Rihui Kang, Jiaxing Wu, Rong Cheng, Meng Li, Luxiao Sang, Hulin Zhang, Shengbo Sang
3D bioprinting technology is widely used in biomedical fields such as tissue regeneration and constructing pathological model. The prevailing printing technique is extrusion-based bioprinting. In this printing method, the bioink needs to meet both printability and functionality, which are often conflicting requirements. Therefore, this study has developed an innovative microvalve-based equipment, incorporating components such as pressure control, a three-dimensional motion platform, and microvalve. Here, we present a droplet-based method for constructing complex three-dimensional structures. By leveraging the rapid switching characteristics of the microvalve, this equipment can achieve precise printing of bio-materials with viscosities as low as 10mPa·s, significantly expanding the biofabrication window for bioinks. This technology is of great significance for 3D bioprinting in tissue engineering and lays a solid foundation for the construction of complex artificial organ tissues.
{"title":"3D bioprinting technology and equipment based on microvalve control","authors":"Rihui Kang, Jiaxing Wu, Rong Cheng, Meng Li, Luxiao Sang, Hulin Zhang, Shengbo Sang","doi":"10.1002/bit.28850","DOIUrl":"https://doi.org/10.1002/bit.28850","url":null,"abstract":"3D bioprinting technology is widely used in biomedical fields such as tissue regeneration and constructing pathological model. The prevailing printing technique is extrusion-based bioprinting. In this printing method, the bioink needs to meet both printability and functionality, which are often conflicting requirements. Therefore, this study has developed an innovative microvalve-based equipment, incorporating components such as pressure control, a three-dimensional motion platform, and microvalve. Here, we present a droplet-based method for constructing complex three-dimensional structures. By leveraging the rapid switching characteristics of the microvalve, this equipment can achieve precise printing of bio-materials with viscosities as low as 10mPa·s, significantly expanding the biofabrication window for bioinks. This technology is of great significance for 3D bioprinting in tissue engineering and lays a solid foundation for the construction of complex artificial organ tissues.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142237021","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}
In eukaryotes, gene expression typically requires individual promoter and terminator for each gene, making the expression of multiple genes tedious and sometimes too difficult to handle. This is especially true for underdeveloped nonmodel organisms with few genetic engineering tools and genetic elements such as Rhodosporidium toruloides. In contrast, polycistronic expression offers advantages such as smaller size and ease of cloning. Here we report the development of a multigene expression system using 2A peptides in R. toruloides. First, twenty‐two 2A peptides were evaluated for their cleavage efficiencies, which ranged from 33.65% to 93.32%. Subsequently, the 2A peptide of ERBV‐1 with the highest efficiency was selected to enable simultaneous expression of four proteins. In addition, we demonstrated the optimization of the α‐linolenic acid biosynthetic pathway using ERBV‐1 peptide mediated polycistronic expression, which increased the α‐linolenic acid production by 104.72%. These results suggest that using ERBV‐1 peptide is an efficient strategy for multigene expression in R. toruloides.
