Na Guo, Shangjun Wang, Christopher Tyler Whitfield, William D. Batchelor, Yifen Wang, David Blersch, Brendan T. Higgins, Yucheng Feng, Mark R. Liles, Luz E. de-Bashan, Yi Wang, Yuechao Ma
Bacillus velezensis FZB42 is a prominent plant growth-promoting rhizobacterium and biocontrol agent known for producing a wide array of antimicrobial compounds. The capability to genetically manipulate this strain would facilitate understanding its metabolism and enhancing its sustainable agriculture applications. In this study, we report the first successful implementation of high-efficiency CRISPR-Cas9 genome editing in B. velezensis FZB42, enabling targeted genetic modifications to gain insights into its plant growth-promotion and biocontrol properties. Deletion of the slrR gene, a key regulator of biofilm formation, resulted in significant alterations in biofilm structure and development, as demonstrated by scanning electron microscopy and quantitative biofilm assays. These findings provide valuable insights into the mechanisms of biofilm formation, which are critical for root colonization and plant growth promotion. Additionally, we overexpressed the bac gene cluster responsible for bacilysin biosynthesis by replacing its native promoter with the strong constitutive promoter P43 and integrating an additional copy of the bacG gene. This genetic manipulation led to a 2.7-fold increase in bacB gene expression and significantly enhanced antibacterial activity against Escherichia coli and Lactobacillus diolivorans. The successful implementation of the CRISPR-Cas9 system for genome editing in FZB42 provides a valuable tool for genetic engineering, with the potential to improve its biocontrol efficacy and broaden its applications in agriculture and other biotechnology areas. Our principles and procedures are broadly applicable to other agriculturally significant microorganisms.
{"title":"High-Efficiency CRISPR-Cas9 Genome Editing Unveils Biofilm Insights and Enhances Antimicrobial Activity in Bacillus velezensis FZB42","authors":"Na Guo, Shangjun Wang, Christopher Tyler Whitfield, William D. Batchelor, Yifen Wang, David Blersch, Brendan T. Higgins, Yucheng Feng, Mark R. Liles, Luz E. de-Bashan, Yi Wang, Yuechao Ma","doi":"10.1002/bit.28933","DOIUrl":"https://doi.org/10.1002/bit.28933","url":null,"abstract":"<i>Bacillus velezensis</i> FZB42 is a prominent plant growth-promoting rhizobacterium and biocontrol agent known for producing a wide array of antimicrobial compounds. The capability to genetically manipulate this strain would facilitate understanding its metabolism and enhancing its sustainable agriculture applications. In this study, we report the first successful implementation of high-efficiency CRISPR-Cas9 genome editing in <i>B. velezensis</i> FZB42, enabling targeted genetic modifications to gain insights into its plant growth-promotion and biocontrol properties. Deletion of the <i>slrR</i> gene, a key regulator of biofilm formation, resulted in significant alterations in biofilm structure and development, as demonstrated by scanning electron microscopy and quantitative biofilm assays. These findings provide valuable insights into the mechanisms of biofilm formation, which are critical for root colonization and plant growth promotion. Additionally, we overexpressed the <i>bac</i> gene cluster responsible for bacilysin biosynthesis by replacing its native promoter with the strong constitutive promoter P43 and integrating an additional copy of the <i>bacG</i> gene. This genetic manipulation led to a 2.7-fold increase in <i>bacB</i> gene expression and significantly enhanced antibacterial activity against <i>Escherichia coli</i> and <i>Lactobacillus diolivorans</i>. The successful implementation of the CRISPR-Cas9 system for genome editing in FZB42 provides a valuable tool for genetic engineering, with the potential to improve its biocontrol efficacy and broaden its applications in agriculture and other biotechnology areas. Our principles and procedures are broadly applicable to other agriculturally significant microorganisms.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"49 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143050932","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}
Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the most widespread infectious diseases, with nearly 2 billion people infected globally. We present an innovative approach for the real-time detection of TB antigens Mpt64 and Ag85B using DNA aptamers in combination with a graphene oxide (GO)-assisted optical microfiber super-sensor. The high surface-to-volume ratio and superior properties of the GO layer significantly enhance the microfiber's fixation capabilities. To validate the clinical applicability of this sensing method, we employed the optical sensor to successfully detect Mpt64 and Ag85B in serum samples within 10 s, achieving limits of detection of 4.23 × 10⁻²⁰ M and 3.11 × 10⁻¹⁹ M, respectively. Due to the high conservation of Mpt64 and Ag85B in human and bovine MTB strains, our detection system can be used to identify MTB in both humans and bovine. These results demonstrate the sensor's high sensitivity for quantifying MTB particles, enabling rapid identification of infected individuals or bovine. Overall, the optical microfiber sensor system offers a promising platform for diagnosing MTB due to its straightforward detection scheme and potential for miniaturization.
