Pub Date : 2024-09-14DOI: 10.1186/s12934-024-02501-2
Yinlu Liu, Qianzhen Dong, Wan Song, Wenwen Pei, Yan Zeng, Min Wang, Yuanxia Sun, Yanhe Ma, Jiangang Yang
Seven-carbon sugars, which rarely exist in nature, are the key constitutional unit of septacidin and hygromycin B in bacteria. These sugars exhibit a potential therapeutic effect for hypoglycaemia and cancer and serve as building blocks for the synthesis of C-glycosides and novel antibiotics. However, chemical and enzymatic approaches for the synthesis of seven-carbon sugars have faced challenges, such as complex reaction steps, low overall yields and high-cost feedstock, limiting their industrial-scale production. In this work, we propose a strain engineering approach for synthesising sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards the pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production. We achieved a sedoheptulose concentration of 24 g/L with a yield of 0.4 g/g glucose in a 1 L fermenter, marking the highest value up to date. The produced sedoheptulose could further function as feedstock for synthesising structural seven-carbon sugars through coupling with enzymatic isomerisation, epimerisation and reduction reactions.
{"title":"Microbial synthesis of sedoheptulose from glucose by metabolically engineered Corynebacterium glutamicum","authors":"Yinlu Liu, Qianzhen Dong, Wan Song, Wenwen Pei, Yan Zeng, Min Wang, Yuanxia Sun, Yanhe Ma, Jiangang Yang","doi":"10.1186/s12934-024-02501-2","DOIUrl":"https://doi.org/10.1186/s12934-024-02501-2","url":null,"abstract":"Seven-carbon sugars, which rarely exist in nature, are the key constitutional unit of septacidin and hygromycin B in bacteria. These sugars exhibit a potential therapeutic effect for hypoglycaemia and cancer and serve as building blocks for the synthesis of C-glycosides and novel antibiotics. However, chemical and enzymatic approaches for the synthesis of seven-carbon sugars have faced challenges, such as complex reaction steps, low overall yields and high-cost feedstock, limiting their industrial-scale production. In this work, we propose a strain engineering approach for synthesising sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards the pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production. We achieved a sedoheptulose concentration of 24 g/L with a yield of 0.4 g/g glucose in a 1 L fermenter, marking the highest value up to date. The produced sedoheptulose could further function as feedstock for synthesising structural seven-carbon sugars through coupling with enzymatic isomerisation, epimerisation and reduction reactions.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1186/s12934-024-02518-7
Yasmine Fathy Mohamed, Rachel C. Fernandez
Bordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines. This work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs). These findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.
百日咳杆菌是百日咳或百日咳的病原体。虽然无细胞百日咳疫苗（aP）和全细胞百日咳疫苗（wP）都能预防疾病，但高致反应性的全细胞百日咳疫苗能更好地预防定植和传播。致反应性主要归因于百日咳杆菌脂寡糖（LOS）中的脂质 A 分子。在 LOS 中，脂质 A 充当疏水锚，与宿主免疫细胞上的 TLR4-MD2 结合，启动 MyD88 依赖性和 TRIF 依赖性途径，从而影响适应性免疫反应。脂质 A 的变体，如单磷脂质 A（MPLA），也可以作为佐剂。佐剂可以克服 aP 疫苗的缺点。这项研究利用来自其他细菌的脂质 A 修饰酶，在百日咳杆菌中产生了一种类似 MPLA 的佐剂菌株。我们创建了具有不同脂质 A 修饰的百日咳杆菌菌株，并使用 MALDI-TOF 对其进行了验证。我们设计了一种六烷基化的单磷酸化脂质 A，它能显著降低人类 TLR4 的活化并激活 TRIF 通路。经修饰的脂寡糖（LOS）可促进 IRF3 磷酸化和 I 型干扰素的产生，与 MPLA 反应类似。我们还生成了另外三种变体，它们具有更强的佐剂特性和更低的内毒素。使用单核细胞活化试验（MAT）进行的热原性研究表明，这四种脂质 A 变体能显著降低外周血单核细胞（PBMCs）中作为发热标志的 IL-6 的反应。这些发现为开发致反应性可能较低的 wP 疫苗以及为现有疫苗配方设计适应性强的佐剂铺平了道路，从而推进了更有效的百日咳免疫策略。
{"title":"Programming Bordetella pertussis lipid A to promote adjuvanticity","authors":"Yasmine Fathy Mohamed, Rachel C. Fernandez","doi":"10.1186/s12934-024-02518-7","DOIUrl":"https://doi.org/10.1186/s12934-024-02518-7","url":null,"abstract":"Bordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines. This work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs). These findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1186/s12934-024-02523-w
Jasmine De Baets, Brecht De Paepe, Marjan De Mey
