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Development of Tat-fused drug binding protein to improve anti-cancer effect of mammalian target of rapamycin inhibitors 开发与 Tat 融合的药物结合蛋白,提高哺乳动物雷帕霉素靶点抑制剂的抗癌效果
IF 3.2 4区 生物学 Q2 Engineering Pub Date : 2024-02-13 DOI: 10.1007/s12257-024-00015-7
Su Yeon Lim, Sugyeong Kim, Hongbin Kim, Hyun-Ouk Kim, Suk-Jin Ha, Kwang Suk Lim

The mammalian target of rapamycin (mTOR) is known to regulate cell growth, protein stability and cell-cycle progression, and many human tumors result from the dysregulation of mTOR signaling. Although various mTOR inhibitors have been developed, effective delivery systems are still needed to enhance the anti-cancer effects of mTOR inhibitors. In this study, we developed the Tat-fused mTOR inhibitor binding domain (Tat-MBD/TMBD) for the enhancement of the anti-cancer effect of mTOR inhibitors, due to the improvement of intracellular uptake. A TMBD/mTOR inhibitors complex spontaneously formed by biological affinity between MBD and mTOR inhibitors without chemical conjugation and modification. We constructed that a recombinant fusion protein expression vector composed of Tat (protein transduction domain) and mTOR inhibitor-binding domain (Tat-MBD) to deliver the mTOR inhibitors. The MBD spontaneously bound with mTOR inhibitors including sirolimus, everolimus, and temsirolimus, resulting in the formation of a TMBD/mTOR inhibitors complex. The enhancement of the delivery efficacy of mTOR inhibitors into various breast cancer cells was confirmed and improved anti-cancer efficacy was observed. We demonstrated the effective delivery systems of mTOR inhibitors without chemical conjugation of mTOR inhibitors.

众所周知,哺乳动物雷帕霉素靶标(mTOR)可调控细胞生长、蛋白质稳定性和细胞周期进展,许多人类肿瘤都是由 mTOR 信号失调引起的。虽然目前已开发出多种 mTOR 抑制剂,但仍需要有效的递送系统来增强 mTOR 抑制剂的抗癌效果。在这项研究中,我们开发了 Tat 融合 mTOR 抑制剂结合域(Tat-MBD/TMBD),通过改善细胞内吸收来增强 mTOR 抑制剂的抗癌效果。TMBD/mTOR抑制剂复合物是MBD和mTOR抑制剂之间通过生物亲和力自发形成的,无需化学共轭和修饰。我们构建了一种由 Tat(蛋白质转导结构域)和 mTOR 抑制剂结合结构域(Tat-MBD)组成的重组融合蛋白表达载体来递送 mTOR 抑制剂。MBD 自发地与包括西罗莫司、依维莫司和替西罗莫司在内的 mTOR 抑制剂结合,形成 TMBD/mTOR 抑制剂复合物。实验证实,TMBD 可增强 mTOR 抑制剂在各种乳腺癌细胞中的递送效果,并提高抗癌疗效。我们证明了 mTOR 抑制剂的有效递送系统,无需对 mTOR 抑制剂进行化学共轭。
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引用次数: 0
Assessment of pulsed dielectrophoretic-field flow fractionation separation coupled with fibre-optic detection on a lab-on-chip as a technique to separate similar bacteria cells 在片上实验室评估脉冲介电泳-场流分馏分离和光纤检测技术,作为一种分离相似细菌细胞的技术
IF 3.2 4区 生物学 Q2 Engineering Pub Date : 2024-02-13 DOI: 10.1007/s12257-024-00001-z
Mohd Firdaus Kamuri, Zurina Zainal Abidin, Mohd Hanif Yaacob, Mohd Nizar Hamidon

This study addresses the challenge of separating bacteria with similar structures such as Escherichia coli and Aeromonas hydrophila. This approach employs pulsed field dielectrophoresis assisted by laminar flow fractionation in a lab-on-a-chip system with integrated optical detection. Bacterial cells passed through 30-µm microelectrodes subjected at 1 MHz and 14 V peak-to-peak in pulsed mode, while fluid flow carried bacteria towards the chamber’s end. The on-and-off electric field at specific pulse intervals expose bacterial cells to diverse forces, including kinetics, dielectrophoresis, gravity, drag, and diffusion, resulting in a net force facilitating their movement. Variations of pulsing time, flow rates, and voltage were investigated to identify the optimal combination for efficient separation. Next, the bacteria were detected using an optical fibre based on their absorbance. Results demonstrated a 30% separation efficiency in 90 min at 9.6 μL min−1 flow rates, 4 s pulsing time, and 40 μS cm−1 medium conductivity. A. hydrophila aggregates experienced greater DEP force and retained at microelectrodes during electric field application compared to E. coli, which moved faster towards optical detection. The separation mechanism with and without electric field was different, and precise control of cell movement during field-off periods is important to minimize uncontrolled diffusion. While the optical detection part has been successful, longer time and separation length are recommended for better separation. A carefully tuned combination of pulsing time, flow rates, voltage, and microelectrode design is crucial for this integrated lab-on-chip system to be efficient for separating and detecting closely related microorganisms.

