Pub Date : 2024-05-22DOI: 10.1016/j.jbiosc.2024.04.005
Only a few reports available about the assimilation of hydrophobic or oil-based feedstock as carbon sources by Lipomyces starkeyi. In this study, the ability of L. starkeyi to efficiently utilize free fatty acids (FFAs) and real biomass like palm acid oil (PAO) as well as crude palm kernel oil (CPKO) for growth and lipid production was investigated. PAO, CPKO, and FFAs were evaluated as sole carbon sources or in the mixed medium containing glucose. L. starkeyi was able to grow on the medium supplemented with PAO and FFAs, which contained long-chain length FAs and accumulated lipids up to 35% (w/w) of its dry cell weight. The highest lipid content and lipid concentration were achieved at 50% (w/w) and 10.1 g/L, respectively, when L. starkeyi was cultured in nitrogen-limited mineral medium (-NMM) supplemented with PAO emulsion. Hydrophobic substrate like PAO could be served as promising carbon source for L. starkeyi.
{"title":"Production of single cell oil by Lipomyces starkeyi from waste plant oil generated by the palm oil mill industry","authors":"","doi":"10.1016/j.jbiosc.2024.04.005","DOIUrl":"10.1016/j.jbiosc.2024.04.005","url":null,"abstract":"<div><p>Only a few reports available about the assimilation of hydrophobic or oil-based feedstock as carbon sources by <em>Lipomyces starkeyi</em>. In this study, the ability of <em>L. starkeyi</em> to efficiently utilize free fatty acids (FFAs) and real biomass like palm acid oil (PAO) as well as crude palm kernel oil (CPKO) for growth and lipid production was investigated. PAO, CPKO, and FFAs were evaluated as sole carbon sources or in the mixed medium containing glucose. <em>L. starkeyi</em> was able to grow on the medium supplemented with PAO and FFAs, which contained long-chain length FAs and accumulated lipids up to 35% (w/w) of its dry cell weight. The highest lipid content and lipid concentration were achieved at 50% (w/w) and 10.1 g/L, respectively, when <em>L. starkeyi</em> was cultured in nitrogen-limited mineral medium (-NMM) supplemented with PAO emulsion. Hydrophobic substrate like PAO could be served as promising carbon source for <em>L. starkeyi</em>.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141079525","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}
Pub Date : 2024-05-18DOI: 10.1016/j.jbiosc.2024.04.006
The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms in vitro. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma in vitro have been conducted. However, in vitro reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the in vitro expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired in vitro expression profiles of these genes. Additionally, we demonstrated that the in vitro expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the in vitro expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.
{"title":"Optimizing in vitro expression balance of central dogma-related genes using parallel reaction monitoring","authors":"","doi":"10.1016/j.jbiosc.2024.04.006","DOIUrl":"10.1016/j.jbiosc.2024.04.006","url":null,"abstract":"<div><p>The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms <em>in vitro</em>. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma <em>in vitro</em> have been conducted. However, <em>in vitro</em> reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the <em>in vitro</em> expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired <em>in vitro</em> expression profiles of these genes. Additionally, we demonstrated that the <em>in vitro</em> expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the <em>in vitro</em> expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957620","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}
Pub Date : 2024-05-07DOI: 10.1016/j.jbiosc.2024.04.003
Kevin Maafu Juma , Yuto Murakami , Kenta Morimoto , Teisuke Takita , Kenji Kojima , Koichiro Suzuki , Itaru Yanagihara , Soichiro Ikuta , Shinsuke Fujiwara , Kiyoshi Yasukawa
Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.