Engineering bacteria with the purpose of optimizing the production of interesting molecules often leads to a decrease in growth due to metabolic burden or toxicity. By delaying the production in time, these negative effects on the growth can be avoided in a process called a two-stage fermentation. During this two-stage fermentation process, the production stage is only activated once sufficient cell mass is obtained. Besides the possibility of using external triggers, such as chemical molecules or changing fermentation parameters to induce the production stage, there is a renewed interest towards autoinducible systems. These systems, such as quorum sensing, do not require the extra interference with the fermentation broth to start the induction. In this review, we discuss the different possibilities of both external and autoinduction methods to obtain a two-stage fermentation. Additionally, an overview is given of the tuning methods that can be applied to optimize the induction process. Finally, future challenges and prospects of (auto)inducible expression systems are discussed. There are numerous methods to obtain a two-stage fermentation process each with their own advantages and disadvantages. Even though chemically inducible expression systems are well-established, an increasing interest is going towards autoinducible expression systems, such as quorum sensing. Although these newer techniques cannot rely on the decades of characterization and applications as is the case for chemically inducible promoters, their advantages might lead to a shift in future inducible expression systems.
{"title":"Delaying production with prokaryotic inducible expression systems","authors":"Jasmine De Baets, Brecht De Paepe, Marjan De Mey","doi":"10.1186/s12934-024-02523-w","DOIUrl":"https://doi.org/10.1186/s12934-024-02523-w","url":null,"abstract":"Engineering bacteria with the purpose of optimizing the production of interesting molecules often leads to a decrease in growth due to metabolic burden or toxicity. By delaying the production in time, these negative effects on the growth can be avoided in a process called a two-stage fermentation. During this two-stage fermentation process, the production stage is only activated once sufficient cell mass is obtained. Besides the possibility of using external triggers, such as chemical molecules or changing fermentation parameters to induce the production stage, there is a renewed interest towards autoinducible systems. These systems, such as quorum sensing, do not require the extra interference with the fermentation broth to start the induction. In this review, we discuss the different possibilities of both external and autoinduction methods to obtain a two-stage fermentation. Additionally, an overview is given of the tuning methods that can be applied to optimize the induction process. Finally, future challenges and prospects of (auto)inducible expression systems are discussed. There are numerous methods to obtain a two-stage fermentation process each with their own advantages and disadvantages. Even though chemically inducible expression systems are well-established, an increasing interest is going towards autoinducible expression systems, such as quorum sensing. Although these newer techniques cannot rely on the decades of characterization and applications as is the case for chemically inducible promoters, their advantages might lead to a shift in future inducible expression systems.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265462","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1186/s12934-024-02519-6
Juan José León, Nía Oetiker, Nicolás Torres, Nicolás Bruna, Evgenii Oskolkov, Pedro Lei, Andrey N. Kuzmin, Kaiwen Chen, Stelios Andreadis, Blaine A. Pfeifer, Mark T. Swihart, Paras N. Prasad, José Pérez-Donoso
Rare-earth sulfide nanoparticles (NPs) could harness the optical and magnetic features of rare-earth ions for applications in nanotechnology. However, reports of their synthesis are scarce and typically require high temperatures and long synthesis times. Here we present a biosynthesis of terbium sulfide (TbS) NPs using microorganisms, identifying conditions that allow Escherichia coli to extracellularly produce TbS NPs in aqueous media at 37 °C by controlling cellular sulfur metabolism to produce a high concentration of sulfide ions. Electron microscopy revealed ultrasmall spherical NPs with a mean diameter of 4.1 ± 1.3 nm. Electron diffraction indicated a high degree of crystallinity, while elemental mapping confirmed colocalization of terbium and sulfur. The NPs exhibit characteristic absorbance and luminescence of terbium, with downshifting quantum yield (QY) reaching 28.3% and an emission lifetime of ~ 2 ms. This high QY and long emission lifetime is unusual in a neat rare-earth compound; it is typically associated with rare-earth ions doped into another crystalline lattice to avoid non-radiative cross relaxation. This suggests a reduced role of nonradiative processes in these terbium-based NPs. This is, to our knowledge, the first report revealing the advantage of biosynthesis over chemical synthesis for Rare Earth Element (REE) based NPs, opening routes to new REE-based nanocrystals.
