Journal of bioscience and bioengineering最新文献_第5页

Characterization of a molting-related chitinase from a land crab, Chiromantes haematocheir 一种陆地蟹（Chiromantes haematocheir）几丁质酶的特性。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-10-07 DOI: 10.1016/j.jbiosc.2025.09.006

Yuma Nagakura, Katsuhide Miyake

Chitinases play an important role in many biological processes, including molting, digestion, and immunity in crustaceans. This study represents an attempt to apply chitinases in the field of biotechnology, detecting chitinase mRNAs from a land crab, Chiromantes haematocheir, via transcriptome analysis and analyzing their properties. Seven chitinase genes were detected from the RNA-seq data of midgut glands. Among these genes, TRINITY_DN29294 transcripts accounted for virtually all of total expression of the chitinases. The 29294 cDNA contained a 1467 bp open reading frame, coded for 488 amino acid residues, and was classified into the GH18 chitolectin chitotriosidase and the group 3 crab chitinase. The expression of the 29294-chitinase mRNA was detected in all tissues, with the highest levels expressed in the midgut glands. The transcripts increased significantly in the early post-molted crab compared to the non-molting crab. These results suggest that 29294-chitinase plays important roles in the molting process. While the recombinant 29294-chitinase over-produced in Escherichia coli did not show any activity, the enzyme expressed in Pichia pastoris exhibited sufficient activity. The 29294-chitinase had its optimal pH 3.0. The optimal temperature was relatively high at 45 °C. The enzyme hydrolyzed both soluble and crystalline substrates.

几丁质酶在甲壳类动物的蜕皮、消化和免疫等生物过程中起着重要作用。本研究尝试将几丁质酶应用于生物技术领域，通过转录组分析检测陆地蟹Chiromantes haematocheir的几丁质酶mrna并分析其性质。从中肠腺的RNA-seq数据中检测到7个几丁质酶基因。在这些基因中，TRINITY_DN29294转录本几乎占几丁质酶总表达量的全部。29294 cDNA包含1467 bp的开放阅读框，编码488个氨基酸残基，被分类为GH18壳聚糖壳三醇苷酶和第3组螃蟹几丁质酶。29294-几丁质酶mRNA在各组织中均有表达，其中以中肠腺表达量最高。与未蜕皮的蟹相比，早期蜕皮蟹的转录本显著增加。上述结果表明，29294-几丁质酶在玉米的蜕皮过程中起着重要作用。在大肠杆菌中过量产生的重组29294-几丁质酶没有表现出任何活性，而在毕赤酵母中表达的酶表现出足够的活性。29294-几丁质酶的最佳pH值为3.0。最佳温度为45℃。这种酶既能水解可溶性底物，也能水解结晶底物。

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引用次数: 0

Design of bispecific antibody Fc region employing a shark-human chimeric and asymmetric format 采用鲨鱼-人嵌合和不对称格式设计双特异性抗体Fc区。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-10-01 DOI: 10.1016/j.jbiosc.2025.09.001

Ryota Munetomo , Aiko Inoue , Masayoshi Onitsuka

Bispecific antibodies (BsAbs) can bind to two antigens simultaneously and have undergone rapid advancements in recent years owing to their ability to enable novel mechanisms of action that are unachievable using conventional monoclonal antibodies (mAbs). However, the structural complexity of BsAbs remains a problem during product development. One of these problems is the presence of impurities and by-products. Although BsAbs with the human Fc region must be assembled using heterogeneous polypeptide chains, undesired by-products from unpaired and mispaired chain components can contaminate them. These by-products are difficult to remove in the purification process because their physicochemical properties resemble those of the target BsAb with correct pairing. Here, we designed a novel Fc region for enhanced BsAbs in which the human CH2 domain on one side of the Fc region was replaced with the C2 domain from an immunoglobulin new antigen receptor (IgNAR) shark antibody. The designed BsAbs with chimeric and asymmetric Fcs exhibited separate pH elution profiles against soluble aggregates in protein A affinity chromatography. An overlapping elution profile corresponding to the by-product homogeneous chain observed in human Fc BsAbs was not detected in shark C2-introduced BsAbs. Although another homogeneous by-product was observed in the designed BsAb, introducing N-glycosylation at C2 significantly improved this problem. Additionally, BsAbs with the designed Fc demonstrated higher stability in both the colloidal and structural aspects. This study is the first approach for the chimeric and asymmetric design of Fc using a shark-derived constant domain and offers a novel alternative for BsAb development.