重组酶聚合酶扩增(RPA)是利用重组酶(Rec)、单链 DNA 结合蛋白(SSB)、链置换 DNA 聚合酶(Pol)和 ATP 再生酶在 41 ℃ 左右进行的 DNA 等温扩增反应。考虑到 RPA 试剂的现场使用,人们非常需要具有长期储存稳定性的冻干 RPA 试剂。在本研究中,作为解决这一问题的方法之一,我们尝试使用一种恒温丙酮酸激酶(PK)。PK 基因是从嗜热菌 Thermotoga maritima(Tma-PK)中分离出来的。Tma-PK 在大肠杆菌中表达并从细胞中纯化。与人类 PK 相比,Tma-PK 具有更高的热稳定性。纯化的 Tma-PK 制剂作为 ATP 再生酶应用于 RPA。含有 Tma-PK 的液体 RPA 试剂与含有人类 PK 的试剂具有相同的性能。含有 Tma-PK 的冻干 RPA 试剂的性能高于人 PK。结合我们以前对来自嗜热细菌苍白球杆菌的恒温 Pol 的 RPA 试剂的研究结果,本研究结果表明,作为冻干 RPA 试剂的成分,恒温酶比中嗜热酶更可取。
{"title":"Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima","authors":"Kevin Maafu Juma , Yuto Murakami , Kenta Morimoto , Teisuke Takita , Kenji Kojima , Koichiro Suzuki , Itaru Yanagihara , Soichiro Ikuta , Shinsuke Fujiwara , Kiyoshi Yasukawa","doi":"10.1016/j.jbiosc.2024.04.003","DOIUrl":"10.1016/j.jbiosc.2024.04.003","url":null,"abstract":"<div><p>Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium <em>Thermotoga maritima</em> (<em>Tma</em>-PK). <em>Tma</em>-PK was expressed in <em>Escherichia coli</em> and purified from the cells. <em>Tma</em>-PK exhibited higher thermostability than human PK. The purified <em>Tma</em>-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with <em>Tma</em>-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with <em>Tma</em>-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, <em>Aeribacillus pallidus</em>, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891630","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}
Pub Date : 2024-04-23DOI: 10.1016/j.jbiosc.2024.03.007
Koichi Tamano , Haruka Takayama
Genome co-editing technology is effective in breeding filamentous fungi for applications in the fermentation industry, achieving site-directed mutagenesis, the status of non-genetically modified organisms (non-GMOs), and wild-type-like growth phenotype. Prior to this study, thiI gene was found as a selectable marker for such genome co-editing in the filamentous fungus Aspergillus oryzae, while it cannot be reused via marker recycling. Therefore, we aimed to identify another marker gene to knock out another target gene via genome co-editing in A. oryzae. In this study, we focused on the membrane transporter gene nrtA (AO090012000623), which promotes uptake of nitrate (NO3-). It is known that, in nrtA knockout strain, chlorate (ClO3-), an analog of nitrate with antifungal activity, cannot be imported into the cytosol, which enables the mutant to grow in the presence of chlorate. Based on this information, knockout of the target gene wA was attempted using both nrtA- and wA-specific single-guide RNAs via genome co-editing with KClO3 supplementation in A. oryzae laboratory strain RIB40 and industrial strain KBN616. Resultantly, wA knockout mutant was generated, and nrtA was identified as a selectable marker. Moreover, this genome co-editing system using nrtA was compatible with that using thiI, and thus, a double knockout mutant of two target genes wA and yA was constructed in RIB40 while maintaining non-GMO status and wild-type-like growth. As nrtA homologs have been found in several industrial Aspergillus species, genome co-editing using homolog genes as selectable markers is plausible, which would contribute to the widespread breeding of industrial strains of Aspergilli.
基因组共编辑技术可有效培育丝状真菌,应用于发酵行业,实现定点诱变、非转基因生物(non-GMOs)地位和类野生型生长表型。在本研究之前,人们发现 thiI 基因是丝状真菌黑曲霉(Aspergillus oryzae)中这种基因组联合编辑的可选择标记,但它不能通过标记循环再利用。因此,我们希望找到另一个标记基因,通过基因组共编辑技术敲除黑曲霉的另一个目标基因。在这项研究中,我们重点研究了膜转运体基因 nrtA(AO090012000623),它能促进硝酸盐(NO3-)的吸收。众所周知,在 nrtA 基因敲除菌株中,具有抗真菌活性的硝酸盐类似物氯酸盐(ClO3-)无法进入细胞质,这使得突变体能够在氯酸盐存在的情况下生长。根据这一信息,我们尝试使用 nrtA 和 wA 特异性单导 RNA,通过基因组联合编辑和 KClO3 补充,在 A. oryzae 实验室菌株 RIB40 和工业菌株 KBN616 中敲除目标基因 wA。结果产生了 wA 基因敲除突变体,并确定 nrtA 为可选择标记。此外,使用 nrtA 的基因组联合编辑系统与使用 thiI 的基因组联合编辑系统兼容,因此在 RIB40 中构建了 wA 和 yA 两个目标基因的双基因敲除突变体,同时保持了非转基因状态和野生型生长。由于在多个工业曲霉菌种中发现了 nrtA 同源物,利用同源基因作为可选择标记进行基因组共编辑是可行的,这将有助于工业曲霉菌种的广泛培育。