这项研究解决了分离大肠杆菌和嗜水气单胞菌等结构相似细菌的难题。这种方法采用脉冲场介电泳,并在集成光学检测功能的片上实验室系统中以层流分馏法辅助。在脉冲模式下,细菌细胞通过 30 微米的微电极,微电极的频率为 1 兆赫,峰-峰值电压为 14 伏特,同时流体流动将细菌带向腔室的末端。特定脉冲间隔的通断电场使细菌细胞受到各种力的作用,包括动力学力、介电泳力、重力、阻力和扩散力,从而产生促进其运动的净力。对脉冲时间、流速和电压的变化进行了研究,以确定高效分离的最佳组合。接下来,使用光纤根据细菌的吸光度对其进行检测。结果表明,在流速为 9.6 μL min-1、脉冲时间为 4 秒、介质电导率为 40 μS cm-1 的条件下,90 分钟内的分离效率为 30%。与大肠杆菌相比,在施加电场的过程中,纤毛虫聚集体受到了更大的去污力,并保留在微电极上,而大肠杆菌则更快地向光学检测方向移动。有电场和无电场时的分离机制是不同的,因此在电场关闭期间精确控制细胞的移动对减少不受控制的扩散非常重要。虽然光学检测部分取得了成功,但为了更好地进行分离,建议延长时间和分离长度。精心调整脉冲时间、流速、电压和微电极设计的组合对于这个集成片上实验室系统高效分离和检测密切相关的微生物至关重要。
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引用次数: 0
Reconstruction of genome-scale metabolic models of non-conventional yeasts: current state, challenges, and perspectives 非常规酵母菌基因组尺度代谢模型的重建:现状、挑战和前景
IF 3.2 4区 生物学 Q2 Engineering Pub Date : 2024-02-13 DOI: 10.1007/s12257-024-00009-5
Eduardo Luís Menezes de Almeida, Eduard J. Kerkhoven, Wendel Batista da Silveira

Non-conventional yeasts are promising cell factories to produce lipids and oleochemicals, metabolites of industrial interest (e.g., organics acids, esters, and alcohols), and enzymes. They can also use different agro-industrial by-products as substrates within the context of a circular economy. Some of these yeasts can also comprise economic and health burdens as pathogens. Genome-scale metabolic models (GEMs), networks reconstructed based on the genomic and metabolic information of one or more organisms, are great tools to understand metabolic functions and landscapes, as well as propose engineering targets to improve metabolite production or propose novel drug targets. Previous reviews on yeast GEMs have mainly focused on the history and the evaluation of Saccharomyces cerevisiae modeling paradigms or the accessibility and usability of yeast GEMs. However, they did not describe the reconstruction strategies, limitations, validations, challenges, and research gaps of non-conventional yeast GEMs. Herein, we focused on the reconstruction of available non-Saccharomyces GEMs, their validation, underscoring the physiological insights, as well as the identification of both metabolic engineering and drug targets. We also discuss the challenges and knowledge gaps and propose strategies to boost their use and novel reconstructions.

非常规酵母是生产脂类和油脂化学品、工业用代谢物(如有机酸、酯和醇)以及酶的有前途的细胞工厂。在循环经济的背景下,它们还可以利用不同的农用工业副产品作为基质。其中一些酵母菌还可以作为病原体造成经济和健康负担。基因组尺度代谢模型(GEM)是根据一个或多个生物体的基因组和代谢信息重建的网络,是了解代谢功能和代谢景观的重要工具,也是提出工程目标以改善代谢物生产或提出新型药物靶点的重要工具。以往关于酵母 GEM 的综述主要集中在酵母建模范式的历史和评估,或酵母 GEM 的可及性和可用性。然而,它们并没有描述非常规酵母 GEM 的重建策略、局限性、验证、挑战和研究空白。在本文中,我们重点讨论了现有非酵母 GEMs 的重建、验证、生理学见解以及代谢工程和药物靶点的鉴定。我们还讨论了所面临的挑战和知识差距,并提出了促进其使用和新型重建的策略。
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引用次数: 0
Enhanced fermentative γ-aminobutyric acid production by a metabolic engineered Corynebacterium glutamicum 经代谢工程改造的谷氨酸棒状杆菌提高了γ-氨基丁酸的发酵产量
IF 3.2 4区 生物学 Q2 Engineering Pub Date : 2024-02-12 DOI: 10.1007/s12257-024-00008-6

