Biotechnology and Bioengineering最新文献

{"title":"A Novel Screening System to Characterize and Engineer Quorum Quenching Lactonases","authors":"Kitty Sompiyachoke,&nbsp;Joseph Bravo,&nbsp;Rakesh Sikdar,&nbsp;Jowan Abdullah,&nbsp;Mikael H. Elias","doi":"10.1002/bit.28928","DOIUrl":"10.1002/bit.28928","url":null,"abstract":"<p><i>N</i>-acyl <span>l</span>-homoserine lactones are signaling molecules used by numerous bacteria in quorum sensing. Some bacteria encode lactonases, which can inactivate these signals. Lactonases were reported to inhibit quorum sensing-dependent phenotypes, including virulence and biofilm. As bacterial signaling is dependent on the type of molecule used, lactonases with high substrate specificity are desirable for selectively targeting species in communities. Lactonases characterized from nature show limited diversity in substrate preference, making their engineering appealing but complicated by the lack of convenient assays for evaluating lactonase activity. We present a medium-throughput lactonase screening system compatible with lysates that couples the ring opening of <i>N</i>-acyl <span>l</span>-homocysteine thiolactones with 5,5-dithio-bis-(2-nitrobenzoic acid) to generate a chromogenic signal. We show that this system is applicable to lactonases from diverse protein families and demonstrate its utility by screening mutant libraries of GcL lactonase from <i>Parageobacillus caldoxylosilyticus</i>. Kinetic characterization corroborated the screening results with thiolactonase and homoserine lactonase activity levels. This system identified GcL variants with altered specificity: up to 1900-fold lower activity for long-chain <i>N-</i>acyl <span>l</span>-homoserine lactone substrates and ~38-fold increase in preference for short-chain substrates. Overall, this new system substantially improves the evaluation of lactonase activity and will facilitate the identification and engineering of quorum quenching enzymes.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"922-935"},"PeriodicalIF":3.5,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28928","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142989379","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":"Creating a Halotolerant Degrader for Efficient Mineralization of p-Nitrophenol-Substituted Organophosphorus Pesticides in High-Saline Wastewater","authors":"Yujie Liu,&nbsp;Weini Xiong,&nbsp;Yuting Jiang,&nbsp;Yan Meng,&nbsp;Wanwan Zhao,&nbsp;Chao Yang,&nbsp;Ruihua Liu","doi":"10.1002/bit.28923","DOIUrl":"10.1002/bit.28923","url":null,"abstract":"<div>\u0000 \u0000 <p>The bioaugmentation performance is severely reduced in the treatment of high-saline pesticide wastewater because the growth and degradation activity of pesticide degraders are significantly inhibited by high salt concentrations. In this study, a heterologous biodegradation pathway comprising the seven genes <i>mpd/pnpABCDEF</i> responsible for the bioconversion of <i>p</i>-nitrophenol (PNP)-substituted organophosphorus pesticides (OPs) into β-oxoadipate and the genes encoding <i>Vitreoscilla</i> hemoglobin (VHb) and green fluorescent protein (GFP) were integrated into the genome of a salt-tolerant chassis <i>Halomonas cupida</i> J9, to generate a genetically engineered halotolerant degrader J9U-MP. RT-PCR assays demonstrated that the nine exogenous genes are successfully transcribed to mRNA in J9U-MP. Gas chromatography analysis of methyl parathion (MP) and its intermediates demonstrated that the expressed MP hydrolase and PNP-degrading enzymes PnpABCD show obvious degradation activity toward the specific substrates in J9U-MP. Stable isotope analysis showed that J9U-MP is able to efficiently convert ¹³C₆-PNP into ¹³CO₂, demonstrating the complete mineralization of MP in high-salt media. J9U-MP is genetically stable during passage culture, and genomic integration of exogenous genes does not negatively influence the growth of J9U-MP. Under oxygen-limited conditions, VHb-expressing J9U-MP does not show obvious growth inhibition and a significant reduction in the MP degradation rate. A real-time monitoring system with enhanced GFP is used to track the motion and activity of J9U-MP during bioremediation. Moreover, 50 mg/L MP and its intermediates (i.e., PNP and HQ) were completely degraded by J9U-MP within 12 h in wastewater supplemented with 60 g/L NaCl. After 3 days of incubation, 25 mg/L ¹³C₆-PNP was converted into ¹³CO₂ by J9U-MP in wastewater supplemented with 60 g/L NaCl. Our results highlight the power of synthetic biology for creating new halotolerant pollutant-mineralizing strains. The strong competitive advantages of J9U-MP in high-salinity and low-oxygen environments make this degrader suitable for in situ bioaugmentation of OP wastewater.