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Biotechnology and Bioengineering: Volume 121, Number 12, December 2024 生物技术与生物工程第 121 卷第 12 号,2024 年 12 月
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-11 DOI: 10.1002/bit.28887
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引用次数: 0
Correction to “A High‐Throughput Expression and Screening Platform for Applications‐Driven PETase Engineering” 对 "应用驱动 PETase 工程的高通量表达和筛选平台 "的更正
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-11 DOI: 10.1002/bit.28882
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引用次数: 0
Mathematical Modeling and Simulation of 1,3‐Propanediol Production by Klebsiella pneumoniae BLh‐1 in a Batch Bioreactor Using Bayesian Statistics 利用贝叶斯统计对批式生物反应器中肺炎克雷伯氏菌 BLh-1 生产 1,3-丙二醇的过程进行数学建模和模拟
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-08 DOI: 10.1002/bit.28883
Nathalia Lobato Moraes, Mailson Batista de Vilhena, Daniele Misturini Rossi, Bruno Marques Viegas
Mathematical modeling and computer simulation are fundamental for optimizing biotechnological processes, enabling cost reduction and scalability, thereby driving advancements in the bioindustry. In this work, mathematical modeling and estimation of fermentative kinetic parameters were carried out to produce 1,3‐propanediol (1,3‐PDO) from residual glycerol and Klebsiella pneumoniae BLh‐1. The Markov chain Monte Carlo method, using the Metropolis‐Hastings algorithm, was applied to experimental data from a batch bioreactor under aerobic and anaerobic conditions. Sensitivity analysis and parameter evolution studies were conducted. The root‐mean‐square error (rRMSE) was chosen as the validation and calibration metric for the developed mathematical model. The results indicated that the average tolerance of glycerol was 174.68 and 44.85 g L−1, the inhibitory products was 150.95 g L−1 for ethanol and 35.56 g L−1 for 1,3‐PDO, and the maximum specific rate of cell growth was 0.189 and 0.275 h−1, for aerobic and anaerobic cultures, respectively. The model presented excellent fits in both crops, with rRMSE values between 0.09 − 33.74% and 3.58 − 31.82%, for the aerobic and anaerobic environment, respectively. With this, it was possible to evaluate and extract relevant information for a better understanding and control of the bioprocess.
数学建模和计算机模拟是优化生物技术过程、降低成本和提高可扩展性的基础,从而推动生物产业的进步。在这项工作中,对利用残余甘油和肺炎克雷伯氏菌 BLh-1 生产 1,3-丙二醇(1,3-PDO)的发酵动力学参数进行了数学建模和估算。使用 Metropolis-Hastings 算法的马尔科夫链蒙特卡罗方法被应用于有氧和厌氧条件下批量生物反应器的实验数据。进行了敏感性分析和参数演变研究。选择均方根误差(rRMSE)作为所开发数学模型的验证和校准指标。结果表明,在有氧和厌氧培养条件下,甘油的平均耐受量分别为 174.68 和 44.85 g L-1,乙醇的抑制产物为 150.95 g L-1,1,3-PDO 的抑制产物为 35.56 g L-1,细胞生长的最大比速率分别为 0.189 和 0.275 h-1。该模型对两种作物的拟合效果都很好,有氧环境和厌氧环境的 rRMSE 值分别为 0.09 - 33.74% 和 3.58 - 31.82%。因此,可以对相关信息进行评估和提取,以便更好地理解和控制生物过程。
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引用次数: 0
Implemention of Innovative Process Analytical Technologies to Characterize Critical Quality Attributes of Co‐Formulated Monoclonal Antibody Products 采用创新工艺分析技术表征共配单克隆抗体产品的关键质量属性
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-08 DOI: 10.1002/bit.28881
Apurva Godbole, Lyufei Chen, Jay Desai, Smita Raghava, Richard Ruzanski, Bhumit Patel, Emmanuel Appiah‐Amponsah, Hanzhou Feng
Characterizing co‐formulated monoclonal antibodies (mAbs) poses significant challenges in the pharmaceutical industry. Due to the high structural similarity of the mAbs, traditional analytical methods, compounded by the lengthy method development process, hinder product development and manufacturing efficiency. There is increasing critical need in the pharmaceutical industry to streamline analytical approaches, minimizing time and resources, ensuring a rapid clinical entry and cost‐effective manufacturing. This study investigates the application of process analytical technologies (PAT) to address such challenges. Our investigation introduces two complementary technologies, on‐line ultra‐performance liquid chromatography (online UPLC) and multimode fluorescence spectroscopy (MMFS), as potential PAT tools tailored for characterizing critical quality attributes (CQA) in co‐formulated mAb products. Specifically, the CQAs under evaluation include the total protein concentration of the mAbs within the co‐formulation and the ratio of mAb A to mAb B. Online UPLC enables direct and automated measurement of the CQAs through physical separation, while MMFS determines them in a non‐destructive and more swift manner based on chemometric modeling. We demonstrate these technologies' comparable performance to conventional methods, alongside substantial benefits such as reduced analytical turnaround time and decreased laboratory efforts. Ultimately, integrating them as innovative PAT tools expedites the delivery of therapeutic solutions to patients and enhances manufacturing efficiency, aligning with the imperative for swift translation of scientific discoveries into clinical benefits.
共配制单克隆抗体(mAbs)的表征给制药行业带来了巨大挑战。由于 mAbs 的结构高度相似,传统的分析方法加上漫长的方法开发过程阻碍了产品开发和生产效率。制药行业越来越迫切需要简化分析方法,最大限度地减少时间和资源,确保产品快速进入临床并实现经济高效的生产。本研究调查了过程分析技术 (PAT) 在应对此类挑战中的应用。我们的研究引入了在线超高效液相色谱(UPLC)和多模荧光光谱(MMFS)这两种互补技术,作为潜在的 PAT 工具,专门用于表征共配制 mAb 产品的关键质量属性(CQA)。在线超高效液相色谱(UPLC)可通过物理分离直接自动测量 CQA,而多模式荧光光谱(MMFS)则基于化学计量建模以非破坏性和更快速的方式确定 CQA。我们展示了这些技术与传统方法相当的性能,以及缩短分析周转时间和减少实验室工作量等实质性优势。最终,将这些技术整合为创新的 PAT 工具,可加快向患者提供治疗方案并提高生产效率,这与将科学发现迅速转化为临床效益的要求不谋而合。
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引用次数: 0
Metabolic Engineering of Nonmodel Yeast Issatchenkia orientalis SD108 for 5-Aminolevulinic Acid Production. 生产 5-氨基乙酰丙酸的非模式酵母 Issatchenkia orientalis SD108 的代谢工程。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-06 DOI: 10.1002/bit.28877
Shih-I Tan, I-Son Ng, Huimin Zhao