双特异性抗体（BsAbs）可以同时结合两种抗原，近年来取得了快速进展，因为它们能够实现传统单克隆抗体（mab）无法实现的新作用机制。然而，在产品开发过程中，bsab结构的复杂性仍然是一个问题。其中一个问题是杂质和副产品的存在。虽然具有人类Fc区的bsab必须使用异质多肽链进行组装，但来自未配对和错配链成分的不希望的副产物会污染它们。这些副产物在纯化过程中很难去除，因为它们的物理化学性质与正确配对的目标BsAb相似。在这里，我们设计了一个新的Fc区域用于增强bsab，其中Fc区域一侧的人CH2结构域被免疫球蛋白新抗原受体（IgNAR）鲨鱼抗体的C2结构域取代。在蛋白A亲和层析中，嵌合Fcs和不对称Fcs的bsab对可溶性聚集体表现出不同的pH洗脱谱。与在人类Fc bsab中观察到的副产物均相链相对应的重叠洗脱谱在鲨鱼c2引入的bsab中未检测到。虽然在设计的BsAb中观察到另一个均匀的副产物，但在C2处引入n -糖基化显着改善了这个问题。此外，具有设计Fc的bsab在胶体和结构方面都表现出更高的稳定性。该研究首次采用鲨鱼衍生的恒定结构域进行Fc嵌合和非对称设计，为BsAb的开发提供了一种新的选择。

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引用次数: 0

Development of a method for quantifying metabolites in Escherichia coli colonies using hyperspectral imaging 利用高光谱成像技术定量大肠杆菌菌落代谢物方法的建立。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-10-01 DOI: 10.1016/j.jbiosc.2025.09.005

Manami Takama , Takatoshi Suematsu , Takayuki Okano , Shumpei Asamizu , Takahiro Bamba , Tomohisa Hasunuma

Fermentation by microorganisms has attracted attention for the synthesis of bulk and fine chemicals with high added value, including pharmaceutical intermediates. To accelerate the development of high-producing microbial strains, a rapid screening method is warranted. This study aimed to develop a novel, nondestructive approach to quantify metabolite production in microbial colonies using hyperspectral imaging (HSI). As a model, we examined the heterologous production of 1,3,5-trihydroxyanthraquinone (AQ256), an anthraquinone with antimicrobial and anticancer activities, using Escherichia coli. Fluorescence spectral data from HSI, along with AQ256 concentrations measured via high-performance liquid chromatography, were used to construct regression models. In addition, red-green-blue (RGB)-based models were developed, as AQ256 exhibits a characteristic reddish-brown color. Four regression models were compared: multiple linear regression, partial least squares regression (PLSR), support vector regression, and random forest regression. Among them, the PLSR model based on HSI data showed the highest prediction accuracy (R² = 0.75 ± 0.23, root mean square error = 0.08 ± 0.02, mean absolute error = 0.07 ± 0.02). In particular, it outperformed the RGB-based model in extrapolation beyond the training data. These findings demonstrate that the HSI-based method enables accurate, nondestructive quantification of metabolites and has strong potential for high-throughput screening of microbial strains that produce various valuable compounds at elevated yields.