{"title":"Development of a multigene expression system using 2A peptides in Rhodosporidium toruloides","authors":"Xiao Guo, Zhenzhen Bai, Huimin Zhao, Shuobo Shi","doi":"10.1002/bit.28843","DOIUrl":"https://doi.org/10.1002/bit.28843","url":null,"abstract":"In eukaryotes, gene expression typically requires individual promoter and terminator for each gene, making the expression of multiple genes tedious and sometimes too difficult to handle. This is especially true for underdeveloped nonmodel organisms with few genetic engineering tools and genetic elements such as <jats:italic>Rhodosporidium toruloides</jats:italic>. In contrast, polycistronic expression offers advantages such as smaller size and ease of cloning. Here we report the development of a multigene expression system using 2A peptides in <jats:italic>R. toruloides</jats:italic>. First, twenty‐two 2A peptides were evaluated for their cleavage efficiencies, which ranged from 33.65% to 93.32%. Subsequently, the 2A peptide of ERBV‐1 with the highest efficiency was selected to enable simultaneous expression of four proteins. In addition, we demonstrated the optimization of the α‐linolenic acid biosynthetic pathway using ERBV‐1 peptide mediated polycistronic expression, which increased the α‐linolenic acid production by 104.72%. These results suggest that using ERBV‐1 peptide is an efficient strategy for multigene expression in <jats:italic>R. toruloides</jats:italic>.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142236281","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}
Chen-Yu Chen, Dana Motabar, Fauziah Rahma Zakaria, Eunkyoung Kim, Benjamin Wu, Gregory F. Payne, William E. Bentley
We report the integration of 3D printing, electrobiofabrication, and protein engineering to create a device that enables near real-time analysis of monoclonal antibody (mAb) titer and quality. 3D printing was used to create the macroscale architecture that can control fluidic contact of a sample with multiple electrodes for replicate measurements. An analysis “chip” was configured as a “snap-in” module for connecting to a 3D printed housing containing fluidic and electronic communication systems. Electrobiofabrication was used to functionalize each electrode by the assembly of a hydrogel interface containing biomolecular recognition and capture proteins. Specifically, an electrochemical thiol oxidation is used to assemble a thiolated polyethylene glycol hydrogel, that in turn is covalently coupled to either a cysteine-tagged protein G that binds the antibody's Fc region or a lectin that binds the glycans of target mAb analytes. We first show the design, assembly, and testing of the hardware device. Then, we show the transition of a step-by-step sensing methodology (e.g., mix, incubate, wash, mix, incubate, wash, measure) into the current method where functionalization, antibody capture, and assessment are performed in situ and in parallel channels. Both titer and glycan analyses were found to be linear with antibody concentration (to 0.2 mg/L). We further found the interfaces could be reused with remarkably similar results. Because the interface assembly and use are simple, rapid, and robust, we suggest this assessment methodology will be widely applicable, including for other biomolecular process development and manufacturing environments.
我们报告了三维打印、电子生物制造和蛋白质工程的整合，从而创造出一种可对单克隆抗体（mAb）滴度和质量进行近乎实时分析的设备。三维打印技术用于创建宏观架构，该架构可控制样品与多个电极的流体接触，以进行重复测量。分析 "芯片 "被配置为 "卡入式 "模块，可连接到包含流体和电子通信系统的 3D 打印外壳。通过组装含有生物分子识别和捕获蛋白的水凝胶界面，利用电生物制造技术对每个电极进行功能化。具体来说，利用电化学硫醇氧化法组装硫醇化聚乙二醇水凝胶，再与结合抗体 Fc 区域的半胱氨酸标记蛋白质 G 或结合目标 mAb 分析物聚糖的凝集素共价偶联。我们首先展示了硬件设备的设计、组装和测试。然后，我们展示了一步步传感方法（例如，混合、孵育、洗涤、混合、孵育、洗涤、测量）向当前方法的转变，在当前方法中，功能化、抗体捕获和评估是在原位和平行通道中进行的。我们发现滴度和聚糖分析与抗体浓度（至 0.2 毫克/升）呈线性关系。我们还发现，接口可以重复使用，而且结果非常相似。由于界面的组装和使用简单、快速、稳健，我们认为这种评估方法将被广泛应用，包括其他生物分子工艺开发和生产环境。