Abstract

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with important physiological functions, which has been widely used in food, pharmaceuticals, and polyamides production. The fermentative GABA production by Corynebacterium glutamicum was recognized as one of the most promising methods. However, the problems of low catalytic activity of the heterologously expressed glutamate decarboxylase (GAD) and the imbalanced carbon flux between cell growth and GABA synthesis severely limited the GABA production by C. glutamicum. This study applied combinational metabolic engineering and catalytic condition optimization strategies to solve these two major obstacles. The secretory expression of GAD was enhanced using a bicistronic-designed expression cassette. This bicistronic expression cassette was further triply inserted into the genome by substituting the ldhA, pqo, and ack genes, thus stabilizing the expression of GAD and reducing the accumulation of by-products of lactate and acetate. A growth-regulated promoter PCP_2836 was applied to dynamically control the expression of odhA, thus controlling the α-oxoglutarate dehydrogenase complex activity for balanced cell growth and GABA production. The glutamate precursor synthesis and pyridoxal 5′-phosphate supply were also strengthened by promoter substitution. Finally, through a two-stage pH-controlled fed-batch fermentation under optimized conditions, the engineered strain reached GABA titer of 81.31 ± 1.31 g/L with a yield and productivity of 0.50 ± 0.01 g/g and 1.36 ± 0.23 g L−1 h−1, which was 4.8%, 13.6%, and 11.2% higher than that of the original strain. This study laid a solid foundation for industrial fermentative GABA production by engineered C. glutamicum.

摘要 γ-氨基丁酸(GABA)是一种非蛋白源氨基酸,具有重要的生理功能,已被广泛应用于食品、药品和聚酰胺的生产。谷氨酸棒杆菌发酵生产 GABA 被认为是最有前途的方法之一。然而,异源表达的谷氨酸脱羧酶(GAD)催化活性低以及细胞生长与 GABA 合成之间的碳通量不平衡等问题严重限制了谷氨酸棒杆菌生产 GABA。本研究采用组合代谢工程和催化条件优化策略来解决这两大障碍。使用双螺旋设计的表达盒增强了 GAD 的分泌表达。通过替换ldhA、pqo和ack基因,进一步将该双螺旋表达盒三重插入基因组,从而稳定了GAD的表达,并减少了副产物乳酸和乙酸的积累。应用生长调控启动子 PCP_2836 动态控制 odhA 的表达,从而控制 α-氧谷氨酸脱氢酶复合物的活性,以平衡细胞生长和 GABA 的产生。通过启动子替换,谷氨酸前体合成和 5′-磷酸吡哆醛供应也得到了加强。最后,通过优化条件下的两阶段 pH 控制饲料批量发酵,工程菌株的 GABA 滴度达到 81.31 ± 1.31 g/L,产量和生产率分别为 0.50 ± 0.01 g/g 和 1.36 ± 0.23 g L-1 h-1,比原始菌株分别高出 4.8%、13.6% 和 11.2%。这项研究为利用工程谷氨酸棒状杆菌发酵生产 GABA 奠定了坚实的基础。
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引用次数: 0
Effects of a novel bioprocess for the cultivation Synechococcus nidulans on Mars on its biochemical composition: focus on the lipidome 用于在火星上培养 Synechococcus nidulans 的新型生物工艺对其生化组成的影响:重点关注脂质组
IF 3.2 4区 生物学 Q2 Engineering Pub Date : 2024-02-12 DOI: 10.1007/s12257-024-00019-3
Mattia Casula, Giacomo Fais, Agnieszka Sidorowicz, Pierluigi Caboni, Giacomo Cao, Alessandro Concas

