Xin Wei, Guang-Lei Liu, Zhe Chi, Bo Jiang, Zhen-Ming Chi","doi":"10.1186/s12934-024-02608-6","DOIUrl":"10.1186/s12934-024-02608-6","url":null,"abstract":"<p><strong>Background: </strong>Xylose from lignocellulose is one of the most abundant and important renewable and green raw materials. It is very important how to efficiently transform xylose into useful bioproducts such as fumaric acid and so on.</p><p><strong>Results: </strong>In this study, it was found that the GC1 strain (∆gox, in which the GOX gene encoding glucose oxidase which could transform glucose into gluconic acid was removed) of A. pullulans var. aubasidani DH177 had the high ability to utilize xylose and corncob-derived xylose with CO₂ fixation derived from CaCO₃ to produce calcium fumarate. Overexpression of the XI gene encoding xylose isomerase, the XK gene encoding xylose kinase and the TKL gene coding for transketolase made the strain TKL-4 produce 73.1 g/L of calcium fumarate from xylose. At the same time, the transcriptional levels of the key ASS gene coding for argininosuccinate synthase and the ASL gene coding for argininosuccinate lyase in the ornithine-urea cycle (OUC) were also obviously enhanced. The results also demonstrated that the TKL-4 strain could produce more calcium fumarate from xylose and corncob-derived xylose than from glucose. During 10-liter fermentation, the TKL-4 strain could produce 88.5 g/L of calcium fumarate from xylose, the productivity was 0.52 g/h/L. Meanwhile, it could yield 85.6 g/L of calcium fumarate from corncob-derived xylose and the productivity was 0.51 g/h/L. During the same fermentation, the TKL-4 strain could transform the mixture containing 75.0 g/L glucose and 45.0 g/L xylose to produce 78.7 ± 1.1 g/L calcium fumarate.</p><p><strong>Conclusions: </strong>This indicated that the TKL-4 strain constructed in this study indeed could actively transform xylose and corncob-derived xylose into calcium fumarate through the green ways.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"327"},"PeriodicalIF":4.3,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11616135/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142780411","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":"Synthesis of TUDCA from chicken bile: immobilized dual-enzymatic system for producing artificial bear bile substitute.","authors":"Tang Shijing, Pan Yinping, Yang Qiong, Lou Deshuai, Zhu Liancai, Tan Jun, Liu Shaoyong, Wang Bochu","doi":"10.1186/s12934-024-02592-x","DOIUrl":"10.1186/s12934-024-02592-x","url":null,"abstract":"<p><p>Bear bile, a valuable animal-derived medicinal substance primarily composed of tauroursodeoxycholic acid (TUDCA), is widely distributed in the medicinal market across various countries due to its significant therapeutic potential. Given the extreme cruelty involved in bear bile extraction, researchers are focusing on developing synthetic bear bile powder as a more humane alternative. This review presents an industrially practical and environmentally friendly process for producing an artificial substitute for bear bile powder using inexpensive and readily available chicken bile powder through an immobilized 7α-,7β-HSDH dual-enzymatic syste. Current technology has facilitated the industrial production of TUDCA from Tauodeoxycholic acid (TCDCA) using chicken bile powder. The review begins by examining the chemical composition, structure, and properties of bear bile, followed by an outline of the pharmacological mechanisms and manufacturing methods of TUDCA, covering chemical synthesis and biotransformation methods, and a discussion on their respective advantages and disadvantages. Finally, the process of converting chicken bile powder into bear bile powder using an immobilized 7α-Hydroxysteroid Dehydrogenases(7α-HSDH) with 7β- Hydroxysteroid Dehydrogenases (7β-HSDH) dual-enzyme system is thoroughly explained. The main objective of this review is to propose a comprehensive strategy for the complete synthesis of artificial bear bile from chicken bile within a controlled laboratory setting.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"326"},"PeriodicalIF":4.3,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11613824/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142770412","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":"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}