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Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography 制备色谱过程中大肠杆菌宿主细胞蛋白质组保留的实验表征与预测
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-12 DOI: 10.1002/bit.28840
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.

纯化重组生产的生物制药需要去除宿主细胞物质,如宿主细胞蛋白(HCP)。对于常见表达宿主大肠杆菌(E. coli)的裂解物,可以鉴定出超过 1500 种独特的蛋白质。目前,人们对制备色谱等纯化操作中单个 HCP 的行为了解有限。因此,我们旨在阐明大肠杆菌菌株 BLR(DE3) 中的单个 HCP 在层析过程中的洗脱行为。了解这种复杂的混合物并掌握每种 HCP 的色谱行为有助于提高合理设计纯化工艺的能力。具体来说,我们使用离子交换色谱(IEX)和疏水相互作用色谱进行了线性梯度实验,并结合基于质谱的蛋白质组学来绘制单个 HCP 的保留图谱。我们结合文献中有关蛋白质位置、功能和相互作用的知识,确定了洗脱行为的趋势。此外,我们还训练了将蛋白质三维结构与 IEX 期间的洗脱行为相关联的定量结构-性质关系模型。对于完整的数据集,构建了一个交叉验证 R2 为 0.55 的模型,如果只考虑单体蛋白质,R2 可提高到 0.70。最终,这项研究为加深对工艺的理解迈出了重要一步。
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引用次数: 0
Biosynthesis of nonnutritive monosaccharide d-allulose by metabolically engineered Escherichia coli from nutritive disaccharide sucrose 代谢工程大肠杆菌从营养性二糖蔗糖生物合成非营养性单糖 d-阿洛糖
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-11 DOI: 10.1002/bit.28842
Ling-Jie Zheng, Wei-Xiang Chen, Shang-He Zheng, Irfan Ullah, Hui-Dong Zheng, Li-Hai Fan, Qiang Guo

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.

蔗糖是食品和饮料中常用的营养甜味剂,因为它在自然界中含量丰富,生产成本低。然而,过量摄入蔗糖会增加患糖尿病和肥胖症等代谢性疾病的风险。d-Allulose 是一种超低卡路里的稀有六碳单糖,甜度高,是蔗糖的理想替代品。在这项研究中,我们以大肠杆菌 JM109 (DE3) 为底盘宿主,开发了从蔗糖生产 d-阿洛糖的细胞工厂。基因 cscA、cscB、cscK、alsE 和 a6PP 共同表达,构建了合成途径。然后,通过引入 ptsG-F 和敲除 ptsG、fruA、ptsI 和 ptsH 来重新规划糖运输途径,从而提高了底物利用率。接着,通过使 pfkA 和 zwf 失活来调节恩布登-梅耶霍夫-帕尔纳斯途径和磷酸戊糖途径的碳通量,使 d-阿洛糖滴度和产量分别提高了 154.2% 和 161.1%。最后,在 5 升发酵罐中进行了放大发酵。d-allulose 滴度达到 11.15 g/L,蔗糖产量为 0.208 g/g。
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引用次数: 0
Cover Image, Volume 121, Number 10, October 2024 封面图片,第 121 卷第 10 期,2024 年 10 月
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-11 DOI: 10.1002/bit.28853
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.

封面图片来自 Melanie Maier 等人撰写的文章《在抗体纯化过程中调整抛光步骤以有效去除会降解聚山梨醇酯的宿主细胞蛋白》,https://doi.org/10.1002/bit.28767。
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引用次数: 0
Biotechnology and Bioengineering: Volume 121, Number 10, October 2024 生物技术与生物工程第 121 卷第 10 号,2024 年 10 月
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-11 DOI: 10.1002/bit.28835
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引用次数: 0
Rational design of short-chain dehydrogenase/reductase for enantio-complementary synthesis of chiral 1,2-diols by successive hydroxymethylation and reduction of aldehydes 合理设计短链脱氢酶/还原酶,通过醛的连续羟甲基化和还原反应对手性 1,2 二醇进行对映互补合成
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-10 DOI: 10.1002/bit.28841
Xiu-Xin Ren, Bing-Mei Su, Xin-Qi Xu, Lian Xu, Juan Lin

Enantiopure 1,2-diols are widely used in the production of pharmaceuticals, cosmetics, and functional materials as essential building blocks or bioactive compounds. Nevertheless, developing a mild, efficient and environmentally friendly biocatalytic route for manufacturing enantiopure 1,2-diols from simple substrate remains a challenge. Here, we designed and realized a step-wise biocatalytic cascade to access chiral 1,2-diols starting from aromatic aldehyde and formaldehyde enabled by a newly mined benzaldehyde lyase from Sphingobium sp. combined with a pair of tailored-made short-chain dehydrogenase/reductase from Pseudomonas monteilii (PmSDR-MuR and PmSDR-MuS) capable of producing (R)- and (S)-1-phenylethane-1,2-diol with 99% ee. The planned biocatalytic cascade could synthesize a series of enantiopure 1,2-diols with a broad scope (16 samples), excellent conversions (94%–99%), and outstanding enantioselectivity (up to 99% ee), making it an effective technique for producing chiral 1,2-diols in a more environmentally friendly and sustainable manner.

