Biotechnology and Bioengineering最新文献

{"title":"Integrated Experimental and Mathematical Modeling to Guide Microbial Biocatalysis: Pseudomonas putida Conversion of L-Phenylalanine to trans-Cinnamic Acid","authors":"Sompot Antimanon,&nbsp;Sheila Ingemann Jensen,&nbsp;John M. Woodley","doi":"10.1002/bit.28897","DOIUrl":"10.1002/bit.28897","url":null,"abstract":"<div>\u0000 \u0000 <p><i>trans</i>-Cinnamic acid (<i>t</i>CA) can be produced by the deamination of L-phenylalanine using phenylalanine ammonia-lyase (PAL). In bioprocesses, optimizing production processes to improve key performance metrics such as titer, rate, and yield has proved challenging. This study investigates <i>t</i>CA production in <i>Pseudomonas putida</i> KT2440 using a whole-cell biocatalyst expressing four different PAL genes. Among these, RmXAL showed the highest titer and rate. Biocatalysis at pH 8.5 and 37°C were identified as the best conditions, giving a <i>t</i>CA concentration of 2.65 g L⁻¹ and a production rate of 0.44 g L⁻¹ h⁻¹. To improve the metrics further, a decoupled bioprocess with various biocatalyst concentrations in the second stage was examined. With a whole-cell biocatalyst concentration of 30 g_DCW L⁻¹, optimal process parameters were achieved, giving a titer of 29.88 g L⁻¹, rate of 5.99 g L⁻¹ h⁻¹, yield on glucose of 0.27 g <i>t</i>CA g glucose⁻¹, and yield on L-phe of 0.75 g <i>t</i>CA g L-phe⁻¹. This study is the first report of a model that enables performance metrics to evaluate a suitable process for <i>t</i>CA production and provides valuable insights into <i>t</i>CA production using a decoupled bioprocess. This would appear applicable to larger-scale production, paving the way for an efficient and sustainable industrial process.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"525-537"},"PeriodicalIF":3.5,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142788702","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":"Design Principles of an Engineered Metastatic Niche for Monitoring of Cancer Progression","authors":"Guillermo Escalona,&nbsp;Ramon Ocadiz-Ruiz,&nbsp;Jeffrey A. Ma,&nbsp;Ian A. Schrack,&nbsp;Brian C. Ross,&nbsp;Alexis K. Morrison,&nbsp;Jacqueline S. Jeruss,&nbsp;Lonnie D. Shea","doi":"10.1002/bit.28895","DOIUrl":"10.1002/bit.28895","url":null,"abstract":"<p>Across many types of cancer, metastatic disease is associated with a substantial decrease in 5-year survival rates relative to only a localized primary tumor. Many patients self-report metastatic disease due to disruption of normal organ or tissue function, and earlier detection could enable treatment with a lower burden of disease. We have previously reported a subcutaneous biomaterial implant for early detection by serving as an engineered metastatic niche, which has been reported to recruit tumor cells before colonization of solid organs. In this report, we investigated the design principles of the scaffold and defined the conditions for use in disease detection. Using the metastatic 4T1 triple-negative breast cancer model, we identified that a porous structure was essential to capture tumor and immune cells. Scaffolds of multiple diameters were investigated for their ability to serve as a metastatic niche, with a porous scaffold with a diameter as small as 2 mm identifying disease accurately. Additionally, scaffolds that had been in vivo for 1–5 weeks were able to identify disease accurately. Finally, the sensitivity of the scaffold relative to liquid biopsies was analyzed, with scaffolds accurately detecting disease at earlier time points than liquid biopsy. Collectively, these studies inform the design principles and use conditions for porous scaffolds to detect metastatic disease.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"631-641"},"PeriodicalIF":3.5,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28895","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142763076","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":"Plant-Made Vaccines Targeting Enteric Pathogens—Safe Alternatives for Vaccination in Developing Countries","authors":"Edgar Trujillo,&nbsp;Carlos Angulo","doi":"10.1002/bit.28876","DOIUrl":"10.1002/bit.28876","url":null,"abstract":"<div>\u0000 \u0000 <p>Enteric diseases by pathogenic organisms are one of the leading causes of death worldwide, particularly in low-income countries. Despite antibiotics, access to clean water and vaccination are the most economically affordable options to prevent those infections and their health consequences. Vaccines, such as those approved for rotavirus and cholera, have played a key role in preventing several enteric diseases. However, vaccines for other pathogens are still in clinical trials. Distribution and cost remain significant barriers to vaccine access in developing regions due to poor healthcare infrastructure, cold-chain requirements, and high production costs. Plant-made vaccines offer a promising alternative to address these challenges. Plants can be easily grown, lowering production costs, and can be administered in oral forms, potentially eliminating cold-chain dependency. Although there are some promising prototypes of vaccines produced in plants, challenges remain, including yields and achieving sufficient immunogenicity. This review aims to describe common enteric pathogens and available vaccines, followed by a strategic summary of plant-made vaccine development and a discussion of plant-made enteric vaccine prototypes. Trends to overcome the key challenges for plant-made vaccines are identified and placed in perspective for the development of affordable and effective vaccines for populations at the highest risk of enteric diseases.