微生物发酵已成为合成高附加值原料药和精细化学品（包括医药中间体）的研究热点。为了加速高产微生物菌株的开发，需要一种快速筛选方法。本研究旨在开发一种新的、无损的方法，利用高光谱成像（HSI）来量化微生物菌落中代谢物的产生。作为模型，我们研究了利用大肠杆菌外源生产具有抗菌和抗癌活性的蒽醌类化合物1,3,5-三羟基蒽醌（AQ256）。HSI的荧光光谱数据与高效液相色谱测定的AQ256浓度一起构建回归模型。此外，基于红-绿-蓝（RGB）的模型被开发出来，因为AQ256表现出红棕色的特征。比较了多元线性回归、偏最小二乘回归、支持向量回归和随机森林回归四种回归模型。其中，基于HSI数据的PLSR模型预测精度最高（R2 = 0.75±0.23，均方根误差= 0.08±0.02，平均绝对误差= 0.07±0.02）。特别是，它在训练数据之外的外推方面优于基于rgb的模型。这些发现表明，基于si的方法能够准确、无损地定量代谢物，并具有高通量筛选微生物菌株的强大潜力，这些微生物菌株能以高产量产生各种有价值的化合物。

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引用次数: 0

Importance rapid initial decline in oxidation–reduction potential, followed by an increase in extracellular electron transport activities, for the rapid onset of indigo reduction 重要的是氧化还原电位的快速初始下降，随后是细胞外电子传递活动的增加，对靛蓝还原的快速开始。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-09-29 DOI: 10.1016/j.jbiosc.2025.08.008

Nowshin Farjana , Hiromitsu Furukawa , Kensuke Igarashi , Souichiro Kato , Isao Yumoto

In most complex microbial systems, the ideal process underlying transitional microbial changes that lead to the formation of functional states is not fully elucidated. To understand the basis for the occurrence of indigo reduction, we analyzed the prerequisites causing transitional shifts in microflora that lead to the indigo-reducing state. To this end, timing of wheat bran (WB) addition, during indigo fermentation process using sukumo (composted leaves of Polygonum tinctorium L.) as the inoculum, substrate, and indigo source, were varied. Early initiation of indigo reduction was achieved through the early proliferation of obligate anaerobic Alkalicella caledoniensis followed by Alkalibacterium spp. or Evansella vedderi. Although it can be predicted that Alkalicella caledoniensis exhibits extracellular electron transport (EET) activity, to promote even effective reduction of indigo, Alkalibacterium spp. or E. vedderi, which have the EET gene sequence series and exert strong metabolic abilities, should emerge using WB. The emergence of Alkalicella caledoniensis was associated with drastic a decrease in bacterial diversity and a concurrent rapid decline in oxidation–reduction potential (ORP). The rate and extent of Alkalicella caledoniensis appearance depended on the rate of ORP reduction. Multivariate analysis (i.e., RDA) revealed that Alkalicella caledoniensis directed the initial drastic changes of microbiota, aligning with the decline in ORP. Prior to these major microbial shifts oxygen consumption by aerobic bacteria utilizing sukumo initiated the ORP decrease. These findings contribute to understanding the approach to steer the initially highly diverse bacterial community during early fermentation toward rapid induction of indigo reduction.

在大多数复杂的微生物系统中，导致功能状态形成的过渡性微生物变化的理想过程尚未完全阐明。为了了解靛蓝还原发生的基础，我们分析了导致靛蓝还原状态的微生物区系过渡变化的先决条件。为此，在以黄蓼（Polygonum tinctorium L.）堆肥叶为接种物、底物和靛蓝源的靛蓝发酵过程中，小麦麸皮（WB）的添加时间不同。靛蓝还原的早期开始是通过专性厌氧caledoniensis的早期增殖实现的，其次是Alkalibacterium sp .或Evansella vedderi。虽然可以预测caledoniensis具有胞外电子传递（extracellular electron transport， EET）活性，但为了促进靛蓝的有效还原，还需要利用WB来研究具有EET基因序列序列且具有较强代谢能力的Alkalibacterium spp.或e.w edderi。caledoniensis的出现伴随着细菌多样性的急剧减少和氧化还原电位（ORP）的快速下降。喀里多碱菌出现的速度和程度取决于ORP还原的速度。多变量分析（即RDA）表明，caledoniensis引导了微生物群最初的剧烈变化，与ORP的下降一致。在这些主要的微生物转变之前，好氧细菌利用sukumo的耗氧量引发了ORP的降低。这些发现有助于理解在早期发酵过程中引导最初高度多样化的细菌群落快速诱导靛蓝还原的方法。