{"title":"Double knockout of two target genes via genome co-editing using a nitrate transporter gene nrtA and a putative thiamine transporter gene thiI as selectable markers in Aspergillus oryzae","authors":"Koichi Tamano , Haruka Takayama","doi":"10.1016/j.jbiosc.2024.03.007","DOIUrl":"10.1016/j.jbiosc.2024.03.007","url":null,"abstract":"<div><p>Genome co-editing technology is effective in breeding filamentous fungi for applications in the fermentation industry, achieving site-directed mutagenesis, the status of non-genetically modified organisms (non-GMOs), and wild-type-like growth phenotype. Prior to this study, <em>thiI</em> gene was found as a selectable marker for such genome co-editing in the filamentous fungus <em>Aspergillus oryzae</em>, while it cannot be reused via marker recycling. Therefore, we aimed to identify another marker gene to knock out another target gene via genome co-editing in <em>A. oryzae</em>. In this study, we focused on the membrane transporter gene <em>nrtA</em> (AO090012000623), which promotes uptake of nitrate (NO<sub>3</sub><sup>-</sup>). It is known that, in <em>nrtA</em> knockout strain, chlorate (ClO<sub>3</sub><sup>-</sup>), an analog of nitrate with antifungal activity, cannot be imported into the cytosol, which enables the mutant to grow in the presence of chlorate. Based on this information, knockout of the target gene <em>wA</em> was attempted using both <em>nrtA</em>- and <em>wA</em>-specific single-guide RNAs via genome co-editing with KClO<sub>3</sub> supplementation in <em>A. oryzae</em> laboratory strain RIB40 and industrial strain KBN616. Resultantly, <em>wA</em> knockout mutant was generated, and <em>nrtA</em> was identified as a selectable marker. Moreover, this genome co-editing system using <em>nrtA</em> was compatible with that using <em>thiI</em>, and thus, a double knockout mutant of two target genes <em>wA</em> and <em>yA</em> was constructed in RIB40 while maintaining non-GMO status and wild-type-like growth. As <em>nrtA</em> homologs have been found in several industrial <em>Aspergillus</em> species, genome co-editing using homolog genes as selectable markers is plausible, which would contribute to the widespread breeding of industrial strains of Aspergilli.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140769868","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}
In recent years, the demand for beers with a variety of flavors has increased considerably owing to the diversification of consumer preferences. Sour beer is characterized by a sour taste unlike normal beer flavor, and previous studies on sour beer have been primarily focused on addressing issues, such as inconsistent product quality and long production time, and on the associated microorganisms. Scientific knowledge regarding the characteristic flavor of sour beer and flavor components is limited. Therefore, in this study, we aimed to clarify the characteristic sensory attributes of sour beer and the component profiles that explain these attributes. Component analysis was performed on 10 traditional sour beers (eight Flanders Red Ales and two Lambics), using untargeted gas chromatography-mass spectrometry with liquid–liquid extraction, liquid chromatography-mass spectrometry targeting amines and anionic compounds. Further, sensory evaluation was conducted by well-trained panelists via quantitative descriptive analysis. Orthogonal partial least squares regression analysis was also conducted to investigate candidate flavor components. Thus, 261 components were identified and our methods could explain the flavor attributes of the examined samples. Comprehensive component profiling data also showed that differences in fermentation method, barrel aging duration, and blending ratio affected beer flavor. Further, Lambics were found to be characterized by citrus and phenolic aroma, while Flanders Red Ales were characterized by solvent-like aroma, sourness complexity, full bodied, graininess, astringency, and bitterness. These findings may serve as a basis for addressing issues related to sour beer production and may facilitate process design for obtaining targeted sour beer flavors.
