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Engineering GID4 for use as an N-terminal proline binder via directed evolution 通过定向进化将 GID4 改造为 N 端脯氨酸结合剂
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-25 DOI: 10.1002/bit.28868
Svetlana P. Ikonomova, Bo Yan, Zhiyi Sun, Rachel B. Lyon, Kelly M. Zatopek, John P. Marino, Zvi Kelman
Nucleic acid sequencing technologies have gone through extraordinary advancements in the past several decades, significantly increasing throughput while reducing cost. To create similar advancement in proteomics, numerous approaches are being investigated to advance protein sequencing. One of the promising approaches uses N-terminal amino acid binders (NAABs), also referred to as recognizers, that selectively can identify amino acids at the N-terminus of a peptide. However, there are only a few engineered NAABs currently available that bind to specific amino acids and meet the requirements of a biotechnology reagent. Therefore, additional NAABs need to be identified and engineered to enable confident identification and, ultimately, de novo protein sequencing. To fill this gap, a human protein GID4 was engineered to create a NAAB for N-terminal proline (Nt-Pro). While native GID4 binds Nt-Pro, its binding is weak (µmol/L) and greatly influenced by the identity of residues following the Nt-Pro. Through directed evolution, yeast-surface display, and fluorescence-activated cell sorting, we identified sequence variants of GID4 with increased binding response to Nt-Pro. Moreover, variants with an A252V mutation showed a reduced influence from residues in the second and third positions of the target peptide when binding to Nt-Pro. The workflow outlined here is shown to be a viable strategy for engineering NAABs, even when starting from native Nt-binding proteins whose binding is strongly impacted by the identity of residues following Nt-amino acid.
过去几十年来,核酸测序技术取得了长足的进步,在降低成本的同时大幅提高了产量。为了在蛋白质组学方面取得类似的进展,目前正在研究多种方法来推进蛋白质测序。其中一种很有前景的方法是使用 N 端氨基酸结合剂(NAABs),它也被称为识别器,可以选择性地识别肽 N 端的氨基酸。然而,目前只有少数工程化的 NAAB 能与特定氨基酸结合,并符合生物技术试剂的要求。因此,需要鉴定和设计更多的 NAABs,以便进行可靠的鉴定,并最终进行全新的蛋白质测序。为了填补这一空白,我们对人类蛋白质 GID4 进行了改造,以创建 N 端脯氨酸(Nt-Pro)的 NAAB。虽然原生 GID4 能与 Nt-Pro 结合,但其结合力很弱(µmol/L),而且在很大程度上受 Nt-Pro 后面残基特性的影响。通过定向进化、酵母表面展示和荧光激活细胞分选,我们发现了对 Nt-Pro 结合反应更强的 GID4 序列变体。此外,带有 A252V 突变的变体在与 Nt-Pro 结合时,目标肽第二和第三位残基的影响减弱。本文概述的工作流程被证明是一种可行的 NAABs 工程设计策略,即使是从原生 Nt 结合蛋白开始,其结合也会受到 Nt 氨基酸后残基身份的强烈影响。
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引用次数: 0
Inhibition Control by Continuous Extractive Fermentation Enhances De Novo 2-Phenylethanol Production by Yeast 连续萃取发酵的抑制控制提高了酵母的新 2-苯基乙醇产量
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-25 DOI: 10.1002/bit.28872
Alessandro Brewster, Arjan Oudshoorn, Marion van Lotringen, Pieter Nelisse, Emily van den Berg, Marijke Luttik, Jean-Marc Daran
Current microbial cell factory methods for producing chemicals from renewable resources primarily rely on (fed-)batch production systems, leading to the accumulation of the desired product. Industrially relevant chemicals like 2-phenylethanol (2PE), a flavor and fragrance compound, can exhibit toxicity at low concentrations, inhibit the host activity, and negatively impact titer, rate, and yield. Batch liquid-liquid (L-L) In Situ Product Removal (ISPR) was employed to mitigate inhibition effects, but was not found sufficient for industrial-scale application. Here, we demonstrated that continuous selective L-L ISPR provides the solution for maintaining the productivity of de novo produced 2PE at an industrial pilot scale. A unique bioreactor concept called “Fermentation Accelerated by Separation Technology” (FAST) utilizes hydrostatic pressure differences to separate aqueous- and extractant streams within one unit operation, where both production and product extraction take place - allowing for the control of the concentration of the inhibiting compound. Controlled aqueous 2PE levels (0.43 ± 0.02 g kg−1) and extended production times (>100 h) were obtained and co-inhibiting by-product formation was reduced, resulting in a twofold increase of the final product output of batch L-L ISPR approaches. This study establishes that continuous selective L-L ISPR, enabled by FAST, can be applied for more economically viable production of inhibiting products.