{"title":"Electrobiofabrication of antibody sensor interfaces within a 3D printed device yield rapid and robust electrochemical measurements of titer and glycan structure","authors":"Chen-Yu Chen, Dana Motabar, Fauziah Rahma Zakaria, Eunkyoung Kim, Benjamin Wu, Gregory F. Payne, William E. Bentley","doi":"10.1002/bit.28839","DOIUrl":"https://doi.org/10.1002/bit.28839","url":null,"abstract":"We report the integration of 3D printing, electrobiofabrication, and protein engineering to create a device that enables near real-time analysis of monoclonal antibody (mAb) titer and quality. 3D printing was used to create the macroscale architecture that can control fluidic contact of a sample with multiple electrodes for replicate measurements. An analysis “chip” was configured as a “snap-in” module for connecting to a 3D printed housing containing fluidic and electronic communication systems. Electrobiofabrication was used to functionalize each electrode by the assembly of a hydrogel interface containing biomolecular recognition and capture proteins. Specifically, an electrochemical thiol oxidation is used to assemble a thiolated polyethylene glycol hydrogel, that in turn is covalently coupled to either a cysteine-tagged protein G that binds the antibody's Fc region or a lectin that binds the glycans of target mAb analytes. We first show the design, assembly, and testing of the hardware device. Then, we show the transition of a step-by-step sensing methodology (e.g., mix, incubate, wash, mix, incubate, wash, measure) into the current method where functionalization, antibody capture, and assessment are performed in situ and in parallel channels. Both titer and glycan analyses were found to be linear with antibody concentration (to 0.2 mg/L). We further found the interfaces could be reused with remarkably similar results. Because the interface assembly and use are simple, rapid, and robust, we suggest this assessment methodology will be widely applicable, including for other biomolecular process development and manufacturing environments.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142237020","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}
James Tang, Matthew Becker, Abraham Lenhoff, Wilfred Chen
Affinity precipitation is a powerful separation method in that it combines the binding selectivity of affinity chromatography with precipitation of captured biomolecules via phase separation triggered by small changes in the environment, e.g., pH, ionic strength, temperature, light, etc. Elastin-like polypeptides (ELPs) are thermally responsive biopolymers composed of pentapeptide repeats VPGVG that undergo reversible phase separation, where they aggregate when temperature and/or salt concentration are increased. Here we describe the generation of an ELP fusion to a soluble streptavidin mutant that enables rapid purification of any Strep-tag II fusion protein of interest. This heterobifunctional protein takes advantage of the native tetrameric structure of streptavidin, leading to binding-induced multivalent crosslinking upon protein capture. The efficient biotin-mediated dissociation of the bound Strep-tag II fusion protein from the streptavidin-ELP capturing scaffold allows for mild elution conditions. We also show that this platform is particularly effective in the purification of a virus-like particle (VLP)-like E2 protein nanoparticle, likely because the high valency of the protein particle causes binding-induced crosslinking and precipitation. Considering the importance of VLP for gene therapy applications, we believe this is a particularly exciting advance. We demonstrated this feasibility by the efficient purification of a VLP-like E2 protein nanoparticle as a surrogate.
亲和沉淀是一种功能强大的分离方法，它将亲和色谱的结合选择性与环境（如 pH 值、离子强度、温度、光线等）微小变化引发的相分离沉淀捕获的生物大分子相结合。弹性蛋白样多肽（ELPs）是由五肽重复序列 VPGVG 组成的热敏性生物聚合物，当温度和/或盐浓度升高时，它们会发生可逆的相分离并聚集在一起。在这里，我们介绍了一种与可溶性链霉亲和素突变体融合的 ELP，它能快速纯化任何感兴趣的链霉-标签 II 融合蛋白。这种异功能蛋白利用了链霉亲和素的原生四聚体结构，从而在蛋白质捕获时产生结合诱导的多价交联。由生物素介导的结合 Strep-tag II 融合蛋白与链霉亲和素-ELP 捕获支架的高效解离使洗脱条件变得温和。我们还发现，该平台在纯化类似病毒颗粒（VLP）的 E2 蛋白纳米颗粒时特别有效，这可能是因为蛋白颗粒的高价引起了结合诱导的交联和沉淀。考虑到 VLP 在基因治疗应用中的重要性，我们认为这是一个特别令人兴奋的进步。我们通过高效纯化 VLP-like E2 蛋白纳米粒子作为代用品，证明了这一可行性。
{"title":"Engineering of heterobifunctional biopolymers for tunable binding and precipitation of Strep-Tag proteins and virus-like nanoparticles","authors":"James Tang, Matthew Becker, Abraham Lenhoff, Wilfred Chen","doi":"10.1002/bit.28845","DOIUrl":"https://doi.org/10.1002/bit.28845","url":null,"abstract":"Affinity precipitation is a powerful separation method in that it combines the binding selectivity of affinity chromatography with precipitation of captured biomolecules via phase separation triggered by small changes in the environment, e.g., pH, ionic strength, temperature, light, etc. Elastin-like polypeptides (ELPs) are thermally responsive biopolymers composed of pentapeptide repeats VPGVG that undergo reversible phase separation, where they aggregate when temperature and/or salt concentration are increased. Here we describe the generation of an ELP fusion to a soluble streptavidin mutant that enables rapid