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引用次数: 0

Enrichment of soy protein-derived peptides that decrease pancreatic lipase activity using heat-treated porous silica gel and their relationship with bile acid binding activity 利用热处理多孔硅胶富集降低胰脂肪酶活性的大豆蛋白衍生肽及其与胆汁酸结合活性的关系。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-09-29 DOI: 10.1016/j.jbiosc.2025.09.002

Yusuke Ishii, Yuta Matsunaga, Hirokazu Akiyama, Kazunori Shimizu, Hiroyuki Honda

Excessive lipid absorption is a key factor in obesity. Lipids are solubilized in the gut via bile acid (BA) micelles, where pancreatic lipase hydrolyzes them for absorption. This study aimed to enrich pancreatic lipase inhibitory (PLI) peptides from food protein hydrolysates and clarify their inhibition mechanisms. We used heat-treated porous silica gel (HTSG) to selectively enrich basic and hydrophobic peptides through adsorption–desorption. While HTSG has previously enriched PLI peptides, the mechanism remained unclear. Since basic and hydrophobic peptides can bind strongly to BAs like taurocholic acid, we explored their BA-binding and PLI activities. Pepsin hydrolysates from casein, soybean, pea, and rice endosperm were tested with 1 mM sodium taurocholate (TCA). TCA increased lipase activity over 2.5-fold. Soybean pepsin hydrolysate (SPH) showed notable PLI activity, further enhanced approxiamtely 3-fold after HTSG treatment (SPH (after)). LC–MS/MS of SPH (after) identified 1461 peptides. Among 38 high-abundance peptides (Z ≥ 2) chemically synthesized, 9 inhibited pancreatic lipase in the presence of TCA. BA-binding activity was assessed via micelle disruption. Seven of the nine peptides disrupted over 50 % of micelles. Docking simulation was conducted and peptides that exhibited PLI activity even without TCA and showed TCA-binding activity were predicted to bind directly to pancreatic lipase. In summary, we identified 9 PLI peptides from SPH, most of which inhibit pancreatic lipase by binding to BAs. HTSG-based enrichment offers a promising strategy to obtain bioactive peptides that may serve as functional ingredients for obesity prevention.

脂质吸收过多是肥胖的一个关键因素。脂质通过胆汁酸（BA）胶束在肠道中溶解，胰脂肪酶将其水解以供吸收。本研究旨在从食物蛋白水解物中富集胰脂肪酶抑制肽，并阐明其抑制机制。我们使用热处理多孔硅胶（HTSG）通过吸附-解吸选择性富集碱性肽和疏水性肽。虽然HTSG先前富集了PLI肽，但其机制尚不清楚。由于碱性肽和疏水性肽可以与牛磺酸等ba强结合，我们研究了它们的ba结合和PLI活性。用1mm牛磺胆酸钠（TCA）检测酪蛋白、大豆、豌豆和水稻胚乳的胃蛋白酶水解物。TCA使脂肪酶活性增加了2.5倍以上。大豆胃蛋白酶水解物（SPH）表现出显著的PLI活性，经HTSG处理后，SPH的PLI活性进一步提高了约3倍。SPH的LC-MS/MS鉴定出1461个多肽。在化学合成的38个高丰度肽（Z≥2）中，9个在TCA存在下抑制胰腺脂肪酶。通过胶束破坏来评估ba的结合活性。9个肽中的7个破坏了超过50%的胶束。对接模拟表明，即使没有TCA也具有PLI活性并显示TCA结合活性的肽可以直接与胰脂肪酶结合。总之，我们从SPH中鉴定出9个PLI肽，其中大多数通过与BAs结合来抑制胰脂肪酶。基于htsg的富集为获得生物活性肽提供了一种有前途的策略，这些活性肽可能作为预防肥胖的功能成分。

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引用次数: 0

Whole-genome draft assemblies of Paracoccus pantotrophus DSM 11073 and Paracoccus sp. AS002: Phylogenetics entails classification as Paracoccus versutus AS002 泛养副球菌DSM 11073和副球菌sp. AS002的全基因组草图组装：系统发育需要分类为反副球菌AS002。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-09-27 DOI: 10.1016/j.jbiosc.2025.09.003

