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Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production

Science China(Life Sciences) 2012年第4期55卷 283-290页

作者： LAI ShuJuan ZHANG Yun LIU Shuwen LIANG Yong SHANG XiuLing CHAI Xin wen tingyi Department of lndustrial Microbiology and Biotechnology Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China Graduate University of Chinese Academy of Sciences Beijing 100049 China

L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.

关键词： Corynebacterium glutamicum L-serine intracellular metabolites metabolic engineering elementary mode analysis

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In vitro assembly of the bacterial actin protein MamK from ＇Candidatus Magnetobacterium casensis＇ in the phylum Nitrospirae

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Protein & Cell 2016年第4期7卷 267-280页

作者： Aihua Deng Wei Lin Nana Shi Jie Wu Zhaopeng Sun Qinyun Sun Hua Bai Yongxin Pan tingyi wen CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China Biogeomagnetism Group Paleomagnetism and Geochronology Laboratory Key Laboratory of Earth and Planetary Physics Institute of Geology and Geophysics Chinese Academy of Sciences Beijing 100029 China University of Chinese Academy of Sciences Beijing 100049 China

Magnetotactic bacteria （MTB）, a group of phylogeneti- cally diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth＇s magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multi- ple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated ＇Candidatus Magnetobacterium casensis＇（Mcas） within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell, These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems.

关键词： magnetotactic bacteria Nitrospirae,bacterial actin MamK assembly mechanism

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整合组学数据的代谢网络模型研究进展

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科学通报 2021年第19期66卷 2393-2404页

作者：王雪亮张芸温廷益中国科学院微生物研究所中国科学院病原微生物与免疫学重点实验室北京100101 中国科学院大学北京100049 中国科学院绿色过程制造创新研究院北京100190 中国科学院大学存济医学院北京100049

基因组规模代谢网络模型(genome-scale metabolic model,GEM)是根据化学计量平衡原理,基于基因-蛋白-反应三者关联,建立包含细胞生长必需的生化反应数学模型.细胞代谢网络存在复杂的调控,通过基因和蛋白表达量及代谢物浓度变化调节代谢... 详细信息

基因组规模代谢网络模型(genome-scale metabolic model,GEM)是根据化学计量平衡原理,基于基因-蛋白-反应三者关联,建立包含细胞生长必需的生化反应数学模型.细胞代谢网络存在复杂的调控,通过基因和蛋白表达量及代谢物浓度变化调节代谢反应通量,而这些数据无法直接反映代谢反应的通量.GEM研究面临的一个挑战是如何整合不直接反映代谢通量的数据类型,并将这些调控过程在代谢模型中进行准确的描述.本文综述了将高通量组学数据整合到代谢化学计量模型中的方法,包括根据基因和蛋白的表达判定代谢反应状态、筛选核心代谢反应集、代谢反应通量边界约束、转录调控网络整合和多时间尺度基因动态表达约束等;比较分析了不同算法的优缺点和应用场景;介绍了GEM与多组学数据整合在解析生物代谢特征、分析遗传和环境扰动、筛选癌症治疗潜在药物靶标和抗代谢药物等方面的应用;展望了组学数据和代谢网络模型集成的发展趋势.

关键词：代谢网络模型转录组学蛋白质组学代谢组学组学数据整合

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A salt, detergent, and solvent tolerant protease from <i>Bacillus</i>sp. B001: Low-cost, easy-purified, and enhanced production by raw material based culture strategy

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Advances in Bioscience and Biotechnology 2013年第12期4卷 1039-1048页

作者： Jie Wu Aihua Deng Nana Shi Shuwen Liu Yong Liang tingyi wen CAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of Microbiology Chinese Academy of Sciences Beijing China CAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of Microbiology Chinese Academy of Sciences Beijing China University of Chinese Academy of Sciences Beijing China

To enhance the protease production and decrease cost, corn flour and soy peptone were screened as cheap raw materials for the production of extracellular proteases by Bacillus strains. Their compositions in the medium suitable for enzyme production of Bacillus sp. B001 were optimized using statistical experiment designs. Under the optimized conditions, the protease production of Bacillus sp. B001 was stable at the stationary stage and reached to 63,200 U/mL, approximately 1.84-fold increase compared with that using the original medium. These improvements could be attributed to the release of the catabolite repression by crude materials corn flour and soy peptone which contained low level of available nutrients. Additionally, a highly pure protease which displayed excellent stability and compatibility with high salinity, commercial laundry detergents, and organic solvents, was rapidly obtained by two-step procedure involving ammonium sulphate precipitation and anion exchange from the fermentation cultures of B001 in the optimized medium. When the culture method applied to other Bacillus strains, their protease yields were all remarkably increased approximately 2.9 to 8.5 folds. In conclusion, a low-cost, easy-purified, and effective producing strategy using the cheap raw materials was developed here, representing a potential application for protease production in various Industrial processes.