purification of any <i>Strep</i>-tag II fusion protein of interest. This heterobifunctional protein takes advantage of the native tetrameric structure of streptavidin, leading to binding-induced multivalent crosslinking upon protein capture. The efficient biotin-mediated dissociation of the bound <i>Strep-</i>tag II fusion protein from the streptavidin-ELP capturing scaffold allows for mild elution conditions. We also show that this platform is particularly effective in the purification of a virus-like particle (VLP)-like E2 protein nanoparticle, likely because the high valency of the protein particle causes binding-induced crosslinking and precipitation. Considering the importance of VLP for gene therapy applications, we believe this is a particularly exciting advance. We demonstrated this feasibility by the efficient purification of a VLP-like E2 protein nanoparticle as a surrogate.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142234052","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}
The influence of extracellular matrix (ECM) stiffness on cell behavior is a well-established phenomenon. Tumor development is associated with the stiffening of the ECM. However, the understanding of the role of biomechanical behavior and mechanotransduction pathways in the oncogenesis of tumor cells remains limited. In this study, we constructed in vitro models using Polydimethylsiloxane substrates to create soft and stiff substrates. We then evaluated the migration of lung cancer cells A549 using video-microscopy and transwell assays. The mechanical properties were assessed through the utilization of atomic force microscopy, Optical Magnetic Twisting Cytometry, and traction force analysis. Additionally, the expression of Calponin 3 (CNN3) was evaluated using reverse transcription‑quantitative PCR and immunofluorescence techniques. Our observations indicate that the presence of a stiff substrate enhances A549 motility, as evidenced by increased stiffness and traction force in A549 cells on the stiff substrate. Furthermore, we observed a decrease in CNN3 expression in A549 cells on the stiff substrate. Notably, when CNN3 was overexpressed, it effectively inhibited the migration and invasion of A549 cells on the stiff substrate. The results of our study provide novel perspectives on the mechanisms underlying cancer cell migration in response to substrate mechanical properties.
{"title":"The modulation of lung cancer cell motility by Calponin 3 is achieved through its ability to sense and respond to changes in substrate stiffness","authors":"Yinxiu Chi, Xianhui Wang, Xiaoyun Shao, Dongliang Zhang, Jingjing Han, Linhong Deng, Liucai Yang, Xuebin Qu","doi":"10.1002/bit.28847","DOIUrl":"https://doi.org/10.1002/bit.28847","url":null,"abstract":"The influence of extracellular matrix (ECM) stiffness on cell behavior is a well-established phenomenon. Tumor development is associated with the stiffening of the ECM. However, the understanding of the role of biomechanical behavior and mechanotransduction pathways in the oncogenesis of tumor cells remains limited. In this study, we constructed in vitro models using Polydimethylsiloxane substrates to create soft and stiff substrates. We then evaluated the migration of lung cancer cells A549 using video-microscopy and transwell assays. The mechanical properties were assessed through the utilization of atomic force microscopy, Optical Magnetic Twisting Cytometry, and traction force analysis. Additionally, the expression of Calponin 3 (CNN3) was evaluated using reverse transcription‑quantitative PCR and immunofluorescence techniques. Our observations indicate that the presence of a stiff substrate enhances A549 motility, as evidenced by increased stiffness and traction force in A549 cells on the stiff substrate. Furthermore, we observed a decrease in CNN3 expression in A549 cells on the stiff substrate. Notably, when CNN3 was overexpressed, it effectively inhibited the migration and invasion of A549 cells on the stiff substrate. The results of our study provide novel perspectives on the mechanisms underlying cancer cell migration in response to substrate mechanical properties.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142234054","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}
Marija Milisavljevic, Teresa Rojas Rodriguez, Keith E. J. Tyo
Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3′ end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships.