对映体纯 1,2-二醇作为重要的基本成分或生物活性化合物被广泛应用于药品、化妆品和功能材料的生产中。然而,开发一条温和、高效、环保的生物催化路线,从简单的底物中生产出不对映的 1,2 二醇,仍然是一项挑战。在这里,我们设计并实现了一种分步生物催化级联法,以芳香醛和甲醛为起始原料,通过一种新近从 Sphingobium sp.结合一对定制的假单胞菌短链脱氢酶/还原酶(PmSDR-MuR 和 PmSDR-MuS),能够生成(R)-和(S)-1-苯基乙烷-1,2-二醇,ee值高达 99%。计划中的生物催化级联法可以合成一系列对映体纯的 1,2-二醇,范围广泛(16 个样品),转化率高(94%-99%),对映体选择性好(ee高达 99%),是一种以更环保、更可持续的方式生产手性 1,2- 二醇的有效技术。
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引用次数: 0
Application of microbubble air flotation to harvest Microcystis sp. from agriculture wastewater: The regulation and mechanisms 应用微气泡气浮收获农业废水中的微囊藻:调节和机制
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-09 DOI: 10.1002/bit.28836
Jianfeng Ye, Zhihao Zhu, Zhaofeng Song, Huiting Xu, Tianchen Xu, Hui Liu

The harvesting of microalgae is the main bottleneck of its large-scale biomass production, and seeking an efficient, green, and low-cost microalgae harvesting technology is one of the urgent problems to be solved. Microbubble air flotation has been proven to be an effective measure, but the mechanisms of microbubbles-algal cell attachment are still unclear. In this study, microbubble air flotation was used as a harvesting method for Microcystis cultured in agricultural wastewater. The process mechanism of microbubble air flotation harvesting microalgae in wastewater was fully revealed from three aspects (the design of bubble formation, the adhesion law, and the recovery rate of microalgae under different working conditions). The results show that the length of the release pipe is the main factor affecting the proportion of microbubbles with a particle size of less than 50 μm. In the process of adhesion, when the particle size of microbubbles is 0.6–1.7 times the size of Microcystis, the adhesion efficiency of microbubbles to Microcystis is the highest. Under the conditions of pressure 0.45 MPa, gas–liquid ratio 5%, and release pipe length 100 cm, the harvesting performance of Microcystis was the best. Microbubble air flotation has better harvesting performance (63.5%, collection rate) of Microcystis with higher density. By understanding the mechanism of microbubble flotation, the technical parameters of microbubble flotation for harvesting energy microalgae are optimized to provide support for the development of efficient and low-cost devices and equipment for collecting microalgae.

微藻收获是大规模生物质生产的主要瓶颈,寻求一种高效、绿色、低成本的微藻收获技术是亟待解决的问题之一。微气泡气浮已被证明是一种有效的措施,但微气泡-藻细胞附着的机理仍不清楚。本研究采用微气泡气浮作为农业废水中培养的微囊藻的收获方法。从气泡形成设计、附着规律和不同工况下微囊藻回收率三个方面全面揭示了微气泡气浮收获废水中微囊藻的过程机理。结果表明,释放管的长度是影响粒径小于 50 μm 的微气泡比例的主要因素。在粘附过程中,当微泡粒径为微囊藻粒径的 0.6-1.7 倍时,微泡对微囊藻的粘附效率最高。在压力为 0.45 兆帕、气液比为 5%、释放管道长度为 100 厘米的条件下,微囊藻的收获效果最好。微气泡气浮对密度较大的微囊藻有较好的收获效果(收集率为 63.5%)。通过对微泡气浮机理的认识,优化了微泡气浮收获能源微藻的技术参数,为开发高效、低成本的微藻收集装置和设备提供了支持。
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引用次数: 0
Auto-transduction in lentiviral vector bioprocessing: A quantitative assessment and a novel inhibition strategy 慢病毒载体生物处理中的自动转导:定量评估和新型抑制策略
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-08 DOI: 10.1002/bit.28834
Thomas Williams-Fegredo, Lee Davies, Carol Knevelman, James Miskin, Kyriacos Mitrophanous, Qasim A. Rafiq

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7–6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.

