Hailing Dai, Guoqiang Chen, Jingjiu Mu, Afreen Shagufta, Lijuan Liu, Fan Wang, Sheng Dong, Xiao Men, Lei Wang, Haibo Zhang
{"title":"Universal Electrode Based on Ferredoxin-NADP+ Oxidoreductase Enables Enzymatic Biofuel Cells With Broad Substrate Spectrum","authors":"Hailing Dai, Guoqiang Chen, Jingjiu Mu, Afreen Shagufta, Lijuan Liu, Fan Wang, Sheng Dong, Xiao Men, Lei Wang, Haibo Zhang","doi":"10.1002/biot.70090","DOIUrl":"https://doi.org/10.1002/biot.70090","url":null,"abstract":"<div>\u0000 \u0000 <p>Enzymatic biofuel cells face substrate limitations due to enzyme specificity of the electrode. A universal electrode was designed by immobilizing ferredoxin-NADP<sup>+</sup> oxidoreductase (FNR) with bacterial cellulose (BC), carbon nanotubes (CNTs), and silver nanowires (AgNWs). The electrode coupled with NADPH-dependent malic enzyme or glucose dehydrogenase generated electricity using malic acid and glucose, respectively. The open-circuit voltage reached 79.36 and 75.8 mV, respectively, and the accumulation of pyruvate and gluconate reached 0.30 and 0.25 mM, respectively, after 12 h. This strategy enables electron transfer from diverse substrates via NADPH.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 8","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144767473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Revolutionizing Caffeic Acid Production: Advanced Microbial Metabolic Engineering and Synthetic Biology Approaches","authors":"Jintao Lu, Beining Wang, Xiqiang Liu, Jung-Kul Lee, Vipin Chandra Kalia, Chunjie Gong","doi":"10.1002/biot.70091","DOIUrl":"https://doi.org/10.1002/biot.70091","url":null,"abstract":"<div>\u0000 \u0000 <p>Caffeic acid, a high-value natural phenolic compound synthesized through plant metabolism, plays a critical role in producing phenylpropanoid derivatives and serves as a direct precursor to several key phenolic acids. As a food additive and medicine, caffeic acid has garnered significant attention for its potential in various applications. Recent advances in synthetic biology and metabolic engineering have enabled its biosynthesis via microbial cell factories. This review summarizes five strategies for optimizing caffeic acid production: caffeic acid biosynthetic pathway, modification of metabolic pathway, systems biology and synthetic biology, cofactor engineering, and modular co-culture. However, caffeic acid production via microbial chassis faces bottlenecks such as limited precursor availability for biosynthesis, toxicity from metabolic intermediates, inefficient cofactor utilization, and over-reliance on conventional host microorganisms. Breaking through these bottlenecks by integrating the five strategies outlined is expected to further increase caffeic acid production.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 8","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144767360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Characterizing the Effect of Volume on Hydrodynamics of Plant Cell Suspensions Using CFD Modeling","authors":"Vidya Muthulakshmi Manickavasagam, Kameswararao Anupindi, Nirav Bhatt, Smita Srivastava","doi":"10.1002/biot.70086","DOIUrl":"https://doi.org/10.1002/biot.70086","url":null,"abstract":"<div>\u0000 \u0000 <p>Biomass productivities in shake flasks are often not reproduced in bioreactors for plant cell cultures due to change in hydrodynamics. Considering shake flask biomass productivity as benchmark, this study employs shake flask geometries as a model system to understand hydrodynamic changes with volume and identify suitable scale-up criteria for plant cell cultivations, with minimal cost and time, given their slow growth time, using computational fluid dynamics (CFD) and experiments. Cultivation of <i>Viola odorata</i> cells in increasing flask volumes (100–3000 mL) revealed no significant change in biomass productivity. CFD analysis indicated that volumetric oxygen mass transfer coefficient (<i>k<sub>L</sub>a</i>), increased up to 1000 mL and then decreased, due to saturation of energy dissipation rates (<i>k<sub>L</sub></i> is a function of energy dissipation rates) and decreasing interfacial area. The unaffected biomass concentration, despite decreased <i>k<sub>L</sub>a</i>, suggests that <i>k<sub>L</sub>a</i> may not be a significant scale-up parameter. Instead, maintaining a constant shear environment, indicated by power per unit volume saturation at higher volumes, was proposed as a suitable scale-up parameter for <i>V. odorata</i> cell cultivation in bioreactors. Moreover, the decrease in velocity difference between fluid layers with increased flask volume, indicated that minimizing velocity gradients in bioreactors could help achieve shake flask biomass productivity.