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"457-480"},"PeriodicalIF":3.5,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142758584","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":"Exploration of the Out-of-Phase Phenomenon in Shake Flasks by CFD Calculations of Volumetric Power Input, kLa Value and Shear Rate at Elevated Viscosity","authors":"Carl Dinter,&nbsp;Andreas Gumprecht,&nbsp;Matthias Alexander Menze,&nbsp;Amizon Azizan,&nbsp;Sven Hansen,&nbsp;Jochen Büchs","doi":"10.1002/bit.28892","DOIUrl":"10.1002/bit.28892","url":null,"abstract":"<p>Culture broth with secreted macromolecules and culture broth of filamentous fungi showing disperse growth exhibit elevated viscosity, usually with shear-thinning flow behavior. High viscosity, however, poses a serious challenge in the production and research of these compounds and organisms. It commonly causes insufficient mixing and oxygen transfer in large- and small-scale bioreactors. Computational Fluid dynamics (CFD) has been proven to be a valuable tool for the computation of important bioprocess parameters. The published literature for small-scale shaken bioreactors, especially shake flasks, however, almost exclusively focuses on water-like viscosity. In this paper, a previously published CFD model for 250 mL shake flasks was used to simulate experiments at high viscosities of up to 100 mPa·s. Compared to experimental data, the CFD model accurately predicted the liquid distribution and computed the volumetric power input with a deviation of less than 7% and the k_La value within a factor of two, compared to the k_La correlation from Henzler and Schedel. Furthermore, a novel approach to compute the shear rate was tested. Lastly, new insights into the out-of-phase phenomenon were gained. The presented data confirms the usefulness of the already established critical phase numbers of 0.91 and 1.26, while underlying the fundamentally smooth transition from in-phase to out-of-phase operating conditions.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"509-524"},"PeriodicalIF":3.5,"publicationDate":"2024-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28892","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142756237","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-Efficiency Genome Editing in Naturally Isolated Aeromonas hydrophila and Edwardsiella Piscicida Using the CRISPR-Cas9 System","authors":"Jun Feng,&nbsp;Yuechao Ma,&nbsp;Dunhua Zhang,&nbsp;Yi Wang","doi":"10.1002/bit.28889","DOIUrl":"10.1002/bit.28889","url":null,"abstract":"<div>\u0000 \u0000 <p><i>Aeromonas hydrophila</i> and <i>Edwardsiella piscicida</i> are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. Additionally, both of them can act as opportunistic pathogens in humans, leading to severe infections. Efficient genome editing tools for these pathogens are essential for understanding their pathogenic mechanisms and physiological behaviors, enabling the development of targeted strategies to control and mitigate their effects. In this study, we adapted the CRISPR-Cas9 system for high-efficiency, marker-less genome editing in multiple naturally isolated strains of these two aquaculture pathogens. We developed a streamlined procedure that successfully generated deletion mutants of the <i>aerA</i> gene (encoding for aerolysin, a pore-forming toxin that plays a critical role in the pathogenicity) and the <i>gfp</i> insertion mutants in three naturally isolated <i>A. hydrophila</i> strains. Additionally, we deleted five putative hemolysin-encoding genes in both <i>A. hydrophila</i> ML10-51K and its <i>∆aerA</i> derivative. The same system was also applied to the naturally isolated <i>E. piscicida</i> S11-285 strain, successfully deleting the <i>ssaV</i> gene (a component of the Type III Secretion System—a critical virulence mechanism in many pathogenic bacteria). The methodologies developed herein could be broadly applied to other pathogenic strains from natural environments, providing valuable tools for studying bacterial pathogenesis and aiding in the development of effective control strategies.