{"title":"Microbial green synthesis of luminescent terbium sulfide nanoparticles using E. Coli: a rare earth element detoxification mechanism","authors":"Juan José León, Nía Oetiker, Nicolás Torres, Nicolás Bruna, Evgenii Oskolkov, Pedro Lei, Andrey N. Kuzmin, Kaiwen Chen, Stelios Andreadis, Blaine A. Pfeifer, Mark T. Swihart, Paras N. Prasad, José Pérez-Donoso","doi":"10.1186/s12934-024-02519-6","DOIUrl":"https://doi.org/10.1186/s12934-024-02519-6","url":null,"abstract":"Rare-earth sulfide nanoparticles (NPs) could harness the optical and magnetic features of rare-earth ions for applications in nanotechnology. However, reports of their synthesis are scarce and typically require high temperatures and long synthesis times. Here we present a biosynthesis of terbium sulfide (TbS) NPs using microorganisms, identifying conditions that allow Escherichia coli to extracellularly produce TbS NPs in aqueous media at 37 °C by controlling cellular sulfur metabolism to produce a high concentration of sulfide ions. Electron microscopy revealed ultrasmall spherical NPs with a mean diameter of 4.1 ± 1.3 nm. Electron diffraction indicated a high degree of crystallinity, while elemental mapping confirmed colocalization of terbium and sulfur. The NPs exhibit characteristic absorbance and luminescence of terbium, with downshifting quantum yield (QY) reaching 28.3% and an emission lifetime of ~ 2 ms. This high QY and long emission lifetime is unusual in a neat rare-earth compound; it is typically associated with rare-earth ions doped into another crystalline lattice to avoid non-radiative cross relaxation. This suggests a reduced role of nonradiative processes in these terbium-based NPs. This is, to our knowledge, the first report revealing the advantage of biosynthesis over chemical synthesis for Rare Earth Element (REE) based NPs, opening routes to new REE-based nanocrystals.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1186/s12934-024-02509-8
Anna Weimer, Laura Pause, Fabian Ries, Michael Kohlstedt, Lorenz Adrian, Jens Krömer, Bin Lai, Christoph Wittmann
Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production. When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol). By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.