In the present work, the possibility to grow the strain Synechococcus nidulans CCALA 188 on Mars using a medium mimicking a one obtainable using in situ available resources, i.e. the so-called Martian medium, under an atmosphere obtainable by pressurization of Mars CO2, is investigated. The goal is to obtain a biomass with high-value products to sustain a crewed mission to Mars. The results show that the replacement of 40% vol of Z-medium with the same volume of Martian medium does not affect the cultivation and leads to a slight improvement of biomass productivity. Under an atmosphere consisting of pure CO2 the growth rate was reduced but the strain managed to adapt by modifying its metabolism. Total proteins and carbohydrates were significantly reduced under Mars-like conditions, while lipids increased when using CO2. A balanced diet rich in antioxidants is crucial for the wealth of astronauts, and in our case, radical scavenging capacities range from 15 to 20 mmolTEAC/kg were observed. Under CO2, a reduction in antioxidant power is observed likely due to a decrease in photosynthetic activity. The lipidome consisted of sulfoquinovosyldiacylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylcholine, phosphatidylglycerol, and triacylglycerol. A significant increase in the latter ones was observed under Mars simulated atmosphere.

在本研究工作中,研究了在火星二氧化碳加压产生的大气环境下,使用模拟可利用现场可用资源获得的培养基(即所谓的火星培养基)在火星上培养 Synechococcus nidulans CCALA 188 菌株的可能性。目标是获得一种具有高价值产品的生物质,以维持火星乘员任务。结果表明,用相同体积的火星培养基替代 40% 的 Z 培养基不会影响培养效果,反而会略微提高生物质的生产率。在纯二氧化碳环境下,生长速度降低,但菌株通过改变新陈代谢得以适应。在类似火星的条件下,总蛋白质和碳水化合物明显减少,而在使用二氧化碳时,脂质则有所增加。富含抗氧化剂的均衡饮食对宇航员的财富至关重要,在我们的实验中,观察到自由基清除能力为 15 至 20 毫摩尔TEAC/千克。在二氧化碳环境下,可能由于光合作用减弱,抗氧化能力下降。脂质体包括磺基喹诺酮酰基二乙酰甘油、单半乳糖酰基二乙酰甘油、二半乳糖酰基二乙酰甘油、磷脂酰胆碱、磷脂酰甘油和三酰甘油。在火星模拟大气中观察到后一种物质明显增加。
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引用次数: 0
Xylanase enzyme from novel strain and its immobilization onto metal organic framework MOF for fruit juice clarification 来自新型菌株的木聚糖酶及其在金属有机框架 MOF 上的固定化用于果汁澄清
IF 3.2 4区 生物学 Q2 Engineering Pub Date : 2024-02-12 DOI: 10.1007/s12257-024-00007-7
Jyoti Kaushal, Madhu Khatri, Gursharan Singh, Shailendra Kumar Arya

Bacillus pumilus, a bacterial strain was isolated from agricultural soil and used for xylanase enzyme (Xy) production under the submerged fermentation technique. The (Xy) enzyme had an optimum temperature at 50℃ (maximum activity from 45–60 °C) and was active at broad pH range (5.0–8.0) with an optimum pH at around 6.3 as evaluated from response surface methodology studies. This enzyme after purification (purification; 2.87 folds, specific activity; 64.3 U/mg) was immobilized onto MOFCu-BTC (a copper ion-based metal organic framework) and was used for clarification of freshly squeezed fruit juice (pineapple and pomegranate). The study revealed an improved catalytic efficiency (Vmax from 1.252.5 to 1.361 U/mL/mg of support) and greater half-life of the immobilized system (77–99 min). The activation energy decreased from that required for the free system (37.59–25.63 kJ/mol). The reusability of the enzyme improved after immobilization where 61% of the enzyme’s activity was retained after 21 cycles of usage. The MOFXy-Cu-BTC system showed improved clarification (47.58–57.97% for pineapple, and 15.34–18.3 for pomegranate) thereby showing its effectiveness in commercial juice clarification process.