{"title":"Graphene Oxide-Assisted Tapered Microfiber Super-Sensor for Rapid Detection of Mycobacterium tuberculosis Antigens","authors":"Ren Liu, Tianhuan Song, Xiaolin Chen, Zhiheng Yu, Cunliang Yang, Xianchao Zhang, Fengli Huang, Hao Jia, Jijun Feng, Yujiong Wang","doi":"10.1002/bit.28929","DOIUrl":"https://doi.org/10.1002/bit.28929","url":null,"abstract":"Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (MTB), is one of the most widespread infectious diseases, with nearly 2 billion people infected globally. We present an innovative approach for the real-time detection of TB antigens Mpt64 and Ag85B using DNA aptamers in combination with a graphene oxide (GO)-assisted optical microfiber super-sensor. The high surface-to-volume ratio and superior properties of the GO layer significantly enhance the microfiber's fixation capabilities. To validate the clinical applicability of this sensing method, we employed the optical sensor to successfully detect Mpt64 and Ag85B in serum samples within 10 s, achieving limits of detection of 4.23 × 10⁻²⁰ M and 3.11 × 10⁻¹⁹ M, respectively. Due to the high conservation of Mpt64 and Ag85B in human and bovine MTB strains, our detection system can be used to identify MTB in both humans and bovine. These results demonstrate the sensor's high sensitivity for quantifying MTB particles, enabling rapid identification of infected individuals or bovine. Overall, the optical microfiber sensor system offers a promising platform for diagnosing MTB due to its straightforward detection scheme and potential for miniaturization.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"29 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143044204","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}
Koen Johannes Anthonius Verhagen, Ilse Henrike Pardijs, Hendrik Matthijs van Klaveren, Sebastian Aljoscha Wahl
Microbes experience dynamic conditions in natural habitats as well as in engineered environments, such as large-scale bioreactors, which exhibit increased mixing times and inhomogeneities. While single perturbations have been studied for several organisms and substrates, the impact of recurring short-term perturbations remains largely unknown. In this study, we investigated the response of Saccharomyces cerevisiae to repetitive gradients of four different sugars: glucose, fructose, sucrose, and maltose. Due to different transport mechanisms and metabolic routes, nonglucose sugars lead to varied intracellular responses. To characterize the impact of the carbon sources and the dynamic substrate gradients, we applied both steady-state and dynamic cultivation conditions, comparing the physiology, intracellular metabolome, and proteome. For maltose, the repeated concentration gradients led to a significant decrease in biomass yield. Under glucose, fructose, and sucrose conditions, S. cerevisiae maintained the biomass yield observed under steady-state conditions. Although the physiology was very similar across the different sugars, the intracellular metabolome and proteome were clearly differentiated. Notably, the concentration of upper glycolytic enzymes decreased for glucose and maltose (up to −60% and −40%, respectively), while an increase was observed for sucrose and fructose when exposed to gradients. Nevertheless, for all sugar gradient conditions, a stable energy charge was maintained, ranging between 0.78 and 0.89. This response to maltose is particularly distinct compared to previous single-substrate pulse experiments or limitation to excess shifts, which led to maltose-accelerated death in earlier studies. At the same time, enzymes of lower glycolysis were elevated. Interestingly, common stress-related proteins (GO term: cellular response to oxidative stress) decreased during dynamic conditions.