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144716477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Characterization and Application of a Thermostable HAD Phosphatase From Thermophilibacter mediterraneus for Glucosamine Production","authors":"Yanmei Qin, Nan Geng, Chun You","doi":"10.1002/biot.70083","DOIUrl":"https://doi.org/10.1002/biot.70083","url":null,"abstract":"<div>\u0000 \u0000 <p>Glucosamine (GlcN), a high-value nutraceutical, is currently produced via environmentally detrimental chitin hydrolysis or inefficient microbial fermentation. While acidic hydrolysis of crustacean chitin raises environmental and allergen concerns, microbial fermentation faces challenges in strain engineering and byproduct formation. One-pot production of GlcN from maltodextrin by an in vitro synthetic enzymatic biosystem (ivSEB) containing glucosamine 6-phosphate phosphatase, which dephosphorylates glucosamine 6-phosphate (GlcN6P) to GlcN, was developed recently. In this study, we identified a thermostable haloacid dehalogenase (HAD) phosphatase, TmHAD, from <i>Thermophilibacter mediterraneus</i> through database mining. Biochemical characterization revealed its remarkable dephosphorylation specificity for GlcN6P, exhibiting 27.6- and 138.0-fold higher activity toward GlcN6P compared to glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), respectively. The enzyme demonstrated Mg<sup>2+</sup>-dependent activity and moderate thermal stability with a half-life of 6.6 h at 45°C. When incorporated into an ivSEB (phosphorylation, isomerization, amination, and dephosphorylation), TmHAD enabled GlcN production from maltodextrin with a molar yield of 44.5%. This biosystem represented an effective complement to current GlcN production methods, with the exceptional substrate specificity and thermal stability of TmHAD making it particularly promising for industrial-scale GlcN manufacturing in vitro.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144716886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Biological Routes for Biohydrogen Production: A Clean and Carbon-Free Fuel","authors":"Minseok Cha, Min-Seo Park, Soo-Jung Kim","doi":"10.1002/biot.70074","DOIUrl":"https://doi.org/10.1002/biot.70074","url":null,"abstract":"<p>Hydrogen (H<sub>2</sub>) is a clean, renewable, and sustainable energy source that holds great promise as an alternative fuel and is expected to play a central role in the future transportation energy economy. However, the hydrogen yield from microorganisms remains insufficient, presenting a significant challenge. Biohydrogen (bio-H<sub>2</sub>) production pathways are well established and can be categorized into four main processes: (1) direct biological photolysis of water by green algae; (2) indirect biological photolysis by cyanobacteria, a combination of green algae and photosynthetic microorganisms, or a separate two-stage photolysis using only green algae; (3) photo-fermentation by purple bacteria, photosynthetic bacteria, or fermentative bacteria; and (4) dark anaerobic fermentation by fermentative bacteria. Among these processes, dark fermentation shows great potential for practical applications, such as organic waste treatment. This review summarizes recent advances in bio-H<sub>2</sub> production, including both fundamental research and applied studies.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70074","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144716885","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":"Combinatorial Strategy of Modular Metabolic Engineering and Fermentation Optimization Jointly Improved Gibberellic Acid Production in Fusarium fujikuroi","authors":"Chun-Yue Weng, Jia-Yi Han, Zhi-Tao Dong, Zhi-Qiang Liu, Yu-Guo Zheng","doi":"10.1002/biot.70088","DOIUrl":"https://doi.org/10.1002/biot.70088","url":null,"abstract":"<div>\u0000 \u0000 <p>Gibberellic acid 3 (GA<sub>3</sub>), a diterpenoid phytohormone industrially biosynthesized by <i>Fusarium fujikuroi</i>, serves as a pivotal plant growth regulator with extensive agricultural applications. Currently, industrial GA<sub>3</sub> production predominantly relies on prolonged submerged microbial fermentation with <i>F. fujikuroi</i> as the main production strain, valued for its native biosynthetic capacity. Nevertheless, large-scale industrialization of GA<sub>3</sub> remains constrained by low production yields. In this