关键词： Alkaline Protease Bacillus Enzyme Production Raw Materials Based Strategy Response Surface Methodology

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L-苏氨酸发酵液有机膜过滤工艺研究

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食品与发酵工业 2009年第2期35卷 87-90页

作者：黄金陈宁温廷益天津科技大学生物工程学院天津300457 中国科学院微生物研究所北京100101

采用有机微滤-超滤膜分离系统,两阶段截留发酵液中残留的菌体、蛋白质和悬浮固体颗粒,建立了微滤-超滤有机膜两步法过滤工艺,解决了一步法超滤过程中的膜通量低的难题。膜通量由8.25 L/(m2.h)提高至32 L/(m2.h),色素去除率、蛋白去除率... 详细信息

采用有机微滤-超滤膜分离系统,两阶段截留发酵液中残留的菌体、蛋白质和悬浮固体颗粒,建立了微滤-超滤有机膜两步法过滤工艺,解决了一步法超滤过程中的膜通量低的难题。膜通量由8.25 L/(m2.h)提高至32 L/(m2.h),色素去除率、蛋白去除率及产品回收率可分别达到58.5%、97.6%和87.0%。另外,采用0.01 mol/L NaOH、自来水和0.1 mol/L HCl间歇替换清洗可有效恢复膜通量。

关键词：有机膜 L-苏氨酸分离超滤

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不同溶氧条件下L-苏氨酸生物合成菌株的代谢流量分析

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微生物学报 2008年第8期48卷 1056-1060页

作者：黄金徐庆阳温廷益陈宁天津科技大学生物工程学院天津300457

【目的】探索L-苏氨酸生物合成机理及影响因素。【方法】建立了大肠杆菌L-苏氨酸的代谢流平衡模型,应用MATLAB软件计算出不同溶氧条件下发酵中后期代谢网络的代谢流分布及理想代谢流分布。【结果】5%溶氧条件下,25.5%碳架进入HMP途径,74... 详细信息

【目的】探索L-苏氨酸生物合成机理及影响因素。【方法】建立了大肠杆菌L-苏氨酸的代谢流平衡模型,应用MATLAB软件计算出不同溶氧条件下发酵中后期代谢网络的代谢流分布及理想代谢流分布。【结果】5%溶氧条件下,25.5%碳架进入HMP途径,74.5%碳架进入糖酵解途径,获得33.9%质量转化率;20%溶氧条件下,58.08%碳架进入HMP途径,41.92%碳架进入糖酵解途径,获得46.5%质量转化率;【结论】与理想代谢流(88.23%质量转化率)相比,应从菌种改造和发酵控制方面通过改变6-磷酸葡萄糖异构酶借以增加HMP途径代谢流量,通过增加磷酸烯醇式丙酮酸羧化反应代谢流提高天冬氨酸族合成代谢流,减少TCA循环代谢流量,从而达到减少副产物生成,增加L-苏氨酸生物合成的目的。

关键词： L-苏氨酸溶氧代谢流分析大肠杆菌

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Genomic insights into the uncultured genus‘Candidatus Magnetobacterium' in the phylum Nitrospirae

Genomic insights into the uncultured genus‘Candidatus Magne...

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中国科学院地质与地球物理研究所2014年度（第14届）学术年会

作者： Wei Lin Aihua Deng Zhang Wang Ying Li tingyi wen Long-Fei Wu Martin Wu Yongxin Pan Biogeomagnetism Group Paleomagnetism and Geochronology LaboratoryKey Laboratory of the Earth’s Deep InteriorInstitute of Geology and GeophysicsChinese Academy of Sciences France-China Bio-Mineralization and Nano-Structures Laboratory Chinese Academy of Sciences CAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of MicrobiologyChinese Academy of Sciences Department of Biology University of VirginiaCharlottesvilleVAUSA State Key Laboratory of Agro-Biotechnology and Laboratoire International Associe Franco-Chinois de Bio-Mineralisation et Nano-Structures College of Biological SciencesChina Agricultural University Laboratoire de Chimie Bacte′rienne Aix-Marseille UniversiteCNRSMarseille Cedex 20France

Magnetotactic bacteria(MTB)of the genus‘Candidatus Magnetobacterium’in phylum Nitrospirae are of great interest because of the formation of hundreds of bullet-shaped magnetite magnetosomes in multiple bundles of chains per *** bacteria are worldwide distributed in aquatic environments and have important roles in the biogeochemical cycles of iron and ***,except for a few short genomic fragments,no genome data are available for this ecologically important genus,and little is known about their metabolic capacity owing to the lack of pure *** we report the first draft genome sequence of 3.42 Mb from an uncultivated strain tentatively named‘*** casensis’isolated from Lake Miyun,*** genome sequence indicates an autotrophic lifestyle using the Wood–Ljungdahl pathway for CO2fixation,which has not been described in any previously known MTB or Nitrospirae *** involved in the denitrification,sulfur oxidation and sulfate reduction have been predicted,indicating its considerable capacity for adaptation to variable geochemical conditions and roles in local biogeochemical ***,we have identified a complete magnetosome gene island containing mam,mad and a set of novel genes(named as man genes)putatively responsible for the formation of bullet-shaped magnetite magnetosomes and the arrangement of multiple magnetosome *** first comprehensive genomic analysis sheds light on the physiology,ecology and biomineralization of the poorly understood‘***’genus.