Upasana Pal , Denise Bachmann , Linda Fenske , Lars Mathias Blank , Till Tiso

The Gram-stain-negative bacterium Paracoccus spp., from the class Alphaproteobacteria and family Rhodobacteraceae, exhibits exceptional metabolic flexibility, diverse substrate utilization, and tolerance to abiotic stressors. To broaden the biotechnological applications of the genus, comprehensive sequencing, phylogenetic, and physiological characterization were performed between two strains of the genus, Paracoccus pantotrophus DSM 11073 and Paracoccus sp. AS002. Illumina sequencing yielded a total genome size of 4.2 Mbp for P. pantotrophus DSM 11073 and 4.8 Mbp for Paracoccus sp. AS002. Through phylogenetic analysis using the EDGAR software, Paracoccus sp. AS002 shared with Paracoccus versutus DSM 582 the same clade in the phylogenetic tree and an ANI score of 98.9 % indicating that Paracoccus sp. AS002 could be reclassified as P. versutus AS002. The study was extended to compare various attributes of the sequenced genomes and highlight the metabolic versatility of the genus Paracoccus. The use of a wide substrate panel demonstrated the metabolic versatility of the strains, including the PET monomer ethylene glycol, the C1 carbon source formic acid, and renewable carbon sources such as ethanol. Additionally, the ability of the strains to produce bioplastic was assessed, with P. pantotrophus DSM 11073 producing 36 % and P. versutus AS002 28 % polyhydroxybutyrate (% cell dry weight) on glucose, and 40 % and 16 % on 60 mM ethylene glycol, respectively. This study demonstrates the value of sequencing bacterial strains for biotechnological applications and highlights EDGAR's effectiveness in phylogenetic analysis, paving the way for using Paracoccus as a microbial chassis in sustainable biotechnological processes supporting the circular bioeconomy.

革兰氏染色阴性的副球菌属，属于α变形菌纲和红杆菌科，表现出特殊的代谢灵活性，多种底物利用和对非生物应激源的耐受性。为了扩大该属的生物技术应用，对两株pantotrophus Paracoccus DSM 11073和Paracoccus sp. AS002进行了全面的测序、系统发育和生理特性研究。Illumina测序结果显示，P. pantotrophus DSM 11073的总基因组大小为4.2 Mbp，副球菌sp. AS002的总基因组大小为4.8 Mbp。通过EDGAR软件进行系统发育分析，发现副球菌sp. AS002与反苏副球菌DSM 582在系统发育树上具有相同的进化支，ANI评分为98.9%，表明副球菌sp. AS002可以重新归类为反苏副球菌AS002。该研究扩展到比较测序基因组的各种属性，并突出副球菌属的代谢多样性。广泛底物面板的使用证明了菌株代谢的多功能性，包括PET单体乙二醇、C1碳源甲酸和可再生碳源如乙醇。此外，对菌株产生生物塑料的能力进行了评估，P. pantotrophus DSM 11073在葡萄糖上产生36%的聚羟基丁酸盐，P. versutus AS002在60 mM乙二醇上产生28%的聚羟基丁酸盐（细胞干重的%），分别为40%和16%。该研究证明了细菌菌株测序在生物技术应用中的价值，并强调了EDGAR在系统发育分析中的有效性，为利用副球菌作为支持循环生物经济的可持续生物技术过程中的微生物基础铺平了道路。

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引用次数: 0

Identification and engineering of a cellobiose transporter KmStl1p to enhance cellobiose utilization in Kluyveromyces marxianus and Saccharomyces cerevisiae 纤维素二糖转运蛋白KmStl1p的鉴定与工程设计以提高马氏克卢维菌和酿酒酵母对纤维素二糖的利用。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-09-20 DOI: 10.1016/j.jbiosc.2025.08.009

Satoshi Ebe , Takuya Abe , Shogo Motozono , Tomoya Kagawa , Riko Kobayashi , Yuki Terauchi , Rinji Akada , Hisashi Hoshida