慢病毒载体是一种高效的基因递送载体,广泛应用于快速发展的细胞和基因治疗领域。随着越来越多的疗法进入后期临床试验阶段并实现商业化,对高效、大规模慢病毒载体生物处理的需求也在不断增长。然而,尽管取得了重大进展,但仍存在一些工艺效率低下的问题。在生产过程中,新合成的慢病毒载体颗粒会对病毒载体生产细胞产生意外的自动转导,这就是效率低下的原因之一,而这一问题在很大程度上仍未得到解决。在这项研究中,我们发现在上游生产过程中产生的功能性慢病毒载体颗粒有 60% 以上因自动转导而丢失,这凸显了行业内可能普遍存在的主要工艺低效问题。在载体生产过程中,通过降低细胞外 pH 值,抑制了以广泛使用的水泡性口炎病毒 G 蛋白为假型的颗粒对细胞的自动转导,从而损害了载体与其目标受体相互作用的能力。将转染后的 pH 值调至 6.7-6.8 后,病毒载体生产细胞群中由慢病毒载体介导的转导引起的载体基因组整合事件减少了七倍,并最终改善了慢病毒载体的生产动力学。所提出的策略具有可扩展性和成本效益,为提高慢病毒载体的生产效率提供了一种与工业相关的方法。
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引用次数: 0
Higher efficiency of vanadate iron in heterogeneous Fenton-like systems to pretreat sugarcane bagasse and its enzymatic saccharification. 在类似芬顿的异质系统中使用钒酸盐铁对甘蔗渣进行预处理和酶法糖化的效率更高。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-05-06 DOI: 10.1002/bit.28733
Ju Liang, Huiying Zeng, Yuting Zhang, Wenbing Zhou, Naidong Xiao

Pretreatment is crucial for effective enzymatic saccharification of lignocellulose such as sugarcane bagasse (SCB). In the present study, SCB was pretreated with five kinds of heterogeneous Fenton-like systems (HFSs), respectively, in which α-FeOOH, α-Fe2O3, Fe3O4, and FeS2 worked as four traditional heterogeneous Fenton-like catalysts (HFCs), while FeVO4 worked as a novel HFC. The enzymatic reducing sugar conversion rate was then compared among SCB after different heterogeneous Fenton-like pretreatments (HFPs), and the optimal HFS and pretreatment conditions were determined. The mechanism underlying the difference in saccharification efficiency was elucidated by analyzing the composition and morphology of SCB. Moreover, the ion dissolution characteristics, variation of pH and Eh values, H2O2 and hydroxyl radical (·OH) concentration of FeVO4 and α-Fe2O3 HFSs were compared. The results revealed that the sugar conversion rate of SCB pretreated with FeVO4 HFS reached up to 58.25%, which was obviously higher than that under other HFPs. In addition, the surface morphology and composition of the pretreated SCB with FeVO4 HFS were more conducive to enzymatic saccharification. Compared with α-Fe2O3, FeVO4 could utilize H2O2 more efficiently, since the dissolved Fe3+ and V5+ can both react with H2O2 to produce more ·OH, resulting in a higher hemicellulose and lignin removal rate and a higher enzymatic sugar conversion rate. It can be concluded that FeVO4 HFP is a promising approach for lignocellulose pretreatment.

预处理对于木质纤维素(如甘蔗渣)的有效酶法糖化至关重要。本研究分别使用五种异相芬顿类催化剂体系(HFS)对甘蔗渣进行预处理,其中α-FeOOH、α-Fe2O3、Fe3O4 和 FeS2 是四种传统的异相芬顿类催化剂(HFC),而 FeVO4 则是一种新型的 HFC。然后比较了不同异相芬顿类预处理(HFP)后 SCB 的酶促还原糖转化率,并确定了最佳 HFS 和预处理条件。通过分析 SCB 的组成和形态,阐明了糖化效率差异的机理。此外,还比较了 FeVO4 和 α-Fe2O3 HFS 的离子溶解特性、pH 值和 Eh 值的变化、H2O2 和羟基自由基(-OH)的浓度。结果表明,用 FeVO4 HFS 预处理的 SCB 糖转化率高达 58.25%,明显高于其他 HFP。此外,用 FeVO4 HFS 预处理的 SCB 表面形态和成分更有利于酶糖化。与 α-Fe2O3 相比,FeVO4 能更有效地利用 H2O2,因为溶解的 Fe3+ 和 V5+ 都能与 H2O2 反应产生更多的 -OH,从而导致更高的半纤维素和木质素去除率以及更高的酶糖转化率。因此,FeVO4 HFP 是一种很有前景的木质纤维素预处理方法。
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引用次数: 0
Molecular mechanisms of natural antifreeze phenomena and their application in cryopreservation 天然防冻现象的分子机制及其在低温保存中的应用
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-29 DOI: 10.1002/bit.28832
Lingyu Shi, Chuanbao Zang, Zhicheng Liu, Gang Zhao