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"606-614"},"PeriodicalIF":3.5,"publicationDate":"2024-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142742795","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":"AtLEC2-Mediated Enhancement of Endoplasmic Reticulum-Targeted Foreign Protein Synthesis in Nicotiana benthamiana Leaves: Insights From Transcriptomic Analysis","authors":"Carolina G. Ocampo,&nbsp;Florencia Vignolles,&nbsp;Marina A. Pombo,&nbsp;Maria Laura Colombo,&nbsp;Hernan G. Rosli,&nbsp;Silvana Petruccelli","doi":"10.1002/bit.28893","DOIUrl":"10.1002/bit.28893","url":null,"abstract":"<div>\u0000 \u0000 <p>Many proteins used in industrial and pharmaceutical applications are typically synthesized within the secretory pathway. While yeast and mammalian cells have been engineered to enhance the production of endomembrane-targeted proteins, similar strategies in plant cells remain underexplored. This study investigates the potential of arabidopsis leafy cotyledon 2 (AtLEC2), a key regulator of seed development, to enhance the production of proteins targeted to the endoplasmic reticulum (ER) in <i>Nicotiana benthamiana</i> leaves. Through transient expression experiments, we demonstrate that AtLEC2 selectively increases the production of ER-targeted GUS without affecting its cytosolic variant. Moreover, leaves agroinfiltrated with AtLEC2 show a significant increase in ER-GFP accumulation compared to controls lacking AtLEC2. Transcriptomic analysis reveals that AtLEC2 promotes ribosome and chloroplast biogenesis, along with the upregulation of genes involved in photosynthesis, translation, and membrane synthesis. Notably, seed-specific poly(A) binding proteins involved in RNA stability and translation initiation, as well as 3-hydroxy-3-methylglutaryl coenzyme A reductase—linked to ER hypertrophy—are highly upregulated. This study establishes a novel connection between AtLEC2 and the enhancement of ER-targeted foreign protein synthesis, paving the way for innovative strategies in plant cellular engineering.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"615-630"},"PeriodicalIF":3.5,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142726180","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":"Deciphering the Catalytic Proficiency and Mechanism of the N-Acetylglucosamine Deacetylase From Pantoea dispersa","authors":"Wentao Yang,&nbsp;Xiao Chen,&nbsp;Li Pang,&nbsp;Hong Tian,&nbsp;Liang Yang,&nbsp;Bo Xia","doi":"10.1002/bit.28894","DOIUrl":"10.1002/bit.28894","url":null,"abstract":"<div>\u0000 \u0000 <p>Glucosamine (GlcN) is a widely utilized amino monosaccharide. It is traditionally synthesized from <i>N</i>-acetylglucosamine (GlcNAc) via chemical processes that pose environmental threats. In pursuit of a greener alternative, our investigation explored biocatalysis with a <i>Pantoea dispersa</i> derived deacetylase (Pd-nagA), showcasing its efficacy as a catalyst in GlcN production. As a result, this work provides a comprehensive characterization of Pd-nagA, scrutinizes its enzymatic behavior, and delves into the deacetylation mechanism in detail. Heterologous expression methods were utilized for the production and isolation of Pd-nagA, followed by a kinetic evaluation highlighting its enzymatic activity. The complex interactions between the enzyme and its substrate were investigated by integrating classical molecular dynamics, quantum mechanics/molecular mechanics simulations, funnel metadynamics, and on-the-fly probability enhanced sampling techniques, thereby elucidating the precise deacetylation pathway. Rigorous computational analysis results demonstrated that Pd-nagA exhibited promising specificity and efficiency for GlcNAc with a high turnover rate. The catalytic residues central to the reaction were identified, and the underlying quantum reaction mechanism was detailed. Our findings suggest an approach to GlcN production using eco-friendly biocatalysis, positioning Pd-nagA at the forefront of industrial application not only because of its remarkable catalytic capabilities but also due to its potential for enzyme optimization.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"495-508"},"PeriodicalIF":3.5,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142726183","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":"Development of a Microphysiological System to Model Human Cancer Metastasis From the Colon to the Liver","authors":"Paula G. Miller,&nbsp;Emina Huang,&nbsp;Robert Fisher,&nbsp;Michael L. Shuler","doi":"10.1002/bit.28890","DOIUrl":"10.1002/bit.28890","url":null,"abstract":"<div>\u0000 \u0000 <p>We describe a novel device to mimic the metastasis of cancer cells from the colon into the liver in a human model. The colon mimic is connected to the liver model by a gravity-driven recirculating unidirectional flow of a blood surrogate and can mimic the five steps of the metastatic cascade: invasion in the colon, intravasation into the bloodstream, systemic transportation, extravasation into the liver, and colonization in the liver. The colon mimic uses established normal colon epithelial organoid cells (NL) and human umbilical vein endothelial cells (HUVEC) plated on opposite sides of a membrane. To better mimic the colon structure the NL side of the membrane is exposed to air to establish an air-liquid interface. The liver mimic consists of human liver sinusoidal endothelial cells (HHSEC) and epithelial hepatic cells (HepG2 C3A) plated in Matrigel on opposite