The yeast Kluyveromyces marxianus, a yeast known for its ability to ferment ethanol at high temperatures, can utilize various sugars including cellobiose, lactose and xylose. This study focused on improving cellobiose utilization by identifying and engineering a cellobiose transporter in K. marxianus. To assess cellobiose utilization capabilities, K. marxianus strains were grown in a cellobiose medium. The strains showed various growth levels, for example, the NCYC2791 strain grew well, while the DMKU3-1042 strain did not. This difference provided a basis for identifying a cellobiose transporter. Thirteen transporter candidate genes from the NCYC2791 genome were expressed in DMKU3-1042. As a result, KmSTL1 overexpression enhanced cell growth in a cellobiose medium. In addition, its disruption in NCYC2791 caused growth defects. To confirm its function, KmSTL1 was co-expressed with a β-glucosidase gene in Saccharomyces cerevisiae EBY.VW1000, which only uptake maltose. This engineered strain grew in cellobiose medium, indicating that KmSTL1 encodes a cellobiose transporter. Expression of GFP-fused KmStl1p in K. marxianus revealed that KmStl1p localized on cell membrane under cellobiose conditions, but was degraded in glucose conditions, suggesting that the transporter is regulated by available sugars. By individually disrupting seven α-arrestin genes in K. marxianus, KmRog3p was identified as a major ubiquitination mediator for KmStl1p degradation. Deletion analysis of KmStl1p revealed that its C-terminus is crucial for recognition by KmRog3p. Furthermore, expressing KmStl1p C-terminus mutants enhanced cellobiose assimilation in both K. marxianus and S. cerevisiae. These findings demonstrate that engineering KmStl1p is an effective strategy to improve cellobiose utilization in yeasts.

马氏克鲁维酵母是一种在高温下发酵乙醇的酵母，它可以利用多种糖，包括纤维素二糖、乳糖和木糖。本研究旨在通过对马氏酵母中纤维素二糖转运体的鉴定和工程改造，提高其对纤维素二糖的利用。为了评估利用纤维素二糖的能力，我们在纤维素二糖培养基中培养了马氏克雷伯氏菌菌株。菌株表现出不同的生长水平，如NCYC2791菌株生长良好，而DMKU3-1042菌株生长不佳。这种差异为鉴定纤维素二糖转运体提供了基础。来自NCYC2791基因组的13个转运体候选基因在DMKU3-1042中表达。结果，KmSTL1过表达增强了细胞在纤维素糖培养基中的生长。此外，它在NCYC2791中的破坏导致生长缺陷。为了证实其功能，KmSTL1在酿酒酵母EBY中与β-葡萄糖苷酶基因共表达。VW1000，只摄取麦芽糖。该工程菌株在纤维二糖培养基中生长，表明KmSTL1编码一种纤维二糖转运蛋白。gfp融合的KmStl1p在K. marxianus中的表达表明，KmStl1p在纤维素二糖条件下定位于细胞膜上，但在葡萄糖条件下被降解，这表明该转运体受有效糖的调节。通过单独破坏马氏K. marxianus的7个α-抑制蛋白基因，KmRog3p被鉴定为KmStl1p降解的主要泛素化介质。KmStl1p的缺失分析表明，其c端对于KmRog3p的识别至关重要。此外，表达KmStl1p c端突变体增强了马氏酵母和酿酒酵母的纤维素糖同化。这些发现表明，对KmStl1p进行工程改造是提高酵母对纤维素糖利用的有效策略。

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引用次数: 0

Optimization of heterologous protein production process using genetically engineered Ogataea minuta toward industrial-scale manufacturing 面向工业规模生产的异源蛋白生产工艺的基因工程优化。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-09-08 DOI: 10.1016/j.jbiosc.2025.08.006

Masashi Tsuda , Yuki Nakatani , Satoshi Baba , Koichi Nonaka , Takehiko Yoko-o , Yasunori Chiba

We have developed the methylotrophic yeast Ogataea minuta as a useful host for producing heterologous proteins. In this study, a double mutant that lacks the Prb1 protease and alcohol oxidase was generated and applied for heterologous protein production. Upon our optimization of the fermentation conditions, such as feeding of carbon and nitrogen sources and pH control, this mutant showed increased production of human serum albumin, resulting in a yield of approximately 7.5 g/L at 21 days in the production phase. The established optimal fermentation condition with the double mutant was successfully applied to manufacture a candidate biologic protein: lipocalin derivative which is designed to attach to the calcitonin gene-related peptide, on an industrial scale. The candidate biologic protein was successfully manufactured at an industrial scale in a 4500 L bioreactor under well-controlled conditions. This first successful case highlights the potential of our O. minuta-based production system, and this study is helpful for a large-scale cultivation of a methylotrophic yeast for protein production.