{"title":"Systems biology of electrogenic Pseudomonas putida - multi-omics insights and metabolic engineering for enhanced 2-ketogluconate production","authors":"Anna Weimer, Laura Pause, Fabian Ries, Michael Kohlstedt, Lorenz Adrian, Jens Krömer, Bin Lai, Christoph Wittmann","doi":"10.1186/s12934-024-02509-8","DOIUrl":"https://doi.org/10.1186/s12934-024-02509-8","url":null,"abstract":"Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production. When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol). By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1186/s12934-024-02489-9
Sumeeta Kumari, Alka Kumari, Asmita Dhiman, Kanti Nandan Mihooliya, Manoj Raje, G. S. Prasad, Anil Kumar Pinnaka
Sophorolipids are glycolipid biosurfactants with potential antibacterial, antifungal, and anticancer applications, rendering them promising for research. Therefore, this study hypothesizes that sophorolipids may have a notable impact on disrupting membrane integrity and triggering the production of reactive oxygen species, ultimately resulting in the eradication of pathogenic microbes. The current study resulted in the isolation of two Metschnikowia novel yeast strains. Sophorolipids production from these strains reached maximum yields of 23.24 g/l and 21.75 g/l, respectively, at the bioreactors level. Biosurfactants sophorolipids were characterized using FTIR and LC–MS techniques and found to be a mixture of acidic and lactonic forms with molecular weights of m/z 678 and 700. Our research elucidated sophorolipids’ mechanism in disrupting bacterial and fungal membranes through ROS generation, confirmed by transmission electron microscopy and FACS analysis. The results showed that these compounds disrupted the membrane integrity and induced ROS production, leading to cell death in Klebsiella pneumoniae and Fusarium solani. In addition, the anticancer properties of sophorolipids were investigated on the A549 lung cancer cell line and found that sophorolipid-11D (SL-11D) and sophorolipid-11X (SL-11X) disrupted the actin cytoskeleton, as evidenced by immunofluorescence microscopy. The A549 cells were stained with Acridine orange/Ethidium bromide, which showed that they underwent necrosis. This was confirmed by flow cytometric analysis using Annexin/PI staining. The SL-11D and SL-11X molecules exhibited low levels of haemolytic activity and in-vitro cytotoxicity in HEK293, Caco-2, and L929 cell lines. In this work, novel yeast species CIG-11DT and CIG-11XT, isolated from the bee’s gut, produce significant yields of sophorolipids without needing secondary oil sources, indicating a more economical production method. Our research shows that sophorolipids disrupt bacterial and fungal membranes via ROS production. They suggest they may act as chemo-preventive agents by inducing apoptosis in lung cancer cells, offering the potential for enhancing anticancer therapies.
{"title":"Unveiling the potential of novel Metschnikowia yeast biosurfactants: triggering oxidative stress for promising antifungal and anticancer activity","authors":"Sumeeta Kumari, Alka Kumari, Asmita Dhiman, Kanti Nandan Mihooliya, Manoj Raje, G. S. Prasad, Anil Kumar Pinnaka","doi":"10.1186/s12934-024-02489-9","DOIUrl":"https://doi.org/10.1186/s12934-024-02489-9","url":null,"abstract":"Sophorolipids are glycolipid biosurfactants with potential antibacterial, antifungal, and anticancer applications, rendering them promising for research. Therefore, this study hypothesizes that sophorolipids may have a notable impact on disrupting membrane integrity and triggering the production of reactive oxygen species, ultimately resulting in the eradication of pathogenic microbes. The current study resulted in the isolation of two Metschnikowia novel yeast strains. Sophorolipids production from these strains reached maximum yields of 23.24 g/l and 21.75 g/l, respectively, at the bioreactors level. Biosurfactants sophorolipids were characterized using FTIR and LC–MS techniques and found to be a mixture of acidic and lactonic forms with molecular weights of m/z 678 and 700. Our research elucidated sophorolipids’ mechanism in disrupting bacterial and fungal membranes through ROS generation, confirmed by transmission electron microscopy and FACS analysis. The results showed that these compounds disrupted the membrane integrity and induced ROS production, leading to cell death in Klebsiella pneumoniae and Fusarium solani. In addition, the anticancer properties of sophorolipids were investigated on the A549 lung cancer cell line and found that sophorolipid-11D (SL-11D) and sophorolipid-11X (SL-11X) disrupted the actin cytoskeleton, as evidenced by immunofluorescence