目前利用可再生资源生产化学品的微生物细胞工厂方法主要依赖于(喂养)批量生产系统,从而导致所需产品的积累。2-phenylethanol (2PE)(一种香精香料化合物)等与工业相关的化学品在低浓度时会表现出毒性,抑制宿主活性,并对滴度、速率和产量产生负面影响。批量液-液(L-L)原位产物去除(ISPR)技术可减轻抑制作用,但不足以满足工业规模的应用。在这里,我们证明了连续选择性液-液 ISPR 为在工业试验规模上保持从头生产 2PE 的生产率提供了解决方案。一种名为 "通过分离技术加速发酵"(FAST)的独特生物反应器概念利用静水压力差在一个单元操作中分离水流和萃取剂流,在此同时进行生产和产品萃取,从而控制抑制性化合物的浓度。通过控制 2PE 的水含量(0.43 ± 0.02 g kg-1)和延长生产时间(100 h),减少了共抑制副产品的形成,从而使批量 L-L ISPR 方法的最终产品产量提高了两倍。这项研究证明,利用 FAST 进行连续选择性 L-L ISPR,可以生产出更具经济效益的抑制性产品。
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引用次数: 0
Real Case Study of 600 m3 Bubble Column Fermentations: Spatially Resolved Simulations Unveil Optimization Potentials for l-Phenylalanine Production With Escherichia coli 600 立方米气泡塔发酵的真实案例研究:空间分辨模拟揭示大肠杆菌生产 l-苯丙氨酸的优化潜力
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-25 DOI: 10.1002/bit.28869
Yannic Mast, Adel Ghaderi, Ralf Takors
Large-scale fermentations (»100 m³) often encounter concentration gradients which may significantly affect microbial activities and production performance. Reliably investigating such scenarios in silico would allow to optimize bioproduction. But related simulations are very rare in particular for large bubble columns. Here, we pioneer the spatially resolved investigation of a 600 m³ bubble column operating for Escherichia coli based l-phenylalanine fed-batch production. Microbial kinetics are derived from experimental data. Advanced Euler-Lagrange (EL) computational fluid dynamics (CFD) simulations are applied to track individual bubble dynamics that result from a recently developed bubble breakage model. Thereon, the complex nonlinear characteristics of hydrodynamics, mass transfer, and microbial activities are simulated for large scale and compared with real data. As a key characteristic, zones for upriser, downcomer, and circulation cells were identified that dominate mixing and mass transfer. This results in complex gradients of glucose, dissolved oxygen, and microbial rates dividing the bioreactor into sections. Consequently, alternate feed designs are evaluated splitting real feed rates in two feeds at different locations. The opposite reversed installation of feed spots and spargers improved the product synthesis by 6.24% while alternate scenarios increased the growth rate by 11.05%. The results demonstrate how sophisticated, spatially resolved simulations of hydrodynamics, mass transfer, and microbial kinetics help to optimize bioreactors in silico.
大规模发酵("100 m³")经常会遇到浓度梯度,这可能会严重影响微生物的活动和生产性能。对这种情况进行可靠的模拟研究可以优化生物生产。但相关模拟非常罕见,尤其是针对大型气泡塔的模拟。在此,我们率先对一个 600 立方米的气泡塔进行了空间分辨研究,该气泡塔以大肠杆菌为基础,进行间歇式苯丙氨酸生产。微生物动力学源于实验数据。先进的欧拉-拉格朗日(EL)计算流体动力学(CFD)模拟用于跟踪最近开发的气泡破裂模型所产生的单个气泡动力学。由此,对流体动力学、传质和微生物活动的复杂非线性特性进行了大规模模拟,并与实际数据进行了比较。作为一个关键特征,确定了主导混合和传质的上行器、下行器和循环池区域。这导致葡萄糖、溶解氧和微生物速率的复杂梯度,将生物反应器划分为不同的区域。因此,我们对替代进料设计进行了评估,在不同位置将实际进料率分成两个进料点。相反,反向安装进料点和喷射器可将产品合成率提高 6.24%,而交替方案可将生长率提高 11.05%。这些结果表明,对流体力学、传质和微生物动力学进行复杂的空间分辨模拟,有助于在硅学中优化生物反应器。
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引用次数: 0
Improving the catalytic performance of carbonyl reductase based on the functional loops engineering. 基于功能环工程改善羰基还原酶的催化性能。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-22 DOI: 10.1002/bit.28864
Tao-Shun Zhou, Xiang-Yang Li, Xiao-Jian Zhang, Xue Cai, Zhi-Qiang Liu, Yu-Guo Zheng

Vibegron functions as a potent and selective β3-adrenergic receptor agonist, with its chiral precursor (2S,3R)-aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)-aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868-fold increase in catalytic efficiency (kcat/Km = 260.3 s-1 mM-1) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild-type EaSDR6 (WT). Structural alignment with WT indicated that loops 137-154 and 182-210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT-1a and M5-1a complex illustrated that M5-1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre-reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased Km value. Under organic-aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large-scale biosynthesis of the vibegron intermediate.