{"title":"A Dive Into Yeast's Sugar Diet—Comparing the Metabolic Response of Glucose, Fructose, Sucrose, and Maltose Under Dynamic Feast/Famine Conditions","authors":"Koen Johannes Anthonius Verhagen, Ilse Henrike Pardijs, Hendrik Matthijs van Klaveren, Sebastian Aljoscha Wahl","doi":"10.1002/bit.28935","DOIUrl":"https://doi.org/10.1002/bit.28935","url":null,"abstract":"Microbes experience dynamic conditions in natural habitats as well as in engineered environments, such as large-scale bioreactors, which exhibit increased mixing times and inhomogeneities. While single perturbations have been studied for several organisms and substrates, the impact of recurring short-term perturbations remains largely unknown. In this study, we investigated the response of <i>Saccharomyces cerevisiae</i> to repetitive gradients of four different sugars: glucose, fructose, sucrose, and maltose. Due to different transport mechanisms and metabolic routes, nonglucose sugars lead to varied intracellular responses. To characterize the impact of the carbon sources and the dynamic substrate gradients, we applied both steady-state and dynamic cultivation conditions, comparing the physiology, intracellular metabolome, and proteome. For maltose, the repeated concentration gradients led to a significant decrease in biomass yield. Under glucose, fructose, and sucrose conditions, <i>S. cerevisiae</i> maintained the biomass yield observed under steady-state conditions. Although the physiology was very similar across the different sugars, the intracellular metabolome and proteome were clearly differentiated. Notably, the concentration of upper glycolytic enzymes decreased for glucose and maltose (up to −60% and −40%, respectively), while an increase was observed for sucrose and fructose when exposed to gradients. Nevertheless, for all sugar gradient conditions, a stable energy charge was maintained, ranging between 0.78 and 0.89. This response to maltose is particularly distinct compared to previous single-substrate pulse experiments or limitation to excess shifts, which led to maltose-accelerated death in earlier studies. At the same time, enzymes of lower glycolysis were elevated. Interestingly, common stress-related proteins (GO term: cellular response to oxidative stress) decreased during dynamic conditions.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"4 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143044275","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}
Gene edited pigs have extensive and important application value in the fields of agriculture and biomedicine. With the increasing demand in medical research and agricultural markets, more and more application scenarios require gene edited pigs to possess two or even more advantageous phenotypes simultaneously. The current production of multi gene edited pigs is inefficient, time-consuming, and costly, and there is an urgent need to develop efficient and accurate multi gene editing application technologies. The polycistronic tRNA-gRNA-processing system (PTG), developed based on endogenous tRNA self-processing systems, has been shown to exhibit efficient multi gene editing in plants. This study aims to combine a PTG strategy with multiple gRNA production functions with an adenine base editor (ABE) to test its feasibility for efficient and precise multi gene base editing in pig cells. The results indicate that the PTG based integrated ABE plasmid can perform efficient base editing at multiple gene loci in pig cells. And while the gene editing efficiency was significantly improved, no indel and sgRNA dependent off target effects caused by DSB were detected. This work permit will provide a solid foundation for the production of multi gene edited pigs with agricultural and medical applications.