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"936-947"},"PeriodicalIF":3.5,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142987686","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":"A Paradigm of Computer Vision and Deep Learning Empowers the Strain Screening and Bioprocess Detection","authors":"Feng Xu,&nbsp;Lihuan Su,&nbsp;Yuan Wang,&nbsp;Kaihao Hu,&nbsp;Ling Liu,&nbsp;Rong Ben,&nbsp;Hao Gao,&nbsp;Ali Mohsin,&nbsp;Ju Chu,&nbsp;Xiwei Tian","doi":"10.1002/bit.28926","DOIUrl":"10.1002/bit.28926","url":null,"abstract":"<div>\u0000 \u0000 <p>High-performance strain and corresponding fermentation process are essential for achieving efficient biomanufacturing. However, conventional offline detection methods for products are cumbersome and less stable, hindering the “Test” module in the operation of “Design-Build-Test-Learn” cycle for strain screening and fermentation process optimization. This study proposed and validated an innovative research paradigm combining computer vision with deep learning to facilitate efficient strain selection and effective fermentation process optimization. A practical framework was developed for gentamicin C1a titer as a proof-of-concept, using computer vision to extract different color space components across various cultivation systems. Subsequently, by integrating data preprocessing with algorithm design, a prediction model was developed using 1D-CNN model with Z-score preprocessing, achieving a correlation coefficient (<i>R</i>²) of 0.9862 for gentamicin C1a. Furthermore, this model was successfully applied for high-yield strain screening and real-time monitoring of the fermentation process and extended to rapid detection of fluorescent protein expression in promoter library construction. The visual sensing research paradigm proposed in this study provides a theoretical framework and data support for the standardization and digital monitoring of color-changing bioprocesses.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"817-832"},"PeriodicalIF":3.5,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142987692","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":"Dynamic Culture of Bioprinted Liver Tumor Spheroids in a Pillar/Perfusion Plate for Predictive Screening of Anticancer Drugs","authors":"Pranav Joshi,&nbsp;Hamilton Silva do Nascimento,&nbsp;Soo-Yeon Kang,&nbsp;Minseong Lee,&nbsp;Manav Goud Vanga,&nbsp;Sang-Hyun Lee,&nbsp;Bosung Ku,&nbsp;Moyses dos Santos Miranda,&nbsp;Moo-Yeal Lee","doi":"10.1002/bit.28924","DOIUrl":"10.1002/bit.28924","url":null,"abstract":"<div>\u0000 \u0000 <p>Recent advancements in three-dimensional (3D) cell culture technologies, such as cell spheroids, organoids, and 3D bioprinted tissue constructs, have significantly improved the physiological relevance of in vitro models. These models better mimic tissue structure and function, closely emulating in vivo characteristics and enhancing phenotypic analysis, critical for basic research and drug screening in personalized cancer therapy. Despite their potential, current 3D cell culture platforms face technical challenges, which include user-unfriendliness in long-term dynamic cell culture, incompatibility with rapid cell encapsulation in biomimetic hydrogels, and low throughput for compound screening. To address these issues, we developed a 144-pillar plate with sidewalls and slits (144PillarPlate) and a complementary 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for dynamic 3D cell culture and predictive compound screening. To accelerate biomimetic tissue formation, small Hep3B liver tumor spheroids suspended in alginate were printed and encapsulated on the 144PillarPlate rapidly by using microsolenoid valve-driven 3D bioprinting technology. The microarray bioprinting technology enabled precise and rapid loading of small spheroids in alginate on the pillar plate, facilitating reproducible and scalable formation of large tumor spheroids with minimal manual intervention. The bioprinted Hep3B spheroids on the 144PillarPlate were dynamically cultured in the 144PerfusionPlate and tested with anticancer drugs to measure drug effectiveness and determine the concentration required to inhibit 50% of the cell viability (IC₅₀ value). The perfusion plate enabled the convenient dynamic culture of tumor spheroids and facilitated the dynamic testing of anticancer drugs with increased sensitivity. It is envisioned that the integration of microarray bioprinting of tumor spheroids onto the pillar plate, along with dynamic 3D cell culture in the perfusion plate, could more accurately replicate tumor microenvironments. This advancement has the potential to enhance the predictive drug screening process in personalized cancer therapy significantly.