维贝琼是一种强效、选择性的β3-肾上腺素能受体激动剂,其手性前体(2S,3R)-氨基羟基酯(1b)对其合成至关重要。本研究将环路工程学应用于藻类Exiguobacterium的羰基还原酶(EaSDR6),以实现(rac)-氨基酮酯1a的不对称还原。与野生型 EaSDR6(WT)相比,变体 M5(A138L/A190V/S193A/Y201F/N204A)的催化效率提高了 868 倍(kcat/Km = 260.3 s-1 mM-1),并对目标产物 1b 具有理想的立体选择性(对映体过量率 >99%,e.e.;非对映体过量率 >99%,d.e.)。与 WT 的结构比对表明,137-154 环和 182-210 环可能在促进催化和底物结合方面起着重要作用。此外,WT-1a 和 M5-1a 复合物的分子动力学(MD)模拟表明,M5-1a 表现出更有效的亲核攻击距离,更容易进入预反应状态。结合自由能的计算分析表明,与 WT 相比,M5 对底物 1a 的亲和力更强,这与其 Km 值的降低是一致的。在有机-水双相条件下,M5 突变体在底物 1a 为 300 g/L 的情况下,12 小时内的转化率大于 99%(据报道,底物负载量最高)。这项研究通过功能环工程提高了羰基还原酶的催化性能,并为大规模生物合成维贝琼中间体建立了一个稳健的框架。
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引用次数: 0
Elevated endoplasmic reticulum pH is associated with high growth and bisAb aggregation in CHO cells. 内质网 pH 值升高与 CHO 细胞的高生长和双抗体聚集有关。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-22 DOI: 10.1002/bit.28866
Kevin S McFarland, Kaitlin Hegadorn, Michael J Betenbaugh, Michael W Handlogten

Chinese hamster ovary (CHO) bioprocesses, the dominant platform for therapeutic protein production, are increasingly used to produce complex multispecific proteins. Product quantity and quality are affected by intracellular conditions, but these are challenging to measure and often overlooked during process optimization studies. pH is known to impact quality attributes like protein aggregation across upstream and downstream processes, yet the effects of intracellular pH on cell culture performance are largely unknown. Recently, advances in protein biosensors have enabled investigations of intracellular environments with high spatiotemporal resolution. In this study, we integrated a fluorescent pH-sensitive biosensor into a bispecifc (bisAb)-producing cell line to investigate changes in endoplasmic reticulum pH (pHER). We then investigated how changes in lactate metabolism impacted pHER, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pHER rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in the stationary phase in all perfusion and fed-batch conditions. pHER decline in the stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Finally, we found protein aggregate levels correlated most closely with pHER which provides new insights into product aggregate formation in CHO processes. An improved understanding of the intracellular changes impacting bioprocesses can ultimately help guide media optimizations, improve bioprocess control strategies, or provide new targets for cell engineering.