从农业土壤中分离出一种细菌菌株--枯草芽孢杆菌(Bacillus pumilus),并采用浸没发酵技术生产木聚糖酶(Xy)。根据响应面方法学研究的评估,木聚糖酶(Xy)的最适温度为 50℃(45-60℃时活性最高),在广泛的 pH 值范围(5.0-8.0)内均有活性,最适 pH 值约为 6.3。纯化后的这种酶(纯化倍数为 2.87 倍,比活性为 64.3 U/mg )被固定在 MOFCu-BTC(一种基于铜离子的金属有机框架)上,用于澄清鲜榨果汁(菠萝和石榴)。研究结果表明,固定化系统的催化效率提高了(Vmax 从 1.252.5 U/mg/支持物提高到 1.361 U/mg/支持物),半衰期延长了(77-99 min)。活化能与游离体系相比有所降低(37.59-25.63 kJ/mol)。固定化后,酶的可再利用性得到了改善,在使用 21 个周期后,61% 的酶活性得以保留。MOFXy-Cu-BTC 系统提高了澄清度(菠萝为 47.58%-57.97%,石榴为 15.34%-18.3%),从而显示了其在商业果汁澄清过程中的有效性。
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引用次数: 0
Biofoundries: Bridging Automation and Biomanufacturing in Synthetic Biology 生物工厂:在合成生物学的自动化和生物制造之间架起桥梁
IF 3.2 4区 生物学 Q2 Engineering Pub Date : 2023-12-11 DOI: 10.1007/s12257-023-0226-x
Dae-Hee Lee, Haseong Kim, Bong-Hyun Sung, Byung Kwan Cho, Seung-Goo Lee

Biofoundries represent advanced automation facilities pivotal for streamlining the Design-Build-Test-Learn (DBTL) paradigm in biomanufacturing and synthetic biology, suitable for both academic research and industrial applications. Nonetheless, establishing such a platform demands significant financial and temporal resources while maintaining a forward-looking perspective on automation, equipment compatibility, and operational efficiency. Despite its challenges, international collaborations between global biofoundries may offer solutions. The automated DBTL framework in biofoundries has transformed the production of bioproducts using engineered microbes. As the field advances, biofoundries are essential for streamlining and standardizing biotechnological processes, addressing efficiency, cost, and consistency challenges.

生物工厂是先进的自动化设施,对于简化生物制造和合成生物学中的 "设计-制造-测试-学习"(DBTL)模式至关重要,既适用于学术研究,也适用于工业应用。然而,建立这样一个平台需要大量的财力和时间资源,同时还要在自动化、设备兼容性和运行效率方面保持前瞻性。尽管挑战重重,但全球生物工厂之间的国际合作可以提供解决方案。生物工厂的自动化 DBTL 框架改变了利用工程微生物生产生物产品的方式。随着该领域的发展,生物熔炉对于简化和标准化生物技术流程、解决效率、成本和一致性挑战至关重要。
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引用次数: 0
Wickerhamomyces ciferrii Auxotroph and Expression Vector for Improved Production of Tetraacetyl Phytosphingosine 枸杞子营养不良菌及其改良生产四乙酰基植物鞘氨酸的表达载体
4区 生物学 Q2 Engineering Pub Date : 2023-10-27 DOI: 10.1007/s12257-023-0128-y
Seok Woo Yoo, Minsun Kim, Boo-Su Park, Jihee Yoon, Min-Kyu Oh
{"title":"Wickerhamomyces ciferrii Auxotroph and Expression Vector for Improved Production of Tetraacetyl Phytosphingosine","authors":"Seok Woo Yoo, Minsun Kim, Boo-Su Park, Jihee Yoon, Min-Kyu Oh","doi":"10.1007/s12257-023-0128-y","DOIUrl":"https://doi.org/10.1007/s12257-023-0128-y","url":null,"abstract":"","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136262118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Convolution Neural Network Approaches for Cancer Cell Image Classification 肿瘤细胞图像分类的卷积神经网络方法
4区 生物学 Q2 Engineering Pub Date : 2023-10-27 DOI: 10.1007/s12257-023-0164-7
Chaeyoung Kim, Sungtae Shin, Sehoon Jeong
{"title":"Convolution Neural Network Approaches for Cancer Cell Image Classification","authors":"Chaeyoung Kim, Sungtae Shin, Sehoon Jeong","doi":"10.1007/s12257-023-0164-7","DOIUrl":"https://doi.org/10.1007/s12257-023-0164-7","url":null,"abstract":"","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136262733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Co-utilization of Maltose and Sodium Acetate via Engineered Corynebacterium glutamicum for Improved Itaconic Acid Production 工程谷氨酸棒状杆菌对麦芽糖和乙酸钠的协同利用改进衣康酸的生产
4区 生物学 Q2 Engineering Pub Date : 2023-10-01 DOI: 10.1007/s12257-023-0091-7
Taghreed Elkasaby, Dao Duy Hanh, Hideo Kawaguchi, Masakazu Toyoshima, Akihiko Kondo, Chiaki Ogino
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引用次数: 0
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Biotechnology and Bioprocess Engineering
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