microscopy. The A549 cells were stained with Acridine orange/Ethidium bromide, which showed that they underwent necrosis. This was confirmed by flow cytometric analysis using Annexin/PI staining. The SL-11D and SL-11X molecules exhibited low levels of haemolytic activity and in-vitro cytotoxicity in HEK293, Caco-2, and L929 cell lines. In this work, novel yeast species CIG-11DT and CIG-11XT, isolated from the bee’s gut, produce significant yields of sophorolipids without needing secondary oil sources, indicating a more economical production method. Our research shows that sophorolipids disrupt bacterial and fungal membranes via ROS production. They suggest they may act as chemo-preventive agents by inducing apoptosis in lung cancer cells, offering the potential for enhancing anticancer therapies.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1186/s12934-024-02511-0
Eman S. E. Aldaby, Amal W. Danial, R. Abdel-Basset
Biofuel can be generated by different organisms using various substrates. The green alga Chlorococcum humicola OQ934050 exhibited the capability to photosynthesize carbonate carbon, maybe via the activity of carbonic anhydrase enzymes. The optimum treatment is C:N ratio of 1:1 (0.2 mmoles sodium carbonate and 0.2 mmoles sodium nitrate) as it induced the highest dry mass (more than 0.5 mg.mL−1). At this combination, biomass were about 0.2 mg/mL−1 carbohydrates, 0.085 mg/mL−1 proteins, and 0.16 mg/mL−1 oil of this dry weight. The C/N ratios of 1:1 or 10:1 induced up to 30% of the Chlorococcum humicola dry mass as oils. Growth and dry matter content were hindered at 50:1 C/N and oil content was reduced as a result. The fatty acid profile was strongly altered by the applied C.N ratios. The defatted leftovers of the grown alga, after oil extraction, were fermented by a newly isolated heterotrophic bacterium, identified as Bacillus coagulans OQ053202, to evolve hydrogen content as gas. The highest cumulative hydrogen production and reducing sugar (70 ml H2/g biomass and 0.128 mg/ml; respectively) were found at the C/N ratio of 10:1 with the highest hydrogen evolution efficiency (HEE) of 22.8 ml H2/ mg reducing sugar. The optimum treatment applied to the Chlorococcum humicola is C:N ratio of 1:1 for the highest dry mass, up to 30% dry mass as oils. Some fatty acids were induced while others disappeared, depending on the C/N ratios. The highest cumulative hydrogen production and reducing sugar were found at the C/N ratio of 10:1.
{"title":"Photosynthesizing carbonate/nitrate into Chlorococcum humicola biomass for biodiesel and Bacillus coagulans-based biohydrogen production","authors":"Eman S. E. Aldaby, Amal W. Danial, R. Abdel-Basset","doi":"10.1186/s12934-024-02511-0","DOIUrl":"https://doi.org/10.1186/s12934-024-02511-0","url":null,"abstract":"Biofuel can be generated by different organisms using various substrates. The green alga Chlorococcum humicola OQ934050 exhibited the capability to photosynthesize carbonate carbon, maybe via the activity of carbonic anhydrase enzymes. The optimum treatment is C:N ratio of 1:1 (0.2 mmoles sodium carbonate and 0.2 mmoles sodium nitrate) as it induced the highest dry mass (more than 0.5 mg.mL−1). At this combination, biomass were about 0.2 mg/mL−1 carbohydrates, 0.085 mg/mL−1 proteins, and 0.16 mg/mL−1 oil of this dry weight. The C/N ratios of 1:1 or 10:1 induced up to 30% of the Chlorococcum humicola dry mass as oils. Growth and dry matter content were hindered at 50:1 C/N and oil content was reduced as a result. The fatty acid profile was strongly altered by the applied C.N ratios. The defatted leftovers of the grown alga, after oil extraction, were fermented by a newly isolated heterotrophic bacterium, identified as Bacillus coagulans OQ053202, to evolve hydrogen content as gas. The highest cumulative hydrogen production and reducing sugar (70 ml H2/g biomass and 0.128 mg/ml; respectively) were found at the C/N ratio of 10:1 with the highest hydrogen evolution efficiency (HEE) of 22.8 ml H2/ mg reducing sugar. The optimum treatment applied to the Chlorococcum humicola is C:N ratio of 1:1 for the highest dry mass, up to 30% dry mass as oils. Some fatty acids were induced while others disappeared, depending on the C/N ratios. The highest cumulative hydrogen production and reducing sugar were found at the C/N ratio of 10:1.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1186/s12934-024-02520-z
Gi Yeon Kim, Jina Yang, Yong Hee Han, Sang Woo Seo
Sugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes. In this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP. Despite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications.