中国仓鼠卵巢(CHO)生物工艺是治疗蛋白质生产的主要平台,越来越多地用于生产复杂的多特异性蛋白质。众所周知,pH 值会影响蛋白质在上游和下游过程中的聚集等质量属性,但细胞内 pH 值对细胞培养性能的影响在很大程度上还不为人所知。最近,蛋白质生物传感器的进步使得人们能够以高时空分辨率研究细胞内环境。在这项研究中,我们将荧光 pH 敏感生物传感器集成到了双胰蛋白酶(bisAb)产生细胞系中,以研究内质网 pH(pHER)的变化。然后,我们研究了乳酸代谢的变化如何影响 pHER、细胞氧化还原以及喂料批次和灌流生物反应器中的产品质量。我们的数据显示,pHER 在指数生长过程中迅速升高到 pH 7.7 的最大值,随后在所有灌流和喂料批次条件下,pHER 在静止期急剧下降。最后,我们发现蛋白质聚合体水平与 pHER 的相关性最为密切,这为了解 CHO 过程中产物聚合体的形成提供了新的视角。提高对影响生物过程的细胞内变化的认识,最终有助于指导培养基优化、改进生物过程控制策略,或为细胞工程提供新的目标。
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引用次数: 0
Adjustment of the main biosynthesis modules to enhance the production of l‐homoserine in Escherichia coli W3110 调整大肠杆菌 W3110 的主要生物合成模块以提高 l-高丝氨酸的产量
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-19 DOI: 10.1002/bit.28861
Kun Niu, Rui Zheng, Miao Zhang, Mao‐Qin Chen, Yi‐Ming Kong, Zhi‐Qiang Liu, Yu‐Guo Zheng
l‐homoserine is an important platform compound of many valuable products. Construction of microbial cell factory for l‐homoserine production from glucose has attracted a great deal of attention. In this study, l‐homoserine biosynthesis pathway was divided into three modules, the glucose uptake and upstream pathway, the downstream pathway, and the energy supply module. Metabolomics of the chassis strain HS indicated that the supply of ATP was inadequate, therefore, the energy supply module was firstly modified. By balancing the ATP supply module, the l‐homoserine production increased by 66% to 12.55 g/L. Further, the results indicated that the upstream pathway was blocked, and increasing the culture temperature to 37°C could solve this problem and the l‐homoserine production reached 21.38 g/L. Then, the downstream synthesis pathways were further strengthened to balance the fluxes, and the l‐homoserine production reached the highest reported level of 32.55 g/L in shake flasks. Finally, fed‐batch fermentation in a 5‐L bioreactor was conducted, and l‐homoserine production could reach to 119.96 g/L after 92 h cultivation, with the yield of 0.41 g/g glucose and productivity of 1.31 g/L/h. The study provides a well research foundation for l‐homoserine production by microbial fermentation with the capacity for industrial application.
l-高丝氨酸是许多有价值产品的重要平台化合物。构建以葡萄糖为原料生产 l-高丝氨酸的微生物细胞工厂引起了广泛关注。本研究将 l-高丝氨酸的生物合成途径分为三个模块,即葡萄糖摄取及上游途径、下游途径和能量供应模块。基质菌株 HS 的代谢组学研究表明 ATP 供应不足,因此首先对能量供应模块进行了改造。通过平衡 ATP 供应模块,l-高丝氨酸的产量增加了 66%,达到 12.55 克/升。此外,结果表明上游途径受阻,将培养温度提高到 37°C 可以解决这一问题,l-高丝氨酸产量达到 21.38 克/升。然后,进一步加强下游合成途径以平衡通量,在摇瓶中,l-高丝氨酸的产量达到了所报道的最高水平,即 32.55 克/升。最后,在 5 升生物反应器中进行饲料批量发酵,经过 92 h 的培养,l-高丝氨酸产量达到 119.96 g/L,葡萄糖产量为 0.41 g/g,生产率为 1.31 g/L/h。该研究为微生物发酵法生产 l-高丝氨酸提供了良好的研究基础,并具有工业应用能力。
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引用次数: 0
Transfer learning Bayesian optimization for competitor DNA molecule design for use in diagnostic assays 用于诊断分析的竞争对手 DNA 分子设计的迁移学习贝叶斯优化方法
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-16 DOI: 10.1002/bit.28854
Ruby Sedgwick, John P. Goertz, Molly M. Stevens, Ruth Misener, Mark van der Wilk
With the rise in engineered biomolecular devices, there is an increased need for tailor-made biological sequences. Often, many similar biological sequences need to be made for a specific application meaning numerous, sometimes prohibitively expensive, lab experiments are necessary for their optimization. This paper presents a transfer learning design of experiments workflow to make this development feasible. By combining a transfer learning surrogate model with Bayesian optimization, we show how the total number of experiments can be reduced by sharing information between optimization tasks. We demonstrate the reduction in the number of experiments using data from the development of DNA competitors for use in an amplification-based diagnostic assay. We use cross-validation to compare the predictive accuracy of different transfer learning models, and then compare the performance of the models for both single objective and penalized optimization tasks.