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"995-1009"},"PeriodicalIF":3.5,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142987684","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":"Manganese Peroxidase Participates in the Liquid–Solid–Gas Triphase Regulation on Microbial Degradation of Lignocellulose in Solid-State Fermentation","authors":"Lei Zhu,&nbsp;Juan Ding,&nbsp;Wenjing Xue,&nbsp;Shu Zhou,&nbsp;Longyu Wang,&nbsp;Ailiang Jiang,&nbsp;Mingwen Zhao,&nbsp;Qin He,&nbsp;Ang Ren","doi":"10.1002/bit.28927","DOIUrl":"10.1002/bit.28927","url":null,"abstract":"<div>\u0000 \u0000 <p>The three-phase structure of solid-state fermentation (SSF) directly affects substrate degradation and fermentation efficiency. However, the mechanism of three-phase regulation on lignocellulose utilization and microbial metabolism is still unclear. Based on comparative transcriptome analysis, a lignocellulose degrading enzyme, manganese peroxidase (GlMnP), which was significantly affected by water stress meanwhile related to triphase utilization, was screened to reveal the mechanism using <i>Ganoderma lucidum</i> as the reference strain. The results showed that GlMnP directly participates in lignocellulose degradation by positively regulating the activity of carboxymethylcellulase (CMCase), filter paper (FPAse), and laccase (LACase) enzymes, and indirectly participates in lignocellulose degradation by negatively regulating the redox levels in microorganisms. In addition, GlMnP can also control microbial glycolysis rate to enhance lignocellulose utilization. The results indicated that GlMnP participates in the liquid–solid–gas triphase regulation on lignocellulose degradation by <i>G. lucidum</i> in SSF.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"908-921"},"PeriodicalIF":3.5,"publicationDate":"2025-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142981671","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 Gene and Protein Switches for Regulation of Lineage-Specifying Transcription Factors","authors":"Ana P. Teixeira,&nbsp;Nik Franko,&nbsp;Martin Fussenegger","doi":"10.1002/bit.28920","DOIUrl":"10.1002/bit.28920","url":null,"abstract":"<div>\u0000 \u0000 <p>Human pluripotent stem cells (hPSCs) can be differentiated in vitro to an increasing number of mature cell types, presenting significant promise for addressing a wide range of diseases and studying human development. One approach to further enhance stem cell differentiation methods would be to coordinate multiple inducible gene or protein switches to operate simultaneously within the same cell, with minimal cross-interference, to precisely regulate a network of lineage-specifying transcription factors (TFs) to guide cell fate decisions. Therefore, in this study, we designed and tested various mammalian gene and protein switches responsive to clinically safe small-molecule inhibitors of viral proteases. First, we leveraged hepatitis C virus and human rhinovirus proteases to control the activity of chimeric transcription factors, enabling gene expression activation exclusively in the presence of protease inhibitors and achieving high fold-inductions in hPSC lines. Second, we built single-chain protein switches regulating the activity of three differentiation-related pancreatic TFs, MafA, Pdx1, and Ngn3, each engineered with a protease cleavage site within its structure and having the corresponding protease fused at one terminus. While variants lacking the protease retained most of the unmodified TF activity, the attachment of the protease significantly decreased the activity, which could be rescued upon addition of the corresponding protease inhibitor. We confirmed the functionality of these protein switches for simultaneously controlling the activity of three TFs with a common input molecule, as well as the orthogonality of each protease-based system to independently regulate two TFs. Finally, we validated these very compact systems for precisely controlling TF activity in hPSCs. Our results suggest that they will be valuable tools for research in both developmental biology and regenerative medicine.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"1051-1061"},"PeriodicalIF":3.5,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142968456","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":"Heterologous Expression and Optimization of Fermentation Conditions for Recombinant Ikarugamycin Production","authors":"Julia K. Evers,&nbsp;Anna Glöckle,&nbsp;Monique Wiegand,&nbsp;Sebastian Schuler,&nbsp;Manuel Einsiedler,&nbsp;Tobias A. M. Gulder","doi":"10.1002/bit.28919","DOIUrl":"10.1002/bit.28919","url":null,"abstract":"<p>Ikarugamycin is a member of the natural product family of the polycyclic tetramate macrolactams (PoTeMs). The compound exhibits a diverse range of biological activities, including antimicrobial, antiprotozoal, anti-leukemic, and anti-inflammatory properties. In addition, it interferes with several crucial cellular functions, such as oxidized low-density lipoprotein uptake in macrophages, Nef-induced CD4 cell surface downregulation, and mechanisms of endocytosis. It is, therefore, used as a tool compound to study diverse biological processes. However, ikarugamycin commercial prices are very high, with up to 1300 € per 1 mg, thus limiting its application. We, therefore, set out to develop a high-yielding recombinant production platform of ikarugamycin by screening different expression vectors, recombinant host strains, and cultivation conditions. Overall, this has led to overproduction levels of more than 100 mg/L, which, together with a straightforward purification protocol, establishes biotechnological access to affordable ikarugamycin enabling its increased use in biomedical research in the future.