study, a systematic multimodular metabolic engineering framework was implemented to enhance GA₃ biosynthesis in <i>F. fujikuroi</i>. The engineering strategy encompassed four synergistic modules: reinforcement of fatty acid biosynthesis, augmentation of acetyl-CoA metabolic flux, optimization of redox cofactor homeostasis, and overexpression of the positive transcriptional regulator. This integrated approach yielded the engineered strain OE: <i>Lae1-AGP3</i> demonstrating a 2.58 g/L GA₃ titer in shake-flask fermentation. Subsequent bioprocess optimization through exogenous fatty acid supplementation further elevated GA<sub>3</sub> production to 2.86 g/L, representing a 10.9% increase. This study demonstrates the feasibility of coordinated metabolic modifications for improving GA<sub>3</sub> biosynthesis in <i>F. fujikuroi</i>, offering practical insights for overcoming productivity limitations in fungal secondary metabolite fermentation processes.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144716884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Isabella Frighetto Bomfiglio, Isabelli Seiler de Medeiros Mendes, Diego Bonatto
{"title":"A Review of DNA Restriction-Free Overlapping Sequence Cloning Techniques for Synthetic Biology","authors":"Isabella Frighetto Bomfiglio, Isabelli Seiler de Medeiros Mendes, Diego Bonatto","doi":"10.1002/biot.70084","DOIUrl":"https://doi.org/10.1002/biot.70084","url":null,"abstract":"<div>\u0000 \u0000 <p>DNA cloning methods are fundamental tools in molecular biology, synthetic biology, and genetic engineering that enable precise DNA manipulation for various scientific and biotechnological applications. This review systematically summarizes the major restriction-free overlapping sequence cloning (RFOSC) techniques currently used in synthetic biology and examines their development, efficiency, practicality, and specific applications. In vitro methods, including Gibson Assembly, Circular Polymerase Extension Cloning (CPEC), Polymerase Incomplete Primer Extension (PIPE), Overlap Extension Cloning (OEC), Uracil DNA Glycosylase-based Cloning (UDG-Cloning), and commercially available techniques such as In-Fusion, have been discussed alongside hybrid approaches such as Ligation-Independent Cloning (LIC), Sequence-Independent Cloning (SLIC), and T5 Exonuclease-Dependent Assembly (TEDA). Additionally, in vivo methods leveraging host recombination machinery, including Yeast Homologous Recombination (YHR), In Vivo Assembly (IVA), Transformation-Associated Recombination (TAR), and innovative approaches such as Phage Enzyme-Assisted Direct Assembly (PEDA), are critically evaluated. The review highlights that method selection should consider individual research projects’ scale, complexity, and specific needs, noting that no single technique is universally optimal. Future trends suggest the increased integration of enzymatic efficiency, host versatility, and automation, broadening the accessibility and capabilities of DNA assembly technologies.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144705592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pik K. Chan, Chao-Hsiang (Richard) Wu, Yaokai Duan, Jiu-Li Song, Chetan T. Goudar
{"title":"Automatic Assay Preparation Platform (A2P2) for Real-Time Critical Quality Attributes Monitoring of Cell Culture Samples","authors":"Pik K. Chan, Chao-Hsiang (Richard) Wu, Yaokai Duan, Jiu-Li Song, Chetan T. Goudar","doi":"10.1002/biot.70082","DOIUrl":"https://doi.org/10.1002/biot.70082","url":null,"abstract":"<div>\u0000 \u0000 <p>As the biopharmaceuticals industry becomes increasingly competitive, improving speed-to-market and achieving “Right First Time” are key factors that propel a company to success. Building upon our foundational work on the Automatic Assay Preparation Platform (A2P2) initially introduced to enhance process analytical technology (PAT), this publication describes its innovative application for real-time sample preparation, acquisition, and monitoring of critical quality attributes (CQAs) for biotherapeutic production. Determination of CQA in cell culture is typically supported by analytical laboratories. Titer is determined before product purification by affinity capture column using a liquid handler. Purified product concentration is then measured before subjecting to a subsequent product quality assay. This process takes up to four separate instruments and several analysts to complete. The A2P2 system, embodying an end-to-end automated and autonomous PAT solution, is configured to streamline titer measurement, product