Biological production of 5-aminolevulinic acid (5-ALA) has received growing attention over the years. However, there is the tradeoff between 5-ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5-ALA. To address this limitation, we engineered an acid-tolerant yeast, Issatchenkia orientalis SD108, for 5-ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5-ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5-ALA was improved from 28 mg/L to 120-, 150-, and 300 mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5-ALA to 510 mg/L, a 13-fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid-tolerant I. orientalis SD108ΔPDC has a high potential for 5-ALA production at a large scale in the future.

近年来,5-氨基乙酰丙酸(5-ALA)的生物生产越来越受到关注。然而,5-ALA 的生物合成与细胞生长之间存在权衡问题,因为 5-ALA 的产生会导致发酵液变酸。为了解决这一限制,我们设计了一种耐酸酵母--东方 Issatchenkia SD108,用于生产 5-ALA。我们首先发现,5-ALA 能促进东方伊沙钦霉 SD108 的细胞生长速度,而且其内源 ALA 合成酶(ALAS)的活性高于其他酵母的同源酶。通过优化质粒设计、超表达转运体和增加基因拷贝数,5-ALA 的滴度分别从 28 mg/L 提高到 120、150 和 300 mg/L。在使用丙酮酸脱羧酶(PDC)基因敲除菌株(SD108ΔPDC)重新定向代谢通量并用尿素培养后,我们将 5-ALA 的滴度提高到了 510 mg/L,提高了 13 倍,这证明了新发现的具有更高活性的 IoALAS 以及对基因敲除菌株进行氮源战略选择的重要性。这项研究表明,耐酸的东方鲑SD108ΔPDC在未来大规模生产5-ALA方面具有很大的潜力。
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引用次数: 0
Oxygen Consumption in Filamentous Pellets of Aspergillus niger: Microelectrode Measurements and Modeling. 黑曲霉丝状颗粒的耗氧量:微电极测量与建模
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-04 DOI: 10.1002/bit.28874
Charlotte Deffur, Anna Dinius, Julian Pagel, Henri Müller, Stefan Schmideder, Heiko Briesen, Rainer Krull