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"974-982"},"PeriodicalIF":3.5,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28919","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142962642","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":"Biotechnology and Bioengineering: Volume 122, Number 2, February 2025","authors":"","doi":"10.1002/bit.28742","DOIUrl":"10.1002/bit.28742","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 2","pages":"243-246"},"PeriodicalIF":3.5,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28742","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142940433","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":"The Effect of Accessibility of Insoluble Substrate on the Overall Kinetics of Enzymatic Degradation","authors":"Zdeněk Petrášek,&nbsp;Bernd Nidetzky","doi":"10.1002/bit.28921","DOIUrl":"10.1002/bit.28921","url":null,"abstract":"<p>The enzymatic reaction kinetics on cellulose and other solid substrates is limited by the access of the enzyme to the reactive substrate sites. We introduce a general model in which the reaction rate is determined by the active surface area, and the resulting kinetics consequently reflects the evolving relationship between the exposed substrate surface and the remaining substrate volume. Two factors influencing the overall surface-to-volume ratio are considered: the shape of the substrate particles, characterized by a single numerical parameter related to its dimensionality, and the distribution of the particle sizes. The model is formulated in a form of simple analytical equations, enabling fast and efficient application to experimental data, and facilitating its incorporation into more detailed and complex models. The application of the introduced formalism exploring its potential to account for the observed reaction rate is demonstrated on two examples: the derivation of particle size distribution from experimentally determined reaction kinetics, and the prediction of reaction slowdown from experimental particle size distribution.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"895-907"},"PeriodicalIF":3.5,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28921","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142935059","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":"Genome-Scale Community Model-Guided Development of Bacterial Coculture for Lignocellulose Bioconversion","authors":"Pritam Kundu,&nbsp;Amit Ghosh","doi":"10.1002/bit.28918","DOIUrl":"10.1002/bit.28918","url":null,"abstract":"<p>Microbial communities have shown promising potential in degrading complex biopolymers, producing value-added products through collaborative metabolic functionality. Hence, developing synthetic microbial consortia has become a predominant technique for various biotechnological applications. However, diverse microbial entities in a consortium can engage in distinct biochemical interactions that pose challenges in developing mutualistic communities. Therefore, a systems-level understanding of the inter-microbial metabolic interactions, growth compatibility, and metabolic synergisms is essential for developing effective synthetic consortia. This study demonstrated a genome-scale community modeling approach to assess the inter-microbial interaction pattern and screen metabolically compatible bacterial pairs for designing the lignocellulolytic coculture system. Here, we have investigated the pairwise growth and biochemical synergisms among six termite gut bacterial isolates by implementing flux-based parameters, i.e., pairwise growth support index (PGSI) and metabolic assistance (PMA). Assessment of the PGSI and PMA helps screen nine beneficial bacterial pairs that were validated by designing a coculture experiment with lignocellulosic substrates. For the cocultured bacterial pairs, the experimentally measured enzymatic synergisms (DES) showed good coherence with model-derived biochemical compatibility (PMA), which explains the fidelity of the in silico predictions. The highest degree of enzymatic synergisms has been observed in <i>C. denverensis</i> P3 and <i>Brevibacterium</i> sp P5 coculture, where the total cellulase activity has been increased by 53%. Hence, the flux-based assessment of inter-microbial interactions and metabolic compatibility helps select the best bacterial coculture system with enhanced lignocellulolytic functionality. The flux-based parameters (PGSI and PMA) in the proposed community modeling strategy will help optimize the composition of microbial consortia for developing synthetic microcosms for bioremediation, bioengineering, and biomedical applications.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"1010-1024"},"PeriodicalIF":3.5,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28918","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142929716","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}