基因编辑猪在农业和生物医学领域具有广泛而重要的应用价值。随着医学研究和农业市场需求的不断增加，越来越多的应用场景要求基因编辑猪同时具有两种甚至更多的优势表型。目前多基因编辑猪的生产效率低、耗时长、成本高，迫切需要开发高效、精准的多基因编辑应用技术。基于内源性tRNA自加工系统开发的多顺反电子tRNA- grna加工系统（poly顺反电子tRNA- grna -processing system， PTG）已被证明在植物中具有高效的多基因编辑功能。本研究旨在将具有多种gRNA产生功能的PTG策略与腺嘌呤碱基编辑器（adenine base editor， ABE）结合起来，验证其在猪细胞中高效、精确地进行多基因碱基编辑的可行性。结果表明，基于PTG的整合ABE质粒可以对猪细胞中的多个基因位点进行高效的碱基编辑。虽然基因编辑效率显著提高，但未检测到DSB引起的indel和sgRNA依赖性脱靶效应。这一工作许可将为生产具有农业和医疗应用的多基因编辑猪提供坚实的基础。
{"title":"Application of Multiple Base-Editing Mediated by Polycistronic tRNA-gRNA-Processing System in Pig Cells","authors":"Wudi Zhao, Xiangxing Zhu, Guobin Huang, Hao Gu, Yanzhen Bi, Dongsheng Tang, Hongyan Ren","doi":"10.1002/bit.28931","DOIUrl":"https://doi.org/10.1002/bit.28931","url":null,"abstract":"Gene edited pigs have extensive and important application value in the fields of agriculture and biomedicine. With the increasing demand in medical research and agricultural markets, more and more application scenarios require gene edited pigs to possess two or even more advantageous phenotypes simultaneously. The current production of multi gene edited pigs is inefficient, time-consuming, and costly, and there is an urgent need to develop efficient and accurate multi gene editing application technologies. The polycistronic tRNA-gRNA-processing system (PTG), developed based on endogenous tRNA self-processing systems, has been shown to exhibit efficient multi gene editing in plants. This study aims to combine a PTG strategy with multiple gRNA production functions with an adenine base editor (ABE) to test its feasibility for efficient and precise multi gene base editing in pig cells. The results indicate that the PTG based integrated ABE plasmid can perform efficient base editing at multiple gene loci in pig cells. And while the gene editing efficiency was significantly improved, no indel and sgRNA dependent off target effects caused by DSB were detected. This work permit will provide a solid foundation for the production of multi gene edited pigs with agricultural and medical applications.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"9 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143020979","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}
Serving as a dedicated process analytical technology (PAT) tool for biomass monitoring and control, the capacitance probe, or dielectric spectroscopy, is showing great potential in robust pharmaceutical manufacturing, especially with the growing interest in integrated continuous bioprocessing. Despite its potential, challenges still exist in terms of its accuracy and applicability, particularly when it is used to monitor cells during stationary and decline phases. In this study, data pre‐processing methods were first evaluated through cross‐validation, where the first‐order derivative emerged as the most effective method to diminish variability in prediction accuracy across different training datasets. Subsequently, a segmented adaptive partial least squares (SA‐PLS) model was developed, and its accuracy and universality were demonstrated through several validation studies using different clones and culture processes. Furthermore, a real‐time viable cell density (VCD) auto‐control system in perfusion culture was established, where the VCD was maintained around the target with notable precision and robustness. This model enhanced the monitoring capabilities of capacitance‐based PAT tools throughout the cultivation, expanded their application in cell‐specific automatic control strategies, and contributed vitally to the advancement of continuous manufacturing paradigms.
{"title":"Real‐Time Auto Controlling of Viable Cell Density in Perfusion Cultivation Aided by In‐Line Dielectric Spectroscopy With Segmented Adaptive PLS Model","authors":"Yunpeng Sun, Qiongqiong Zhang, Yunfei He, Dongliang Chen, Zheyu Wang, Xiang Zheng, Mingyue Fang, Hang Zhou","doi":"10.1002/bit.28930","DOIUrl":"https://doi.org/10.1002/bit.28930","url":null,"abstract":"Serving as a dedicated process analytical technology (PAT) tool for biomass monitoring and control, the capacitance probe, or dielectric spectroscopy, is showing great potential in robust pharmaceutical manufacturing, especially with the growing interest in integrated continuous bioprocessing. Despite its potential, challenges still exist in terms of its accuracy and applicability, particularly when it is used to monitor cells during stationary and decline phases. In this study, data pre‐processing methods were first evaluated through cross‐validation, where the first‐order derivative emerged as the most effective method to diminish variability in prediction accuracy across different training datasets. Subsequently, a segmented adaptive partial least squares (SA‐PLS) model was developed, and its accuracy and universality were demonstrated through several validation studies using different clones and culture processes. Furthermore, a real‐time viable cell density (VCD) auto‐control system in perfusion culture was established, where the VCD was maintained around the target with notable precision and robustness. This model enhanced the monitoring capabilities of capacitance‐based PAT tools throughout the cultivation, expanded their application in cell‐specific automatic control strategies, and contributed vitally to the advancement of continuous manufacturing paradigms.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"11 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142989318","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}
Process models are increasingly used to support upstream process development in the biopharmaceutical industry for process optimization, scale‐up and to reduce experimental effort. Parametric unstructured models based on biological mechanisms are highly promising, since they do not require large amounts of data. The critical part in the application is the certainty of the parameter estimates, since uncertainty of the parameter estimates propagates to model predictions and can increase the risk associated with those predictions. Currently Fisher‐Information‐Matrix based approximations or Monte‐Carlo approaches are used to estimate parameter confidence intervals and regularization approaches to decrease parameter uncertainty. Here we apply profile likelihood to determine parameter identifiability of a recent upstream process model. We have investigated the effect of data amount on identifiability and found out that addition of data reduces non‐identifiability. The likelihood profiles of nonidentifiable parameters were then used to uncover structural model changes. These changes effectively alleviate the remaining non‐identifiabilities except for a single parameter out of 21 total parameters. We present the first application of profile likelihood to a complete upstream process model. Profile likelihood is a highly suitable method to determine parameter confidence intervals in upstream process models and provides reliable estimates even with nonlinear models and limited data.