Filamentous fungi cultivated as biopellets are well established in biotechnology industries. A distinctive feature of filamentous fungi is that hyphal growth and fungal morphology affect product titers and require tailored process conditions. Within the pellet, mass transfer, substrate consumption, and biomass formation are intricately linked to the local hyphal fraction and pellet size. This study combined oxygen concentration measurements with microelectrode profiling and three-dimensional X-ray microtomography measurements of the same fungal pellets for the first time. This allowed for the precise correlation of micromorphological information with local oxygen concentrations of two Aspergillus niger strains (hyperbranching and regular branching). The generated results showed that the identified oxygen-penetrated outer pellet regions exhibited a depth of 90-290 µm, strain-specific, with the active part percentage in the pellet ranging from 18% to 69%, without any difference between strains. Using a 1D continuum diffusion consumption model, the oxygen concentration in the pellets was computed depending on the local hyphal fraction. The best simulation results were achieved by individually estimating the oxygen-related biomass yield coefficient of the consumption term within each examined pellet, with an average estimated value of 1.95 (± 0.72) kg biomass per kg oxygen. The study lays the foundation for understanding oxygen supply in fungal pellets and optimizing processes and pellet morphologies accordingly.

作为生物颗粒培养的丝状真菌已在生物技术产业中得到广泛应用。丝状真菌的一个显著特点是,菌丝生长和真菌形态会影响产品滴度,需要定制的工艺条件。在颗粒内部,传质、基质消耗和生物质形成与局部的菌丝部分和颗粒大小密切相关。本研究首次将氧气浓度测量与同一真菌颗粒的微电极剖面测量和三维 X 射线显微层析测量相结合。这使得两种黑曲霉菌株(超分支和规则分支)的微观形态信息与局部氧浓度之间建立了精确的关联。生成的结果表明,已确定的氧穿透外颗粒区域的深度为 90-290 微米,因菌株而异,颗粒中活性部分的百分比从 18% 到 69% 不等,不同菌株之间没有任何差异。利用一维连续扩散消耗模型,计算了颗粒中的氧气浓度,该浓度取决于局部的头状花序部分。通过单独估算每个受检颗粒内消耗项的氧气相关生物量产量系数,获得了最佳模拟结果,平均估算值为每千克氧气 1.95 (± 0.72) 千克生物量。这项研究为了解真菌球团中的氧气供应以及相应地优化工艺和球团形态奠定了基础。
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引用次数: 0
Urea-Loaded PLGA Microspheres as Chemotaxis Stimulants for Helicobacter pylori. 作为幽门螺旋杆菌趋化刺激剂的尿素负载聚乳酸聚乳酸(PLGA)微球
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-03 DOI: 10.1002/bit.28870
Prasanth Shanmughan, Pravin Subrahmaniyan, Dhruv Bhatnagar, Srinithi Ranganathan, Pushkar P Lele

Helicobacter pylori cells undergo chemotaxis toward several small molecules, called chemo-attractants, including urea produced by the epithelial cells of the stomach. The biophysical mechanisms of chemotaxis are not well understood in H. pylori. Here, we developed point sources of urea by encapsulating it in Poly(lactic-co-glycolic acid) or PLGA microbeads for H. pylori chemotaxis studies. Microscopy and Dynamic Light Scattering characterization indicated that the PLGA particles had an average diameter of < 0.8 μm. The particles were relatively stable and had a net negative surface charge. Absorbance measurements indicated that the beads released ~70% of the urea over a 2-week period, with most of the release occurring within the first 24-h period. Varying pH (2.0-7.0) had little effect on the rate of urea release. A diffusion model predicted that such beads could generate sufficient urea gradients to chemotactically attract H. pylori cells. Single-bead single-cell chemotaxis assays confirmed the predictions, revealing that H. pylori continued to be attracted to beads even after most of the urea had been released in the first 24 h. Our work highlights a novel use of PLGA microbeads as delivery vehicles for stimulating a chemotaxis response in H. pylori, with potential applications in bacterial eradication strategies.