利用 DNA 作为信息存储和信号分子记录的高密度存储介质，在生物技术领域引起了越来越多的关注。最近，利用不依赖模板的 DNA 聚合酶--末端脱氧核苷酸转移酶（TdT）的活性，在酶法 DNA 合成、DNA 数字数据存储和基于 DNA 的分子记录方面取得了进展。TdT 将脱氧核苷酸添加到单链 DNA 的 3′端，生成单链 DNA 的随机序列。TdT 的酶活性可使用多种二价阳离子，并随着反应环境的变化而改变脱氧核苷酸的掺入频率。然而，我们对这些特性的序列-结构-功能关系了解有限，这反过来又限制了我们调节 TdT 以进一步推动基于 TdT 的工具的能力。迄今为止，大多数 TdT 文献探讨的是鼠类、牛类或人类 TdT 的活性；对 TdT 序列和结构的研究主要集中在严格保守的残基上，这些残基在功能上对 TdT 活性至关重要。在这里，我们通过调查 TdT 的天然多样性来探索非保守的 TdT 序列空间。我们描述了来自不同生物体的各种 TdT 同源物，并确定了赋予同源物之间 TdT 行为差异的几个 TdT 残基/区域。本研究中的观察结果可以为靶向 TdT 文库设计规则，并与筛选试验相结合，以调节 TdT 的特性。此外，这些数据还有助于指导对潜在相关残基的进一步研究。总之，我们描述了以前文献中没有研究过的 TdT 的特性，并为 TdT 序列与功能的关系提供了新的见解。
{"title":"Elucidating sequence-function relationships in a template-independent polymerase to enable novel DNA recording applications","authors":"Marija Milisavljevic, Teresa Rojas Rodriguez, Keith E. J. Tyo","doi":"10.1002/bit.28838","DOIUrl":"https://doi.org/10.1002/bit.28838","url":null,"abstract":"Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3′ end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142231836","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}
Roxana Disela, Tim Neijenhuis, Olivier Le Bussy, Geoffroy Geldhof, Marieke Klijn, Martin Pabst, Marcel Ottens
Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry‐based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure–property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross‐validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.
{"title":"Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography","authors":"Roxana Disela, Tim Neijenhuis, Olivier Le Bussy, Geoffroy Geldhof, Marieke Klijn, Martin Pabst, Marcel Ottens","doi":"10.1002/bit.28840","DOIUrl":"https://doi.org/10.1002/bit.28840","url":null,"abstract":"Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host <jats:italic>Escherichia coli</jats:italic> (<jats:italic>E. coli</jats:italic>) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from <jats:italic>E. coli</jats:italic> strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry‐based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure–property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross‐validated <jats:italic>R</jats:italic><jats:sup>2</jats:sup> of 0.55 was constructed, that could be improved to a <jats:italic>R</jats:italic><jats:sup>2</jats:sup> of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142231604","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}
Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance in nature and low production costs. However, excessive intake of sucrose increases the risk of metabolic disorders, including diabetes and obesity. Therefore, there is a growing demand for the development of nonnutritive sweeteners with almost no calories. d-Allulose is an ultra-low-calorie, rare six-carbon monosaccharide with high sweetness, making it an ideal alternative to sucrose. In this study, we developed a cell factory for d-allulose production from sucrose using Escherichia coli JM109 (DE3) as a chassis host. The genes cscA, cscB, cscK, alsE, and a6PP were co-expressed for the construction of the synthesis pathway. Then, the introduction of ptsG-F and knockout of ptsG, fruA, ptsI, and ptsH to reprogram sugar transport pathways resulted in an improvement in substrate utilization. Next, the carbon fluxes of the Embden-Meyerhof-Parnas and the pentose phosphate pathways were regulated by the inactivation of pfkA and zwf, achieving an increase in d-allulose titer and yield of 154.2% and 161.1%, respectively. Finally, scaled-up fermentation was performed in a 5 L fermenter. The titer of d-allulose reached 11.15 g/L, with a yield of 0.208 g/g on sucrose.