工艺模型越来越多地用于支持生物制药行业的上游工艺开发，以优化工艺，扩大规模并减少实验工作。基于生物机制的参数化非结构化模型是非常有前途的，因为它们不需要大量的数据。应用程序中的关键部分是参数估计的确定性，因为参数估计的不确定性会传播到模型预测中，并可能增加与这些预测相关的风险。目前，基于Fisher - Information - Matrix的近似或Monte - Carlo方法用于估计参数置信区间和正则化方法以减少参数的不确定性。在这里，我们应用概要似然来确定最近的上游过程模型的参数可识别性。我们研究了数据量对可识别性的影响，发现数据的增加减少了不可识别性。然后使用不可识别参数的可能性概况来揭示结构模型的变化。除了21个参数中的一个参数外，这些变化有效地缓解了剩余的不可识别性。我们提出了剖面似然的第一个应用到一个完整的上游过程模型。轮廓似然是确定上游过程模型中参数置信区间的一种非常合适的方法，即使在非线性模型和有限数据下也能提供可靠的估计。
{"title":"Reducing Structural Nonidentifiabilities in Upstream Bioprocess Models Using Profile‐Likelihood","authors":"Heiko Babel, Ola Omar, Albert Paul, Joachim Bär","doi":"10.1002/bit.28922","DOIUrl":"https://doi.org/10.1002/bit.28922","url":null,"abstract":"Process models are increasingly used to support upstream process development in the biopharmaceutical industry for process optimization, scale‐up and to reduce experimental effort. Parametric unstructured models based on biological mechanisms are highly promising, since they do not require large amounts of data. The critical part in the application is the certainty of the parameter estimates, since uncertainty of the parameter estimates propagates to model predictions and can increase the risk associated with those predictions. Currently Fisher‐Information‐Matrix based approximations or Monte‐Carlo approaches are used to estimate parameter confidence intervals and regularization approaches to decrease parameter uncertainty. Here we apply profile likelihood to determine parameter identifiability of a recent upstream process model. We have investigated the effect of data amount on identifiability and found out that addition of data reduces non‐identifiability. The likelihood profiles of nonidentifiable parameters were then used to uncover structural model changes. These changes effectively alleviate the remaining non‐identifiabilities except for a single parameter out of 21 total parameters. We present the first application of profile likelihood to a complete upstream process model. Profile likelihood is a highly suitable method to determine parameter confidence intervals in upstream process models and provides reliable estimates even with nonlinear models and limited data.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"7 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142989317","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}
Ricardo Correia, Taja Zotler, Francisco Ferraz, Bárbara Fernandes, Miguel Graça, Gorben P. Pijlman, Paula M. Alves, António Roldão
The insect cell‐baculovirus expression vector system (IC‐BEVS) has been an asset to produce biologics for over 30 years. With the current trend in biotechnology shifting toward process intensification and integration, developing intensified processes such as continuous production is crucial to hold this platform as a suitable alternative to others. However, the implementation of continuous production has been hindered by the lytic nature of this expression system and the process‐detrimental virus passage effect. In this study, we implemented a multi‐stage bioreactor setup for continuous production of influenza hemagglutinin‐displaying virus‐like particles (HA‐VLPs) using IC‐BEVS. A setup consisting of one Cell Growth Bioreactor simultaneously feeding non‐infected insect cells to three parallel Production Bioreactors operated at different residence times (RT) (18, 36, and 54 h) was implemented; Production Bioreactors were continuously harvested. Two insect cell lines (neutral pH–adapted High Five and Sf9) and two recombinant baculovirus (rBAC) constructs (one that originates from a bacmid, rBACbacmid, and another of non‐bacteria origin, rBACflashbac) were tested. Combining rBACflashbac with Sf9 cells was the most