幽门螺杆菌细胞对几种小分子(称为趋化吸引剂)具有趋化作用,其中包括胃上皮细胞产生的尿素。幽门螺杆菌趋化的生物物理机制尚不十分清楚。在此,我们通过将尿素封装在聚乳酸-共-乙醇酸或聚乳酸-丙烯酸微珠中,开发了尿素点源,用于幽门螺杆菌趋化研究。显微镜和动态光散射表征表明,PLGA 颗粒的平均直径为
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引用次数: 0
Design an Energy-Conserving Pathway for Efficient Biosynthesis of 1,5-Pentanediol and 5-Amino-1-Pentanol. 为 1,5-戊二醇和 5-氨基-1-戊醇的高效生物合成设计节能途径。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-31 DOI: 10.1002/bit.28875
Lin Ma, Chong Xie, Yu Zhang, Wenna Li, Ning An, Xiaolin Shen, Jia Wang, Xinxiao Sun, Qipeng Yuan

1,5-Pentanediol (1,5-PDO) is an important five-carbon alcohol, widely used in polymer and pharmaceutical industries. Considering the substantial energy (ATP and NADPH) requirements of previous pathways, an energy-conserving artificial pathway with a higher theoretical yield (0.75 mol/mol glucose) was designed and constructed in this study. In this pathway, lysine is converted into 1,5-PDO by decarboxylation, two transamination, and two reduction reactions. For the purpose of full pathway construction, 5-aminopetanal reductase and 5-amino-1-pentanol (5-APO) transaminase were identified and characterized. By implementing strategies such as modular optimization of gene expression, enhancing lysine biosynthesis and increasing NADPH supply, the engineered strains were able to produce 1502.8 mg/L 5-APO and 726.2 mg/L 1,5-PDO in shake flasks and 11.7 g/L 1,5-PDO in a 3 L bioreactor. This work provides a new and promising pathway for the efficient production of 5-APO and 1,5-PDO.

1,5-戊二醇(1,5-PDO)是一种重要的五碳醇,广泛应用于聚合物和制药行业。考虑到以前的途径需要大量能量(ATP 和 NADPH),本研究设计并构建了一种理论产量(0.75 摩尔/摩尔葡萄糖)更高的节能人工途径。在这一途径中,赖氨酸通过脱羧、两次转氨基和两次还原反应转化为 1,5-PDO。为了构建完整的途径,对 5-氨基戊醛还原酶和 5-氨基-1-戊醇(5-APO)转氨酶进行了鉴定和表征。通过模块化优化基因表达、加强赖氨酸生物合成和增加 NADPH 供应等策略,工程菌株能够在摇瓶中产生 1502.8 mg/L 5-APO 和 726.2 mg/L 1,5-PDO,在 3 L 生物反应器中产生 11.7 g/L 1,5-PDO。这项工作为高效生产 5-APO 和 1,5-PDO提供了一种新的、前景广阔的途径。
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引用次数: 0
Protein Scaffold-Mediated Multi-Enzyme Self-Assembly and Ordered Co-Immobilization of Flavin-Dependent Halogenase-Coenzyme Cycle System for Efficient Biosynthesis of 6-Cl-L-Trp. 蛋白支架介导的多酶自组装和黄素依赖性卤化酶-辅酶循环系统的有序协同固定,以实现 6-Cl-L-Trp 的高效生物合成。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-28 DOI: 10.1002/bit.28871
Han-Yu Liu, Pan Ning, Feng Qian, Yao-Wu Wang, Hai-Min Zhang, Pu Wang