{"title":"Biosynthesis of nonnutritive monosaccharide d-allulose by metabolically engineered Escherichia coli from nutritive disaccharide sucrose","authors":"Ling-Jie Zheng, Wei-Xiang Chen, Shang-He Zheng, Irfan Ullah, Hui-Dong Zheng, Li-Hai Fan, Qiang Guo","doi":"10.1002/bit.28842","DOIUrl":"https://doi.org/10.1002/bit.28842","url":null,"abstract":"Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance in nature and low production costs. However, excessive intake of sucrose increases the risk of metabolic disorders, including diabetes and obesity. Therefore, there is a growing demand for the development of nonnutritive sweeteners with almost no calories. <span>d</span>-Allulose is an ultra-low-calorie, rare six-carbon monosaccharide with high sweetness, making it an ideal alternative to sucrose. In this study, we developed a cell factory for <span>d</span>-allulose production from sucrose using <i>Escherichia coli</i> JM109 (DE3) as a chassis host. The genes <i>cscA</i>, <i>cscB</i>, <i>cscK</i>, <i>alsE</i>, and <i>a6PP</i> were co-expressed for the construction of the synthesis pathway. Then, the introduction of <i>ptsG-F</i> and knockout of <i>ptsG</i>, <i>fruA</i>, <i>ptsI</i>, and <i>ptsH</i> to reprogram sugar transport pathways resulted in an improvement in substrate utilization. Next, the carbon fluxes of the Embden-Meyerhof-Parnas and the pentose phosphate pathways were regulated by the inactivation of <i>pfkA</i> and <i>zwf</i>, achieving an increase in <span>d</span>-allulose titer and yield of 154.2% and 161.1%, respectively. Finally, scaled-up fermentation was performed in a 5 L fermenter. The titer of <span>d</span>-allulose reached 11.15 g/L, with a yield of 0.208 g/g on sucrose.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142166363","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}
Melanie Maier, Stefan Schneider, Linus Weiss, Simon Fischer, Daniel Lakatos, Joey Studts, Matthias Franzreb
The cover image is based on the Article Tailoring polishing steps for effective removal of polysorbate-degrading host cell proteins in antibody purification by Melanie Maier et al., https://doi.org/10.1002/bit.28767.
{"title":"Cover Image, Volume 121, Number 10, October 2024","authors":"Melanie Maier, Stefan Schneider, Linus Weiss, Simon Fischer, Daniel Lakatos, Joey Studts, Matthias Franzreb","doi":"10.1002/bit.28853","DOIUrl":"10.1002/bit.28853","url":null,"abstract":"<p>The cover image is based on the Article <i>Tailoring polishing steps for effective removal of polysorbate-degrading host cell proteins in antibody purification</i> by Melanie Maier et al., https://doi.org/10.1002/bit.28767.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28853","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142166418","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 121, Number 10, October 2024","authors":"","doi":"10.1002/bit.28835","DOIUrl":"10.1002/bit.28835","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":null,"pages":null},"PeriodicalIF":3.5,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28835","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142170489","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}
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