Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli

BackgroundPoly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed.ResultS17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content.ConclusionThe impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.How to cite: Wu H, Li S, Ji M, et al. Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.04.005.     

Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BackgroundNonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach.ResultsWe found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions.ConclusionsWe developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L^-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.How to cite: Oestreich AM, Suli LI, Gerlach D. et al. Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli. Electron J Biotechnol 2021;52. https://doi.org/10.1016/j.ejbt.2021.05.001     

湿法冶金清洁生产技术国家工程实验室

湿法冶金清洁生产技术国家工程实验室于2008年11月由国家发展和改革委员会批准成立。工程实验室以中科院过程工程研究所为依托单位,中科院为主管单位,建设期为3年。工程实验室的运行和管理实行理事会领导下的实验室主任负责制．组织有效的财务、人事、科研项目的管理机制,建立技术合作交流和成果转化机制、产学研合作的长效机制。通过承担国家重大工程技术项目、与企业合作开发产业化技术,     

Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives

BackgroundIn order to produce an effective callus in Echinacea purpurea L.; determination of the explant type and growth regulators that best respond to callus induction and the optimization of the culture conditions to increase the amount of caffeic acid derivatives (CADs) in the obtained callus. CADs contents of callus cultures of E. purpurea were evaluated by establishing an effective callus induction system in vitro.ResultsVarious medium containing different growth regulators were tested using leaf, petiole, cotyledon and root as the explants. The best callus development was achieved in MS medium with 1.0 mg l⁻¹ 2,4-D + 2.0 mg l⁻¹ BAP in leaf, 1.0 mg l⁻¹ NAA + 0.5 mg l⁻¹ TDZ in petiole, 2.0 mg l⁻¹ NAA + 1.0 mg l⁻¹ TDZ in cotyledon and 0.5 mg l⁻¹ NAA + 0.5 mg l⁻¹ BAP in roots. Upon optimisation of callus growth, each type of explant was cultured for 4, 6, 8 and 10 weeks in medium for the analyses of caftaric acid, chlorogenic acid, caffeic acid and chicoric acid contents. The highest amounts of caftaric acid (4.11 mg/g) and chicoric acid (57.89 mg/g) were found from petiole explants and chlorogenic acid (8.83 mg/g) from root explants at the end of the 10-week culture time.ConclusionsAs a result of the present study, the production of caffeic acid derivatives was performed by providing the optimization of E. purpurea L. callus cultures. Effective and repeatable protocols established in this study may offer help for further studies investigating the production of caffeic acid derivatives in vitro.How to cite: Tanur Erkoyuncu M, Yorgancilar M. Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.02.003.     

基于工业工程的气缸盖生产线改进

针对气缸盖的机械加工生产线,运用工业工程的程序分析法、秒表时间研究方法对生产线的加工顺序、加工时间进行记录,从而掌握整个生产线的状况。在此基础上,运用工业工程的ECRS原则、动作经济原则,并结合加工工艺技术分析,对生产线提出并实施了改进措施。在投资不大的情况下,提升了生产线的产能,保证了过程质量,降低了劳动强度,使生产线节拍更趋平衡。对大批量机械加工生产线的改进有一定的借鉴作用。     

大肠杆菌氨基酸转运蛋白的研究进展

总结了大肠杆菌氨基酸的原初主动转运蛋白、次级主动转运蛋白和易化扩散载体在其种类、功能、结构和转运机制方面的研究进展.详细介绍了氨基酸与势能离子相伴的同向共转运和反向共转运,并提出了大肠杆菌氨基酸转运研究中存在问题及展望.     

现代工程前沿图谱与中国自主创新策略

运用科学知识图谱方法,考察和分析信息、生物、环境三个国际工程前沿主要领域的学术群体与研究动态,并在阐述和估计现代工程前沿对人类未来各个方面可能产生巨大影响的基础上,提出我国走向现代工程前沿、基于技术科学与工程科学的自主创新策略。     

Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris

BackgroundLaccases are copper-containing enzymes which have been used as green biocatalysts for many industrial processes. Although bacterial laccases have high stabilities which facilitate their application under harsh conditions, their activities and production yields are usually very low. In this work, we attempt to use a combinatorial strategy, including site-directed mutagenesis, codon and cultivation optimization, for improving the productivity of a thermo-alkali stable bacterial laccase in Pichia pastoris.ResultsA D500G mutant of Bacillus licheniformis LS04 laccase, which was constructed by site-directed mutagenesis, demonstrated 2.1-fold higher activity when expressed in P. pastoris. The D500G variant retained similar catalytic characteristics to the wild-type laccase, and could efficiently decolorize synthetic dyes at alkaline conditions. Various cultivation factors such as medium components, pH and temperature were investigated for their effects on laccase expression. After cultivation optimization, a laccase activity of 347 ± 7 U/L was finally achieved for D500G after 3 d of induction, which was about 9.3 times higher than that of wild-type enzyme. The protein yield under the optimized conditions was about 59 mg/L for D500G.ConclusionsThe productivity of the thermo-alkali stable laccase from B. licheniformis expressed in P. pastoris was significantly improved through the combination of site-directed mutagenesis and optimization of the cultivation process. The mutant enzyme retains good stability under high temperature and alkaline conditions, and is a good candidate for industrial application in dye decolorization.     