Flavin-dependent halogenase (FDH) is highly prized in pharmaceutical and chemical industries for its exceptional capacity to produce halogenated aromatic compounds with precise regioselectivity. This study has devised a multi-enzyme self-assembly strategy to construct an effective and reliable in vitro coenzyme cycling system tailored for FDHs. Initially, tri-enzyme self-assembling nanoclusters (TESNCs) were developed, comprising glucose dehydrogenase (GDH), flavin reductase (FR) and FDH. The TESNCs exhibited enhanced thermal stability and conversion efficiency compared to free triple enzyme mixtures during the conversion of L-Trp to 6-Cl-L-Trp, resulting in a 2.1-fold increase in yield. Subsequently, an ordered co-immobilization of GDH, FR, and FDH was established, further amplifying the stability and catalytic efficiency of the FDH coenzyme cycle system. Compared to the free TESNCs, the immobilized TESNCs demonstrated a 4.2-fold increase in catalytic efficiency in a 5 mL reaction system. This research provides an effective strategy for developing a robust and efficient coenzyme recycling system for FDHs.

黄素依赖性卤化酶(FDH)因其能够以精确的区域选择性生产卤代芳香族化合物而备受制药和化工行业的青睐。本研究设计了一种多酶自组装策略,为 FDH 构建了一个有效可靠的体外辅酶循环系统。最初,研究人员开发了由葡萄糖脱氢酶(GDH)、黄素还原酶(FR)和FDH组成的三酶自组装纳米簇(TESNCs)。在将 L-Trp 转化为 6-Cl-L-Trp 的过程中,与游离三酶混合物相比,TESNCs 表现出更高的热稳定性和转化效率,使产量增加了 2.1 倍。随后,建立了 GDH、FR 和 FDH 的有序共固定,进一步提高了 FDH 辅酶循环系统的稳定性和催化效率。与游离 TESNCs 相比,固定化 TESNCs 在 5 mL 反应体系中的催化效率提高了 4.2 倍。这项研究为开发稳健高效的 FDH 辅酶循环系统提供了有效策略。
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引用次数: 0
Defining Golden Batches in Biomanufacturing Processes From Internal Metabolic Activity to Detect Process Changes That May Affect Product Quality. 从内部代谢活动定义生物制造过程中的黄金批次,以检测可能影响产品质量的过程变化。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-27 DOI: 10.1002/bit.28873
Xin Bush, Erica J Fratz-Berilla, Casey L Kohnhorst, Roberta King, Cyrus Agarabi, David N Powers, Nicholas Trunfio

Cellular metabolism plays a role in the observed variability of a drug substance's Critical Quality Attributes (CQAs) made by biomanufacturing processes. Therefore, here we describe a new approach for monitoring biomanufacturing processes that measures a set of metabolic reaction rates (named Critical Metabolic Parameters (CMP) in addition to the macroscopic process conditions currently being used as Critical Process Parameters (CPP) for biomanufacturing. Constraint-based systems biology models like Flux Balance Analysis (FBA) are used to estimate metabolic reaction rates, and metabolic rates are used as inputs for multivariate Batch Evolution Models (BEM). Metabolic activity was reproducible among batches and could be monitored to detect a deliberately induced macroscopic process shift (i.e., temperature change). The CMP approach has the potential to enable "golden batches" in biomanufacturing processes to be defined from the internal metabolic activity and to aid in detecting process changes that may impact the quality of the product. Overall, the data suggested that monitoring of metabolic activity has promise for biomanufacturing process control.

细胞代谢在生物制造过程中观察到的药物关键质量属性(CQA)的变化中起着重要作用。因此,我们在此介绍一种监测生物制造过程的新方法,除了目前用作生物制造关键工艺参数(CPP)的宏观工艺条件外,还测量一组代谢反应速率(称为关键代谢参数(CMP))。通量平衡分析(FBA)等基于约束的系统生物学模型被用来估算代谢反应速率,代谢速率被用作多元批量进化模型(BEM)的输入。新陈代谢活动在不同批次之间具有可重复性,并且可以通过监测来检测特意诱导的宏观过程转变(即温度变化)。CMP 方法有可能使生物制造过程中的 "黄金批次 "从内部代谢活动中定义出来,并有助于检测可能影响产品质量的过程变化。总之,数据表明,代谢活动监测有望用于生物制造过程控制。
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引用次数: 0
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