{"title":"New insights into the characteristic flavor components of traditional sour beers such as Lambic and Flanders Red Ale beers","authors":"Kyoya Onishi , Masahiro Furuno , Asuka Mori , Eiichiro Fukusaki","doi":"10.1016/j.jbiosc.2024.04.002","DOIUrl":"10.1016/j.jbiosc.2024.04.002","url":null,"abstract":"<div><p>In recent years, the demand for beers with a variety of flavors has increased considerably owing to the diversification of consumer preferences. Sour beer is characterized by a sour taste unlike normal beer flavor, and previous studies on sour beer have been primarily focused on addressing issues, such as inconsistent product quality and long production time, and on the associated microorganisms. Scientific knowledge regarding the characteristic flavor of sour beer and flavor components is limited. Therefore, in this study, we aimed to clarify the characteristic sensory attributes of sour beer and the component profiles that explain these attributes. Component analysis was performed on 10 traditional sour beers (eight Flanders Red Ales and two Lambics), using untargeted gas chromatography-mass spectrometry with liquid–liquid extraction, liquid chromatography-mass spectrometry targeting amines and anionic compounds. Further, sensory evaluation was conducted by well-trained panelists via quantitative descriptive analysis. Orthogonal partial least squares regression analysis was also conducted to investigate candidate flavor components. Thus, 261 components were identified and our methods could explain the flavor attributes of the examined samples. Comprehensive component profiling data also showed that differences in fermentation method, barrel aging duration, and blending ratio affected beer flavor. Further, Lambics were found to be characterized by citrus and phenolic aroma, while Flanders Red Ales were characterized by solvent-like aroma, sourness complexity, full bodied, graininess, astringency, and bitterness. These findings may serve as a basis for addressing issues related to sour beer production and may facilitate process design for obtaining targeted sour beer flavors.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140768223","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}
Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production.
抗体药物在诊断和治疗中发挥着重要作用。然而,由于细胞的异质性,从哺乳动物细胞中生产抗体具有挑战性,这可以通过应用基于液滴的微流控平台进行高通量筛选(HTS)来解决。在这里,我们设计了一种基于二硫键氧化还原反应水凝胶珠(氧化还原-HBs)的集成系统,该系统是通过酶法水凝胶化制备而成的,可对分泌高水平抗体的中国仓鼠卵巢(CHO)细胞进行分隔、筛选、选择、检索和回收。此外,还用蛋白 G 作为抗体结合模块对氧化还原-HBs 进行了功能化处理,以捕获封装细胞分泌的抗体。作为概念验证,以免疫球蛋白 G(IgG)为抗体、绿色荧光蛋白(GFP)为报告分子的细胞被封装到功能化氧化还原桥中,称为 CHO(IgG/GFP)。此外,还利用酪胺放大系统用蛋白 L 结合物辣根过氧化物酶标记了分泌抗体的细胞,从而实现了对珠子内捕获的抗体的荧光染色。然后将氧化还原-HBs 应用于荧光激活的液滴分选,通过还原二硫键降解选定的氧化还原-HBs,从而回收目标细胞。结果表明,所开发的 HTS 平台具有选择可用于生物制药生产的单细胞的潜力。
{"title":"Design and validation of functionalized redox-responsive hydrogel beads for high-throughput screening of antibody-secreting mammalian cells","authors":"Diah Anggraini Wulandari , Kyosuke Tsuru , Kosuke Minamihata , Rie Wakabayashi , Go Egami , Yoshinori Kawabe , Masamichi Kamihira , Masahiro Goto , Noriho Kamiya","doi":"10.1016/j.jbiosc.2024.04.001","DOIUrl":"10.1016/j.jbiosc.2024.04.001","url":null,"abstract":"<div><p>Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140757189","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}
Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.
{"title":"Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage","authors":"Takao Ohashi , Yurika Mabira , Yutaro Mitsuyoshi , Hiroyuki Kajiura , Ryo Misaki , Takeshi Ishimizu , Kazuhito Fujiyama","doi":"10.1016/j.jbiosc.2024.03.006","DOIUrl":"10.1016/j.jbiosc.2024.03.006","url":null,"abstract":"<div><p>Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from <em>Aspergillus aculeatus</em> (<em>Aa</em>RhgA) in the fission yeast <em>Schizosaccharomyces pombe</em> and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant <em>Aa</em>RhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the <em>AarhgA</em> gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in <em>Arabidopsis thaliana</em>. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140775987","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}
Pub Date : 2024-04-20DOI: 10.1016/j.jbiosc.2024.03.008
Ayana Yamaguchi , Yoshihide Hashimoto , Jun Negishi
Cell culture models that mimic tissue environments are useful for cell and extracellular matrix (ECM) function analysis. Decellularized tissues with tissue-specific ECM are expected to be applied as cell culture scaffolds, however, it is often difficult for seeded cells to permeate their structures. In this study, we evaluated the adhesion and proliferation of mouse fibroblasts seeded onto decellularized bone marrow scaffolds that we fabricated from adult and fetal porcine. Decellularized fetal bone marrow displays more cell attachment and faster cell proliferation than decellularized adult bone marrow. Our findings suggest that decellularized fetal bone marrow is useful as a cell culture scaffold with bone marrow ECM and structure.