我们已经开发了甲基营养酵母Ogataea minuta作为产生异源蛋白的有用宿主。在这项研究中，产生了一个缺乏Prb1蛋白酶和酒精氧化酶的双突变体，并将其用于异源蛋白的生产。通过优化发酵条件，如添加碳源和氮源以及控制pH值，该突变体的人血清白蛋白产量增加，在生产阶段的第21天产量约为7.5 g/L。利用双突变体所建立的最佳发酵条件，成功地在工业规模上生产了一种候选生物蛋白：脂钙素衍生物，该衍生物被设计为附着在降钙素基因相关肽上。在控制良好的条件下，在4500l生物反应器中成功地实现了候选生物蛋白的工业化生产。这第一个成功的案例突出了我们基于O. minuta的生产系统的潜力，该研究有助于大规模培养用于蛋白质生产的甲基营养酵母。

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引用次数: 0

Sterilizable autoantigen immobilized column platform for broad-spectrum removal of pathogenic autoantibodies in autoimmune diseases 可灭菌的自身抗原固定化柱平台，用于自身免疫性疾病中病原性自身抗体的广谱去除。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-09-04 DOI: 10.1016/j.jbiosc.2025.08.007

Midori Futami , Eri Kurozumi , Masaya Kamo , Soudai Taguchi , Tomoaki Nakai , Junichiro Futami

Blood purification using immunoadsorbent columns is a therapeutic strategy for removing pathogenic autoantibodies in autoimmune diseases. Currently available columns have limitations: Trp/Phe columns offer cost-effectiveness and sterilizability, but lack antigen specificity and have limited capacity to remove diverse pathogenic autoantibodies; whereas Protein A/peptide/anti-human IgG columns target all antibodies, regardless of pathogenicity, limiting specificity, and often require sterile production due to low stability under sterilization conditions, except for peptide ligands. Full-length autoantigen-immobilized immunoadsorbent columns have great potential to specifically adsorb targeted autoantibodies, because autoantibodies recognize diverse epitopes that vary among individuals. However, it is challenging to prepare biologically active autoantigens on a large scale and maintain the quality of antigen-immobilized columns after sterilization. This study introduced a novel approach for preparing sterilizable antigen-immobilized columns that target autoantibodies, excluding those with conformational epitope specificity. Two type I transmembrane protein-coding extracellular domains associated with autoimmunity and their rabbit antisera were used as models. Recombinant human contactin-associated protein-like 2 (Caspr2) and muscle-specific tyrosine-protein kinase receptor (MuSK) were expressed as bacterial inclusion bodies. These compounds were solubilized and purified using Cys-specific chemical cationization. Columns immobilized with water-soluble S-cationized Caspr2 or MuSK effectively captured specific antibodies from rabbit antisera against each antigen, retaining their capacity after standard sterilization. This approach offers a promising solution for developing immunoadsorbent columns with enhanced specificity and sterilizability and is applicable to various autoantibody-related disorders.