甘蔗糖蜜富含蔗糖、葡萄糖和果糖，因其丰富且成本低廉，为工业发酵提供了一种前景广阔的碳源。然而，同时利用多种糖类和碳代谢抑制（CCR）带来了挑战。尽管蔗糖富含营养，但除 W 菌株外，人们对蔗糖在大肠杆菌中的代谢仍然知之甚少，这阻碍了蔗糖在微生物发酵中的应用。本研究对大肠杆菌 W 进行了改造，以提高糖的消耗率并克服 CCR。这是通过整合合成设计的 csc 操作子以及优化葡萄糖和果糖共同利用途径实现的。这些进步促进了甘蔗糖蜜在生产 3-hydroxypropionic acid（3-HP）过程中的高效利用，为可持续生化生产过程做出了贡献。在本研究中，我们解决了与大肠杆菌 W 中糖代谢相关的挑战，重点是提高蔗糖消耗和改善葡萄糖-果糖协同利用。通过对蔗糖利用系统进行有针对性的工程改造，我们调控了 csc 操作子元件 cscB、cscK、cscA 和 cscR 的表达，从而加快了蔗糖的消耗速度。我们的研究结果表明，在删除 cscR 的情况下，csc 基因的单核苷酸表达可使蔗糖的利用率达到最佳，而不会对生长造成明显的负担。此外，我们还通过调节 FruR 与果糖 PTS 调控子的结合动态，成功地缓解了果糖代谢抑制，使葡萄糖和果糖的共同利用接近等效。为了验证我们的工程菌株在工业上的适用性，我们研究了甘蔗糖蜜中 3-HP 的生产。通过整合异源基因和优化代谢途径，我们的 3-HP 滴度比以前的研究有所提高。此外，甘油醛-3-磷酸脱氢酶（gapA）抑制有助于碳通量的重新分配，提高了糖蜜向 3-HP 的转化。尽管在蔗糖代谢方面存在局限性，但重新设计的大肠杆菌 W 菌株善于利用甘蔗糖蜜，是工业发酵的宝贵财富。其合成的 csc 操作子提高了蔗糖的消耗，而减少 CCR 则改善了葡萄糖-果糖的共同利用。这些改进加上 gapA 的抑制，旨在有效地将甘蔗糖蜜转化为 3-HP，解决工业应用中蔗糖和果糖代谢的局限性。
{"title":"Synthetic redesign of Escherichia coli W for faster metabolism of sugarcane molasses","authors":"Gi Yeon Kim, Jina Yang, Yong Hee Han, Sang Woo Seo","doi":"10.1186/s12934-024-02520-z","DOIUrl":"https://doi.org/10.1186/s12934-024-02520-z","url":null,"abstract":"Sugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes. In this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP. Despite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200127","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1186/s12934-024-02514-x
Anna Ścigaczewska, Tomasz Boruta, Weronika Grzesiak, Marcin Bizukojć
The influence of talc microparticles on metabolism and morphology of S. rimosus at various initial organic nitrogen concentrations was investigated. The shake flask cultivations were conducted in the media with yeast extract (nitrogen source) concentration equal to 1 g YE L− 1 and 20 g YE L− 1. Two talc microparticle concentrations of 5 g TALC L− 1 and 10 g TALC L− 1 were tested in microparticle-enhanced cultivation (MPEC) runs. A high nitrogen concentration of 20 g YE L− 1 promoted the development of small agglomerates (pellets) of projected area lower than 105 µm2 and dispersed pseudohyphae. A low nitrogen concentration of 1 g YE L− 1 led to the limitation of S. rimosus growth and, in consequence, the development of the smaller number of large pseudohyphal agglomerates (pellets) of projected area higher than 105 µm2 compared to the culture containing a high amount of nitrogen source. In both cases talc microparticles were embedded into pellets and caused the decrease in their sizes. The lower amount of talc (5 g TALC L− 1) usually caused the weaker effect on S. rimosus morphology and metabolite production than the higher one. This correlation between the microparticles effect on morphology and metabolism of S. rimosus was especially noticeable in the biosynthesis of oxytetracycline, 2-acetyl-2-dicarboxamide oxytetracycline (ADOTC) and spinoxazine A. Compared to the control run, in MPEC their levels increased 4-fold, 5-fold and 1.6-fold respectively. The addition of talc also improved the production of 2-methylthio-cis-zeatin, lorneic acid J and milbemycin A3.