Cell-enclosing gelatin-based microcapsule production for tissue engineering using a microfluidic flow-focusing system

Gelatin-based microcapsule production using a microfluidic system and the feasibility of the resultant microcapsules for constructing spherical tissues surrounded by heterogeneous cells were studied. The first cell-encapsulation and subsequent cell-enclosing microparticle encapsulation were achieved using a microfluidic flow-focusing droplet production system. A hollow-core structure of about 150 μm in diameter was developed by incubating the resultant microparticles at 37 °C, which induced thermal melting of the enclosed unmodified gelatin microparticles. Mammalian cells filled the hollow-cores after 4 days of incubation. A cell layer on the cell-enclosing microcapsules was developed by simply suspending the microcapsules in medium containing adherent fibroblast cells. This method may prove useful for the generation of gelatin microcapsules using a microfluidic system for formation of artificial tissue constructs.     

Dielectrophoretic microfluidic device for the continuous sorting of Escherichia coli from blood cells

Microfluidic diagnostic devices promise faster disease identification by purifying and concentrating low-abundance analytes from a flowing sample. The diagnosis of sepsis, a whole body inflammatory response often caused by microbial infections of the blood, is a model system for pursuing the advantages of microfluidic devices over traditional diagnostic protocols. Traditional sepsis diagnoses require large blood samples and several days to culture and identify the low concentration microbial agent. During these long delays while culturing, the physician has little or no actionable information to treat this acute illness. We designed a microfluidic chip using dielectrophoresis to sort and concentrate the target microbe from a flowing blood sample. This design was optimized using the applicable electrokinetic and hydrodynamic theories. We quantify the sorting efficiency of this device using growth-based assays which show 30% of injected microbes are recovered viable, consistent with the electroporation of target cells by the dielectrophoretic cell sorters. Finally, the results illustrate the device is capable of a five-fold larger microbe concentration in the target analyte stream compared to the waste stream at a continuous sample flow rate of 35 μl∕h.     

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

BackgroundAn effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated.ResultsRecombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1.ConclusionsInsertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.     

大肠杆菌的mRNA的翻译效率决定于Shine-Dalgarno的局部结构

在大肠杆菌中,多胺对mRNA翻译的影响程度决定于Shine-Dalgarno (SD)序列局部结构。与信使RNA其它区域松散配对的SD序列的局部结构受多胺的扰动较大,其局部解链的概率也较大,因而SD序列结合到核糖体RNA上并启动蛋白质翻译的概率也较大,相反,则启动mRNA翻译的概率就会较小。基于此,建立一个简单数学模型来描述这一现象,即蛋白质的表达水平是与由多胺刺激而引起mRNA的SD序列局部暴露的概率相关的。结果显示,模型与实验结果相符的很好。     

Unique fluorophilic pores engineering within porous aromatic frameworks for trace perfluorooctanoic acid removal

Perfluorooctanoic acid(PFOA),a representative of per/polyfluorinated alkyl substances,has become a persistent water pollutant of widespread concern due to its biological toxicity and refractory property.In this work,we design and synthesize two porous aromatic frameworks(PAF) of PAF-CF₃ and PAF-C₂F₅ using fluorine-containing alkyl based monomers in tetrahedral geometry.Both PAFs exhibit nanosized pores(～1.0 nm) of high surface areas(over 800 m² g^-...     

Vif基因体外表达载体构建及其在无细胞系统中的表达

随着-omics（组学）时代的到来,无细胞蛋白质合成系统以具有快速、方便、易于高通量等优点,正在被广泛地研究和应用。本文选取HIV病毒感染因子Vif作为目标蛋白,构建了适宜体外表达的Vif表达载体pIVEX2.4c-Vif,并将其在大肠杆菌无细胞蛋白质合成系统中进行表达,为下一步进行高通量药物筛选奠定了一定的基础。     

Escherichia coli GutM4 produces 2,5-diketopiperazines and inhibits human pathogens in vitro

BackgroundIt has been a very common practice to use probiotics or their metabolites as alternative antimicrobial strategies for the treatment and prevention of infections as rampant and indiscriminate use of antibiotics causes the development of antibiotic-resistant pathogens. The objective of this study was to select a potential antimicrobial probiotic strain of Escherichia coli from the human gastrointestinal tract and investigate the production of diketopiperazines that contribute to the antimicrobial activity.ResultsE. coli GutM4 was isolated from the feces of a healthy adult. E. coli GutM4 showed significant antagonistic activity against 10 indicator pathogens, and this activity was no less than that of the reference strain E. coli Nissle 1917 against eight of the indicator pathogens. Moreover, E. coli GutM4 produced antagonistic substances containing trypsin-targeted peptide bonds because the inhibitory effects of E. coli GutM4 supernatant significantly decreased upon treatment with trypsin. Consistent with the antagonistic activity and peptide compounds of E. coli GutM4, 14 2,5-diketopiperazines were isolated from the fermented broth of E. coli GutM4, including 12 cyclo(Pro-Phe), 3 cyclo(Pro-Tyr), and 5 cyclo(4-hydroxyl-Pro-Leu), which are reported to have antipathogenic activity.ConclusionE. coli GutM4 produces 2,5-diketopiperazines that are partly involved in antagonistic action against human pathogens in vitro.     

移动通信技术标准化的国家战略与企业战略

在移动通信发展过程中,技术标准的竞争是现代移动通信市场竞争的最重要方面。从移动通信技术标准化的发展历程来看,国家战略与企业战略存在着一定的相互影响和相互作用,其中国家战略发挥着关键的作用。本文通过理论分析和国际经验的比较研究,得出了中国3G技术标准化国家战略的政策性含义。