利用免疫吸附柱净化血液是一种清除自身免疫性疾病病原性自身抗体的治疗策略。目前可用的色谱柱有局限性：色氨酸/苯丙氨酸色谱柱具有成本效益和灭菌性，但缺乏抗原特异性，并且去除多种致病性自身抗体的能力有限；而蛋白A/肽/抗人IgG柱可靶向所有抗体，无论其致病性、限制性特异性如何，除肽配体外，由于在灭菌条件下稳定性较低，通常需要无菌生产。全长自身抗原固定免疫吸附柱具有很大的潜力来特异性吸附靶向自身抗体，因为自身抗体识别不同个体的不同表位。然而，大规模制备具有生物活性的自身抗原，并在灭菌后保持抗原固定化柱的质量是一个挑战。本研究介绍了一种制备可灭菌抗原固定化柱的新方法，该柱针对自身抗体，不包括那些具有构象表位特异性的抗体。以两个与自身免疫相关的I型跨膜蛋白编码胞外结构域及其兔抗血清为模型。重组人接触蛋白样2 （Caspr2）和肌肉特异性酪氨酸蛋白激酶受体（MuSK）以细菌包涵体的形式表达。这些化合物被溶解和纯化使用cys特异性化学阳离子化。用水溶性s阳离子Caspr2或MuSK固定的色谱柱有效捕获兔抗血清中针对每种抗原的特异性抗体，在标准灭菌后保持其能力。该方法为开发具有增强特异性和灭菌性的免疫吸附柱提供了一种有前途的解决方案，适用于各种自身抗体相关疾病。

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引用次数: 0

Rapid and practical microbial detection method for brewery hygiene management using the EZ-Fluo system EZ-Fluo系统用于啤酒厂卫生管理的快速实用微生物检测方法。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-09-03 DOI: 10.1016/j.jbiosc.2025.08.002

Maika Kitazawa , Nobuchika Takesue , Kazumaru Iijima , Koji Suzuki , Masaki Shimokawa

The early detection of microorganisms in the brewery environment is crucial for taking quick corrective actions and minimizing the risk of microbial contamination. However, hygiene tests using traditional culture methods for visual detection have poor turnaround times. In this study, we optimized the test conditions for the EZ-Fluo Rapid Detection System for brewery hygiene management. In this system, microorganisms are trapped on a membrane filter, cultured on agar, and detected after brief incubation using fluorescence-based technology with a dedicated detector. Among several alternatives, the EZ-Fluo system was chosen for its cost-effectiveness. We determined the appropriate test conditions, including the incubation temperature, fluorescent reagent, and temperature for the fluorescence reaction, using two slow growing environmental indicator species, Acetobacter pasteurianus and Pseudomonas fluorescens. Using the EZ-Fluo system under our optimized detection conditions (hereafter referred to as the EZF-ODC method), nine indicator species common to breweries were detected at a recovery rate of 54.0–117.2 % after only 30 h of incubation. No significant difference was found in colony-forming units among the analysts (p > 0.05). In brewery hygiene tests, the EZF-ODC method (30-h incubation) and the traditional culture method (72-h incubation) yielded comparable detection profiles, with positive or negative results matching in 94.6 % of the 222 samples. The EZF-ODC method can provide results by the following day, representing a significant improvement over the 3 days needed for traditional methods and enabling the rapid implementation of hygiene control measures to maintain a clean brewery environment and possibly expedite production planning.

啤酒厂环境中微生物的早期检测对于采取快速纠正措施和最大限度地减少微生物污染的风险至关重要。然而，使用传统培养方法进行视觉检测的卫生检测周转时间较短。在本研究中，我们优化了用于啤酒厂卫生管理的EZ-Fluo快速检测系统的测试条件。在该系统中，微生物被捕获在膜过滤器上，在琼脂上培养，并在使用专用检测器的荧光技术进行短暂孵育后进行检测。在几个备选方案中，EZ-Fluo系统因其成本效益而被选中。以巴氏醋酸杆菌和荧光假单胞菌两种生长缓慢的环境指示菌为实验对象，确定适宜的实验条件，包括培养温度、荧光试剂和荧光反应温度。在优化的检测条件下（以下简称EZF-ODC法），采用EZ-Fluo系统，仅需30 h，即可检测出9种啤酒常用指示剂，回收率为54.0 ~ 117.2%。分析者之间的菌落形成单位无显著差异（p < 0.05）。在啤酒厂卫生检测中，EZF-ODC法（孵育30小时）和传统培养法（孵育72小时）的检测结果相当，222份样品中有94.6%的阳性或阴性结果匹配。EZF-ODC方法可以在第二天提供结果，这比传统方法需要3天的时间有了显著的改进，并且可以快速实施卫生控制措施，以保持清洁的啤酒厂环境，并可能加快生产计划。

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引用次数: 0