efficient approach, allowing consistent HA‐VLPs titers (34 ± 14 HA titer/mL) and rBAC titers (108–109 pfu/mL) throughout the period of continuous operation (20 days). Cell growth kinetics and viability varied across RT, and higher RT was associated with increased expression of HA‐VLPs, independent of the cell line and rBAC used; RT of 54 h allowed to maximize titers. The presence of particles resembling HA‐VLPs was confirmed by transmission electron microscopy throughout the continuous operation. This work showcases the implementation of a process for continuous production of a promising class of biotherapeutics (i.e., VLPs), and paves the way for establishing continuous, integrated setups using the IC‐BEVS expression system.
{"title":"Continuous Production of Influenza VLPs Using IC‐BEVS and Multi‐Stage Bioreactors","authors":"Ricardo Correia, Taja Zotler, Francisco Ferraz, Bárbara Fernandes, Miguel Graça, Gorben P. Pijlman, Paula M. Alves, António Roldão","doi":"10.1002/bit.28925","DOIUrl":"https://doi.org/10.1002/bit.28925","url":null,"abstract":"The insect cell‐baculovirus expression vector system (IC‐BEVS) has been an asset to produce biologics for over 30 years. With the current trend in biotechnology shifting toward process intensification and integration, developing intensified processes such as continuous production is crucial to hold this platform as a suitable alternative to others. However, the implementation of continuous production has been hindered by the lytic nature of this expression system and the process‐detrimental virus passage effect. In this study, we implemented a multi‐stage bioreactor setup for continuous production of influenza hemagglutinin‐displaying virus‐like particles (HA‐VLPs) using IC‐BEVS. A setup consisting of one Cell Growth Bioreactor simultaneously feeding non‐infected insect cells to three parallel Production Bioreactors operated at different residence times (RT) (18, 36, and 54 h) was implemented; Production Bioreactors were continuously harvested. Two insect cell lines (neutral pH–adapted High Five and <jats:italic>Sf</jats:italic>9) and two recombinant baculovirus (rBAC) constructs (one that originates from a bacmid, rBAC<jats:sub>bacmid</jats:sub>, and another of non‐bacteria origin, rBAC<jats:sub>flashbac</jats:sub>) were tested. Combining rBAC<jats:sub>flashbac</jats:sub> with <jats:italic>Sf</jats:italic>9 cells was the most efficient approach, allowing consistent HA‐VLPs titers (34 ± 14 HA titer/mL) and rBAC titers (10<jats:sup>8</jats:sup>–10<jats:sup>9</jats:sup> pfu/mL) throughout the period of continuous operation (20 days). Cell growth kinetics and viability varied across RT, and higher RT was associated with increased expression of HA‐VLPs, independent of the cell line and rBAC used; RT of 54 h allowed to maximize titers. The presence of particles resembling HA‐VLPs was confirmed by transmission electron microscopy throughout the continuous operation. This work showcases the implementation of a process for continuous production of a promising class of biotherapeutics (i.e., VLPs), and paves the way for establishing continuous, integrated setups using the IC‐BEVS expression system.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"56 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142989319","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}
Kitty Sompiyachoke, Joseph Bravo, Rakesh Sikdar, Jowan Abdullah, Mikael H. Elias
N‐acyl l‐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 N‐acyl l‐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 Parageobacillus caldoxylosilyticus. 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 N‐acyl l‐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.