purification, purified product concentration measurement, and CQA assay within a single run sequence using a modified ultrahigh performance liquid chromatography (UHPLC<span>)</span> instrument. By utilizing Chromeleon's System Suitability Tests (SST) and Intelligence Run Control (IRC) feature and the Agilent injector program, A2P2 significantly reduces analysts’ hands-on time and shortens result turnaround time for improved efficiency. This evolution of the A2P2 system not only reduces laboratory footprint but also positions it as an ideal solution for real-time bioprocess monitoring of CQA, further advancing our commitment to accelerating the delivery of safe and effective biotherapeutics.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144705439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Challenges Associated With the Use of Metal and Metal Oxide Nanoparticles as Antimicrobial Agents: A Review of Resistance Mechanisms and Environmental Implications","authors":"Mpho Phehello Ngoepe, Stiaan Schoeman, Saartjie Roux","doi":"10.1002/biot.70066","DOIUrl":"https://doi.org/10.1002/biot.70066","url":null,"abstract":"<p>The use of metal and metal oxide nanoparticles has been suggested as a means of combating antibiotic-resistant bacteria (ARB). This is due to the ability of nanoparticles to target numerous sites inside the bacterial cell. Microbes can, however, develop a resistance to hazardous environments. Soil microorganisms have evolved resistance to specific metals in soil by employing alternative survival strategies, like those adopted against antibiotics. Because of this survival mechanism, bacteria have been able to develop defense mechanisms to deal with metallic nanoparticles. Resistance has evolved in human pathogens to therapies that use metallic nanoparticles, such as silver nanoparticles. Metallic nanoparticles and antibiotics have currently been proven to be ineffective against several infections. Due to these concerns, scientists are investigating whether nanoparticles might cause environmental harm and potentially breed microbes that are resistant to both inorganic and organic nanoparticles. The increased use of inorganic nanoparticles has thus been shown to result in contaminations in wastewater, facilitating horizontal gene transfer among bacterial populations. The resistance mechanism of metallic nanoparticles, role in antibiotic resistance, and a potential solution to the environment's toxicity from nanoparticles are all discussed in this review.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70066","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144705610","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}
Débora Trichez, Thályta Pacheco, Clara Vida G. C. Carneiro, Jessica C. Bergmann, João Ricardo M. de Almeida
{"title":"Metabolic Engineering of Komagataella phaffii and Process Optimization for Biosynthesis of 1,2,4-Butanetriol From Xylose","authors":"Débora Trichez, Thályta Pacheco, Clara Vida G. C. Carneiro, Jessica C. Bergmann, João Ricardo M. de Almeida","doi":"10.1002/biot.70085","DOIUrl":"https://doi.org/10.1002/biot.70085","url":null,"abstract":"<p>1,2,4-butanetriol (BTO) is a four-carbon polyol used as a precursor for synthesizing pharmaceuticals, polymers, and energetic plasticizers. The present study demonstrates the microbial production of BTO from xylose by engineered <i>Komagataella phaffii</i> yeast strains for the first time. The pathway was established through the overexpression of the enzymes xylose dehydrogenase (XylB), xylonate dehydratase (XylD), and 2-ketoacid decarboxylase (KDC). Two xylonate dehydratase genes, <i>xylD-CC</i> from <i>Caulobacter crescentus</i> and <i>xylD-HL</i> from <i>Halomonas lutea</i>, were evaluated in the constructions, both enabling BTO production. Furthermore, to improve BTO production, a central composite design analysis (CCD) was employed, identifying the best cultivation conditions to improve yeast performance. Under these optimized conditions, the engineered <i>K. phaffii</i> strain produced 1.3 g/L of BTO, achieving a 147% increase compared to the initial setup. Although further genetic engineering efforts are required to enhance BTO production, this study provides insights into potential improvement targets and highlights <i>K. phaffii</i> as a promising platform for the bio-based synthesis of chemical compounds like BTO.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70085","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144705611","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}