Cryopreservation presents a critical challenge due to cryo-damage, such as crystallization and osmotic imbalances that compromise the integrity of biological tissues and cells. In contrast, various organisms in nature exhibit remarkable freezing tolerance, leveraging complex molecular mechanisms to survive extreme cold. This review explores the adaptive strategies of freeze-tolerant species, including the regulation of specific genes, proteins, and metabolic pathways, to enhance survival in low-temperature environments. We then discuss recent advancements in cryopreservation technologies that aim to mimic these natural phenomena to preserve cellular and tissue integrity. Special focus is given to the roles of glucose metabolism, microRNA expression, and cryoprotective protein modulation in improving cryopreservation outcomes. The insights gained from studying natural antifreeze mechanisms offer promising directions for advancing cryopreservation techniques, with potential applications in medical, agricultural, and conservation fields. Future research should aim to further elucidate these molecular mechanisms to develop more effective and reliable cryopreservation methods.

由于结晶和渗透失衡等低温损伤会损害生物组织和细胞的完整性,因此低温保存是一项严峻的挑战。与此相反,自然界中的各种生物却表现出卓越的耐冻能力,它们利用复杂的分子机制在极寒环境中生存下来。本综述探讨了耐冻物种的适应策略,包括对特定基因、蛋白质和代谢途径的调控,以提高它们在低温环境中的生存能力。然后,我们讨论了旨在模拟这些自然现象以保存细胞和组织完整性的低温保存技术的最新进展。我们将特别关注葡萄糖代谢、microRNA 表达和低温保护蛋白调节在改善低温保存结果方面的作用。从研究天然防冻机制中获得的启示为冷冻保存技术的发展提供了前景广阔的方向,并有可能应用于医学、农业和自然保护领域。未来的研究应旨在进一步阐明这些分子机制,以开发出更有效、更可靠的低温保存方法。
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引用次数: 0
Insights into yeast response to chemotherapeutic agent through time series genome-scale metabolic models 通过时间序列基因组尺度代谢模型深入了解酵母对化疗药物的反应
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-28 DOI: 10.1002/bit.28833
Muhammed E. Karabekmez

Organism-specific genome-scale metabolic models (GSMMs) can unveil molecular mechanisms within cells and are commonly used in diverse applications, from synthetic biology, biotechnology, and systems biology to metabolic engineering. There are limited studies incorporating time-series transcriptomics in GSMM simulations. Yeast is an easy-to-manipulate model organism for tumor research. Here, a novel approach (TS-GSMM) was proposed to integrate time-series transcriptomics with GSMMs to narrow down the feasible solution space of all possible flux distributions and attain time-series flux samples. The flux samples were clustered using machine learning techniques, and the clusters' functional analysis was performed using reaction set enrichment analysis. A time series transcriptomics response of Yeast cells to a chemotherapeutic reagent—doxorubicin—was mapped onto a Yeast GSMM. Eleven flux clusters were obtained with our approach, and pathway dynamics were displayed. Induction of fluxes related to bicarbonate formation and transport, ergosterol and spermidine transport, and ATP production were captured. Integrating time-series transcriptomics data with GSMMs is a promising approach to reveal pathway dynamics without any kinetic modeling and detects pathways that cannot be identified through transcriptomics-only analysis. The codes are available at https://github.com/karabekmez/TS-GSMM.

生物特异性基因组尺度代谢模型(GSMMs)可以揭示细胞内的分子机制,通常用于合成生物学、生物技术、系统生物学和代谢工程等多种应用领域。将时间序列转录组学纳入 GSMM 模拟的研究还很有限。酵母是一种易于操作的肿瘤研究模式生物。本文提出了一种新方法(TS-GSMM),将时间序列转录组学与 GSMMs 结合起来,缩小所有可能通量分布的可行解空间,并获得时间序列通量样本。利用机器学习技术对通量样本进行聚类,并利用反应集富集分析对聚类进行功能分析。酵母细胞对化疗试剂--多柔比星--的时间序列转录组学反应被映射到酵母GSMM上。我们的方法获得了 11 个通量簇,并显示了通路动态。我们捕捉到了与碳酸氢盐形成和运输、麦角甾醇和亚精胺运输以及 ATP 生产相关的通量诱导。将时间序列转录组学数据与 GSMMs 相结合是一种很有前途的方法,它能揭示通路动态,而无需任何动力学建模,并能检测到仅通过转录组学分析无法确定的通路。代码可在 https://github.com/karabekmez/TS-GSMM 网站上获取。
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引用次数: 0
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