N -酰基-高丝氨酸内酯是许多细菌在群体感应中使用的信号分子。一些细菌编码内酯酶，可以使这些信号失活。据报道，内酯酶抑制群体感应依赖的表型，包括毒力和生物膜。由于细菌信号依赖于所使用的分子类型，因此具有高底物特异性的内酯酶是选择性靶向群落中物种的理想选择。从自然界提取的内酯酶在底物偏好方面表现出有限的多样性，这使得它们在工程上具有吸引力，但由于缺乏方便的内酯酶活性评估方法而变得复杂。我们提出了一种中等通量内酯酶筛选系统，该系统与裂解物兼容，该裂解物将N -酰基1 -同型半胱氨酸硫代内酯的开环与5,5 -二硫代双硫代（2 -硝基苯甲酸）偶联，以产生显色信号。我们证明了该系统适用于来自不同蛋白家族的内酯酶，并通过筛选caldoxylosilyticus副杆菌GcL内酯酶突变文库证明了它的实用性。动力学表征与硫代内酯酶和同丝氨酸内酯酶活性水平证实了筛选结果。该系统以改变的特异性识别GcL变异：长链N -酰基1 -高丝氨酸内酯底物的活性降低了1900倍，而短链底物的活性增加了38倍。总的来说，这个新系统大大提高了内酯酶活性的评价，并将促进群体猝灭酶的鉴定和工程。
{"title":"A Novel Screening System to Characterize and Engineer Quorum Quenching Lactonases","authors":"Kitty Sompiyachoke, Joseph Bravo, Rakesh Sikdar, Jowan Abdullah, Mikael H. Elias","doi":"10.1002/bit.28928","DOIUrl":"https://doi.org/10.1002/bit.28928","url":null,"abstract":"<jats:italic>N</jats:italic>‐acyl <jats:sc>l</jats:sc>‐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 <jats:italic>N</jats:italic>‐acyl <jats:sc>l</jats:sc>‐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 <jats:italic>Parageobacillus caldoxylosilyticus</jats:italic>. 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 <jats:italic>N‐</jats:italic>acyl <jats:sc>l</jats:sc>‐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.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"37 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-01-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yujie Liu, Weini Xiong, Yuting Jiang, Yan Meng, Wanwan Zhao, Chao Yang, Ruihua Liu
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 mpd/pnpABCDEF responsible for the bioconversion of p-nitrophenol (PNP)-substituted organophosphorus pesticides (OPs) into β-oxoadipate and the genes encoding Vitreoscilla hemoglobin (VHb) and green fluorescent protein (GFP) were integrated into the genome of a salt-tolerant chassis Halomonas cupida 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.
{"title":"Creating a Halotolerant Degrader for Efficient Mineralization of p-Nitrophenol-Substituted Organophosphorus Pesticides in High-Saline Wastewater","authors":"Yujie Liu, Weini Xiong, Yuting Jiang, Yan Meng, Wanwan Zhao, Chao Yang, Ruihua Liu","doi":"10.1002/bit.28923","DOIUrl":"https://doi.org/10.1002/bit.28923","url":null,"abstract":"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 <sup>13</sup>C<sub>6</sub>-PNP into <sup>13</sup>CO<sub>2</sub>, 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 <sup>13</sup>C<sub>6</sub>-PNP was converted into <sup>13</sup>CO<sub>2</sub> 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.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"41 1","pages":""},"PeriodicalIF":3.8,"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}
Feng Xu, Lihuan Su, Yuan Wang, Kaihao Hu, Ling Liu, Rong Ben, Hao Gao, Ali Mohsin, Ju Chu, Xiwei Tian
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 (R2) 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.
{"title":"A Paradigm of Computer Vision and Deep Learning Empowers the Strain Screening and Bioprocess Detection","authors":"Feng Xu, Lihuan Su, Yuan Wang, Kaihao Hu, Ling Liu, Rong Ben, Hao Gao, Ali Mohsin, Ju Chu, Xiwei Tian","doi":"10.1002/bit.28926","DOIUrl":"https://doi.org/10.1002/bit.28926","url":null,"abstract":"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><sup>2</sup>) 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.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"37 1","pages":""},"PeriodicalIF":3.8,"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}
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