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Thermodynamic theory of flotation for a complex multiphase solid -liquid system and high-entropy flotation

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International Journal of Minerals,Metallurgy and Materials 2024年第6期31卷 1177-1197页

作者： Shuming wen State Key Laboratory of Complex Nonferrous Metal Resources Clean Utilization Faculty of Land Resource EngineeringKunming University of Science and TechnologyKunming 650093China

The flotation of complex solid–liquid multiphase systems involve interactions among multiple components,the core problem facing flotation ***,the combined use of multicomponent flotation reagents to improve mineral flotation has become an important issue in studies on the efficient use of refractory mineral ***,studying the flotation of complex solid–liquid systems is extremely difficult,and no systematic theory has been developed to *** addition,the physical mechanism associated with combining reagents to improve the flotation effect has not been unified,which limits the development of flotation theory and the progress of flotation *** this study,we applied theoretical thermodynamics to a solid–liquid flotation system and used changes in the entropy and Gibbs free energy of the reagents adsorbed on the mineral surface to establish thermodynamic equilibrium equations that de-scribe interactions among various material components while also introducing adsorption equilibrium constants for the flotation reagents adsorbed on the mineral *** homogenization effect on the mineral surface in pulp solution was determined using the chemical potentials of the material components of the various mineral surfaces required to maintain *** flotation effect can be improved through synergy among multicomponent flotation reagents;its physical essence is the thermodynamic law that as the number of compon-ents of flotation reagents on the mineral surface increases,the surface adsorption entropy change increases,and the Gibbs free energy change of adsorption *** to the results obtained using flotation thermodynamics theory,we established high-entropy flotation theory and a technical method in which increasing the types of flotation reagents adsorbed on the mineral surface,increasing the adsorption entropy change of the flotation reagents,decreasing the Gibbs free energy change,and improving the adsorption efficiency

关键词： multicomponent flotation reagent adsorption entropy change equations of thermodynamic equilibrium adsorption equilibri-um constant high-entropy flotation

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Goal-Oriented Control Systems(GOCS):From HOW to WHAT

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IEEE/CAA Journal of Automatica Sinica 2024年第4期11卷 816-819页

作者： wen-Hua Chen the Department of Aeronautical and Automotive Engineering Loughborough UniversityLeicestershireLE113TUU.K.

Brief:New control theory is required to underpin safe design and deployment of future highly automated systems to deal with uncertain environments and complicated tasks,enabled by AI and other advanced ***-Oriented Control Systems offer potential to transform the control system design from currently instructing a control system how to perform a task to specifying what is to be achieved.

关键词： automated instru system

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Flow characteristics and bubble statistics during the fragmentation process of the ingested main cavity in plunging breaking waves

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Journal of Hydrodynamics 2024年第3期36卷 546-555页

作者： wen-bin Zhang Wei-wen Zhao De-cheng Wan Computational Marine Hydrodynamics Lab(CMHL) School of Naval ArchitectureOcean and Civil EngineeringShanghai Jiao Tong UniversityShanghai200240China

Plunging breaking waves play an important role in the exchange of heat,momentum,and mass between the atmosphere and *** this paper,a series of direct numerical simulations is conducted to investigate the fragmentation process of the ingested main cavity in plunging breaking *** two-phase Navier-Stokes equations are solved using the finite-volume method based on adaptive refinement *** free surface is captured using a geometrical volume of fluid *** 2-D,3-D simulations are *** flow fields at different stages of wave breaking are presented and quantitative analysis for bubbles is *** 2-D instantaneous vorticity field and local velocity field are visualized to discuss the general flow characteristics during the fragmentation *** a 2-D parametric study is conducted to investigate the differences in the flow characteristics during the fragmentation process under different wave parameters including initial wave steepness(ε),Bond number(Bo),and Reynolds number(Re).3-D vortex structures are shown to further investigate the mechanisms behind the differences in the flow *** bubble size distributions under two different initial wave steepness are also discussed with their relationship to the fragmentation process of the ingested main *** research offers a significant understanding of the distinct procedures and fundamental dynamics involved in wave breaking,enhancing our comprehension of this intricate event.

关键词： Plunging breaking waves multiphase flow bubble statistics

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