{"title":"Fabrication of a cell culture scaffold that mimics the composition and structure of bone marrow extracellular matrix","authors":"Ayana Yamaguchi , Yoshihide Hashimoto , Jun Negishi","doi":"10.1016/j.jbiosc.2024.03.008","DOIUrl":"10.1016/j.jbiosc.2024.03.008","url":null,"abstract":"<div><p>Cell culture models that mimic tissue environments are useful for cell and extracellular matrix (ECM) function analysis. Decellularized tissues with tissue-specific ECM are expected to be applied as cell culture scaffolds, however, it is often difficult for seeded cells to permeate their structures. In this study, we evaluated the adhesion and proliferation of mouse fibroblasts seeded onto decellularized bone marrow scaffolds that we fabricated from adult and fetal porcine. Decellularized fetal bone marrow displays more cell attachment and faster cell proliferation than decellularized adult bone marrow. Our findings suggest that decellularized fetal bone marrow is useful as a cell culture scaffold with bone marrow ECM and structure.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140780952","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}
A new extracellular protease from Bacillus subtilis strain MPK with collagenolytic activity was isolated and purified. Fish skin which otherwise would be treated as waste is used as substrate for the production of protease. Using various techniques such as ammonium sulphate precipitation and ion exchange chromatography, protease was purified and characterized subsequently. Protease of approximately 61 kDa molecular weight was purified by 135.7-fold with 18.42% enzyme recovery. The protease showed effective properties like pH and temperature stability over a broad range with optimum pH 7.5 and temperature 60 °C. Km and Vmax were found to be 1.92 mg ml−1 and 1.02 × 10−4 mol L−1 min−1, respectively. The protease exhibited stability in various ions, surfactants, inhibitors and organic solvents. Subsequently, the protease was successfully utilized for collagen hydrolysis to generate collagen peptides; thus, the produced protease would be a potential candidate for multifaceted applications in food and pharmaceutical industries due to its significant characteristics and collagenolytic properties.
{"title":"Purification, characterization and application of collagenolytic protease from Bacillus subtilis strain MPK","authors":"Madhuri Vijay Bhuimbar , Chidambar Balbhim Jalkute , Prashant Kishor Bhagwat , Padma Babulal Dandge","doi":"10.1016/j.jbiosc.2024.03.003","DOIUrl":"10.1016/j.jbiosc.2024.03.003","url":null,"abstract":"<div><p>A new extracellular protease from <em>Bacillus subtilis</em> strain MPK with collagenolytic activity was isolated and purified. Fish skin which otherwise would be treated as waste is used as substrate for the production of protease. Using various techniques such as ammonium sulphate precipitation and ion exchange chromatography, protease was purified and characterized subsequently. Protease of approximately 61 kDa molecular weight was purified by 135.7-fold with 18.42% enzyme recovery. The protease showed effective properties like pH and temperature stability over a broad range with optimum pH 7.5 and temperature 60 °C. <em>K</em><sub>m</sub> and <em>V</em><sub>max</sub> were found to be 1.92 mg ml<sup>−1</sup> and 1.02 × 10<sup>−4</sup> mol L<sup>−1</sup> min<sup>−1</sup>, respectively. The protease exhibited stability in various ions, surfactants, inhibitors and organic solvents. Subsequently, the protease was successfully utilized for collagen hydrolysis to generate collagen peptides; thus, the produced protease would be a potential candidate for multifaceted applications in food and pharmaceutical industries due to its significant characteristics and collagenolytic properties.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140780655","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}
6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform.
{"title":"Characterization of unique EDTA-insensitive methylthioalkylmalate synthase from Eutrema japonicum and its potential application in synthetic biology","authors":"Dheeradhach Medhanavyn , Toshiya Muranaka , Shuhei Yasumoto","doi":"10.1016/j.jbiosc.2024.02.009","DOIUrl":"10.1016/j.jbiosc.2024.02.009","url":null,"abstract":"<div><p>6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from <em>Eutrema japonicum</em>, commonly referred to as Japanese wasabi. <em>E</em><em>utrema</em> <em>japonicum</em> MAMs (EjMAMs) were expressed in an <em>E</em><em>scherichia</em> <em>coli</em> expression system, subsequently purified, and <em>in vitro</em> enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used <em>in silico</em> analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined <em>in vivo</em> biosynthesis in <em>E. coli</em> containing <em>Arabidopsis thaliana</em> branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting <span>l</span>-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property <em>in vitro</em> and highest activity on converting <span>l</span>-methionine to 2-(2-MT)EM <em>in vivo</em> which displays high potential for isothiocyanate biosynthesis in <em>E. coli</em> platform.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140756220","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}