随着工程生物分子设备的增多,对定制生物序列的需求也在增加。通常情况下,需要为特定应用制作许多类似的生物序列,这意味着需要进行大量的实验室实验来优化这些序列,有时实验成本之高令人望而却步。本文介绍了一种转移学习实验设计工作流程,使这一开发变得可行。通过将迁移学习代用模型与贝叶斯优化相结合,我们展示了如何通过在优化任务之间共享信息来减少实验总数。我们利用开发用于基于扩增的诊断检测的 DNA 竞争对手的数据,展示了实验数量的减少。我们使用交叉验证来比较不同迁移学习模型的预测准确性,然后比较这些模型在单一目标和惩罚优化任务中的表现。
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引用次数: 0
Reshaping the substrate‐binding pocket of acyl‐ACP reductase to enhance the production of sustainable aviation fuel in Escherichia coli 重塑酰基-ACP 还原酶的底物结合袋,提高大肠杆菌可持续航空燃料的产量
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-16 DOI: 10.1002/bit.28863
Jiahu Han, Takuya Matsumoto, Ryosuke Yamada, Hiroyasu Ogino
To reduce carbon emissions and address environmental concerns, the aviation industry is exploring the use of sustainable aviation fuel (SAF) as an alternative to traditional fossil fuels. In this context, bio‐alkane is considered a potentially high‐value solution. The present study focuses on the enzymes acyl‐acyl carrier protein [ACP] reductase (AAR) and aldehyde‐deformylating oxygenase (ADO), which are crucial enzymes for alka(e)ne biosynthesis. By using protein engineering techniques, including semi‐rational design and site‐directed mutagenesis, we aimed to enhance the substrate specificity of AAR and improve alkane production efficiency. The co‐expression of a modified AAR (Y26G/Q40M mutant) with wild‐type ADO in Escherichia coli significantly increased alka(e)ne production from 28.92 mg/L to 167.30 mg/L, thus notably demonstrating a 36‐fold increase in alkane yield. This research highlights the potential of protein engineering in optimizing SAF production, thereby contributing to the development of more sustainable and efficient SAF production methods and promoting greener air travel.
为了减少碳排放和解决环境问题,航空业正在探索使用可持续航空燃料(SAF)来替代传统化石燃料。在这种情况下,生物烷烃被认为是一种潜在的高价值解决方案。本研究的重点是酰基-酰基载体蛋白[ACP]还原酶(AAR)和醛脱甲酰氧合酶(ADO),它们是烯烃生物合成的关键酶。我们利用蛋白质工程技术,包括半理性设计和定点突变,旨在增强 AAR 的底物特异性,提高烷烃的生产效率。在大肠杆菌中共同表达修饰的AAR(Y26G/Q40M突变体)和野生型ADO后,烷烃产量从28.92毫克/升显著增加到167.30毫克/升,从而证明烷烃产量增加了36倍。这项研究凸显了蛋白质工程在优化烷烃生产方面的潜力,从而有助于开发更可持续、更高效的烷烃生产方法,促进绿色航空旅行。
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引用次数: 0
Cover Image, Volume 121, Number 11, November 2024 封面图片,第 121 卷第 11 期,2024 年 11 月
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-15 DOI: 10.1002/bit.28867
Jimmy Boman, Tjaša Marušič, Tina Vodopivec Seravalli, Janja Skok, Fredrik Pettersson, Kristina Šprinzar Nemec, Henrik Widmark, Rok Sekirnik

The cover image is based on the Article Quality by design approach to improve quality and decrease cost of in vitro transcription of mRNA using design of experiments by Jimmy Boman and Tjaša Marušič et al., https://doi.org/10.1002/bit.28806.

封面图片基于 Jimmy Boman 和 Tjaša Marušič 等人撰写的《利用实验设计提高 mRNA 体外转录质量并降低成本的设计质量方法》一文,https://doi.org/10.1002/bit.28806。
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引用次数: 0
Biotechnology and Bioengineering: Volume 121, Number 11, November 2024 生物技术与生物工程第 121 卷第 11 号,2024 年 11 月
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-15 DOI: 10.1002/bit.28856
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引用次数: 0
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