sides of a membrane. Labeled colorectal cancer cells/clusters (CA) from organoids are introduced into an established normal colon epithelial cell (NL) layer from the same patient before assembly of the system or alternatively NL organoids and fluorescently labeled CA organoids from the same patient were prepared as a ratio of 10:1 NL:CA and established together before assembly of the system. Cell viability is greater than 85% in this system. We demonstrate that over 5 days of operation that the five steps of the metastatic cascade are replicated. This novel device allows an in vitro estimate of metastatic capability (as measured by using percentages of the labeled areas per device through ImageJ) in response to selected variables. In this study, the metastatic capability depends on the source of cancer cells (e.g., the patient), the clumping of cancer cells, glucose concentration, and oxygen levels (hypoxia). For the first time, this new in vitro system mimics all five steps of the metastatic cascade in a single device and provides a new device to probe and observe the process of metastasis in a human-based model in only 5 days. The rapid observation is due to the use of a high concentration of cancer cells in the colon (e.g. 10%) and the absence of the immune system. Our device makes it possible to probe aspects of each step of metastasis and interactions between steps.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 3","pages":"481-494"},"PeriodicalIF":3.5,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142713357","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":"Sequence and Configuration of a Novel Bispecific Antibody Format Impacts Its Production Using Chinese Hamster Ovary (CHO) Cells","authors":"Hirra Hussain,&nbsp;Angelica M. S. Ozanne,&nbsp;Tulshi Patel,&nbsp;Davide Vito,&nbsp;Mark Ellis,&nbsp;Matthew Hinchliffe,&nbsp;David P. Humphreys,&nbsp;Paul E. Stephens,&nbsp;Bernie Sweeney,&nbsp;James White,&nbsp;Alan J. Dickson,&nbsp;C. Mark Smales","doi":"10.1002/bit.28879","DOIUrl":"10.1002/bit.28879","url":null,"abstract":"<p>There are a number of new format antibody-inspired molecules with multiple antigen binding capabilities in development and clinical evaluation. Here, we describe the impact of the sequence and configuration of a unique bispecific antibody format (termed BYbe) using a panel of four BYbe's and the three IgG1s from which they were derived on their production in a Chinese hamster ovary (CHO) cell expression system. Following transfection and selection, one bispecific antibody format yielded fewer mini-pools in comparison to the other bispecific cell pools. When the top 12 expressing stable mini-pools of all BYbe configurations and sequences were evaluated, both the dsscFv sequence and antibody chain configuration or placement directly impacted productivity. The cell-specific productivity (qP, pg/cell/day) was lower in all BYbe cell pools compared to the IgG1 cell lines. However, when the actual molecules/cell/day produced were considered, three of the four bispecific cell pools outproduced the parental IgG1 cell pools. While gene copy number did not correlate to productivity, mRNA analysis showed that for specific BYbe formats there was a strong correlation with productivity. In summary, we describe how bispecific antibody format configuration impacts the cell line construction process and yield of product from CHO cells.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 2","pages":"435-444"},"PeriodicalIF":3.5,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28879","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142713104","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":"Advances in Artificially Designed Antibacterial Active Antimicrobial Peptides","authors":"Ying Guo,&nbsp;Muhammad Haris Raza Farhan,&nbsp;Fei Gan,&nbsp;Xiaohan Yang,&nbsp;Yuxin Li,&nbsp;Lingli Huang,&nbsp;Xu Wang,&nbsp;Guyue Cheng","doi":"10.1002/bit.28886","DOIUrl":"10.1002/bit.28886","url":null,"abstract":"<div>\u0000 \u0000 <p>Antibacterial resistance has emerged as a significant global concern, necessitating the urgent development of new antibacterial drugs. Antimicrobial peptides (AMPs) are naturally occurring peptides found in various organisms. Coupled with a wide range of antibacterial activity, AMPs are less likely to develop drug resistance and can act as potential agents for treating bacterial infections. However, their characteristics, such as low activity, instability, and toxicity, hinder their clinical application. Consequently, researchers are inclined towards artificial design and optimization based on natural AMPs. This review discusses the research advancements in the field of artificial designing and optimization of various AMPs. Moreover, it highlights various strategies for designing such peptides, aiming to provide valuable insights for developing novel AMPs.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 2","pages":"247-264"},"PeriodicalIF":3.5,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142685988","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}