研究了滑石微粒在不同初始有机氮浓度下对菱形芽孢杆菌新陈代谢和形态的影响。摇瓶培养是在酵母提取物（氮源）浓度为 1 g YE L- 1 和 20 g YE L- 1 的培养基中进行的。在微粒子强化培养（MPEC）运行中测试了 5 g TALC L- 1 和 10 g TALC L- 1 两种浓度的滑石微粒子。20 g YE L- 1 的高浓度氮促进了投影面积小于 105 µm2 的小团聚体（颗粒）和分散的假菌丝的发育。1 g YE L- 1 的低氮浓度限制了 S. rimosus 的生长，因此，与含有大量氮源的培养物相比，投影面积大于 105 µm2 的大型假头状菌团（颗粒）的数量较少。在这两种情况下，滑石粉微粒都会嵌入颗粒中，并导致其体积减小。较低的滑石粉用量（5 g TALC L-1）通常比较高的滑石粉用量对菱形虫的形态和代谢物产生的影响要小。与对照组相比，在 MPEC 中，它们的含量分别增加了 4 倍、5 倍和 1.6 倍。添加滑石粉还能提高 2-甲硫基-顺式泽汀、龙葵酸 J 和米尔贝霉素 A3 的产量。
{"title":"Analysis of secondary metabolites and morphology in Streptomyces rimosus microparticle-enhanced cultivation (MPEC) at various initial organic nitrogen concentrations","authors":"Anna Ścigaczewska, Tomasz Boruta, Weronika Grzesiak, Marcin Bizukojć","doi":"10.1186/s12934-024-02514-x","DOIUrl":"https://doi.org/10.1186/s12934-024-02514-x","url":null,"abstract":"The influence of talc microparticles on metabolism and morphology of S. rimosus at various initial organic nitrogen concentrations was investigated. The shake flask cultivations were conducted in the media with yeast extract (nitrogen source) concentration equal to 1 g YE L− 1 and 20 g YE L− 1. Two talc microparticle concentrations of 5 g TALC L− 1 and 10 g TALC L− 1 were tested in microparticle-enhanced cultivation (MPEC) runs. A high nitrogen concentration of 20 g YE L− 1 promoted the development of small agglomerates (pellets) of projected area lower than 105 µm2 and dispersed pseudohyphae. A low nitrogen concentration of 1 g YE L− 1 led to the limitation of S. rimosus growth and, in consequence, the development of the smaller number of large pseudohyphal agglomerates (pellets) of projected area higher than 105 µm2 compared to the culture containing a high amount of nitrogen source. In both cases talc microparticles were embedded into pellets and caused the decrease in their sizes. The lower amount of talc (5 g TALC L− 1) usually caused the weaker effect on S. rimosus morphology and metabolite production than the higher one. This correlation between the microparticles effect on morphology and metabolism of S. rimosus was especially noticeable in the biosynthesis of oxytetracycline, 2-acetyl-2-dicarboxamide oxytetracycline (ADOTC) and spinoxazine A. Compared to the control run, in MPEC their levels increased 4-fold, 5-fold and 1.6-fold respectively. The addition of talc also improved the production of 2-methylthio-cis-zeatin, lorneic acid J and milbemycin A3.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Herpes simplex virus type 1 (HSV-1) is a major cause of viral encephalitis, genital mucosal infections, and neonatal infections. Lactococcus lactis (L. lactis) has been proven to be an effective vehicle for delivering protein antigens and stimulating both mucosal and systemic immune responses. In this study, we constructed a recombinant L. lactis system expressing the protective antigen glycoprotein D (gD) of HSV-1. To improve the stability and persistence of antigen stimulation of the local mucosa, we inserted the immunologic adjuvant interleukin (IL)-2 and the Fc fragment of IgG into the expression system, and a recombinant L. lactis named NZ3900-gD-IL-2-Fc was constructed. By utilizing this recombinant L. lactis strain to elicit an immune response and evaluate the protective effect in mice, the recombinant L. lactis vaccine induced a significant increase in specific neutralizing antibodies, IgG, IgA, interferon-γ, and IL-4 levels in the serum of mice. Furthermore, in comparison to the mice in the control group, the vaccine also enhanced the proliferation levels of lymphocytes in response to gD. Moreover, recombinant L. lactis expressing gD significantly boosted nonspecific immune reactions in mice through the activation of immune-related genes. Furthermore, following the HSV-1 challenge of the murine lung mucosa, mice inoculated with the experimental vaccine exhibited less lung damage than control mice. Our study presents a novel method for constructing a recombinant vaccine using the food-grade, non-pathogenic, and non-commercial bacterium L. lactis. The findings indicate that this recombinant vaccine shows promise in preventing HSV-1 infection in mice.
{"title":"Immunogenicity and protective efficacy of a recombinant lactococcus lactis vaccine against HSV-1 infection","authors":"Shaoju Qian, Ruixue Li, Yeqing He, Hexi Wang, Danqiong Zhang, Aiping Sun, Lili Yu, Xiangfeng Song, Tiesuo Zhao, Zhiguo Chen, Zishan Yang","doi":"10.1186/s12934-024-02517-8","DOIUrl":"https://doi.org/10.1186/s12934-024-02517-8","url":null,"abstract":"Herpes simplex virus type 1 (HSV-1) is a major cause of viral encephalitis, genital mucosal infections, and neonatal infections. Lactococcus lactis (L. lactis) has been proven to be an effective vehicle for delivering protein antigens and stimulating both mucosal and systemic immune responses. In this study, we constructed a recombinant L. lactis system expressing the protective antigen glycoprotein D (gD) of HSV-1. To improve the stability and persistence of antigen stimulation of the local mucosa, we inserted the immunologic adjuvant interleukin (IL)-2 and the Fc fragment of IgG into the expression system, and a recombinant L. lactis named NZ3900-gD-IL-2-Fc was constructed. By utilizing this recombinant L. lactis strain to elicit an immune response and evaluate the protective effect in mice, the recombinant L. lactis vaccine induced a significant increase in specific neutralizing antibodies, IgG, IgA, interferon-γ, and IL-4 levels in the serum of mice. Furthermore, in comparison to the mice in the control group, the vaccine also enhanced the proliferation levels of lymphocytes in response to gD. Moreover, recombinant L. lactis expressing gD significantly boosted nonspecific immune reactions in mice through the activation of immune-related genes. Furthermore, following the HSV-1 challenge of the murine lung mucosa, mice inoculated with the experimental vaccine exhibited less lung damage than control mice. Our study presents a novel method for constructing a recombinant vaccine using the food-grade, non-pathogenic, and non-commercial bacterium L. lactis. The findings indicate that this recombinant vaccine shows promise in preventing HSV-1 infection in mice.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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