scholarly journals Metabolomic and proteomic analyses of a quiescent Escherichia coli cell factory reveal the mechanisms behind its production efficiency

2016 ◽  
Author(s):  
Nicholas M. Thomson ◽  
Tomokazu Shirai ◽  
Marco Chiapello ◽  
Akihiko Kondo ◽  
Krishnan J. Mukherjee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Production Efficiency ◽  
Tricarboxylic Acid Cycle ◽  
Expression Patterns ◽  
Cell Factory ◽  
Efficient Production ◽  
Chemical Production ◽  
Cell Biomass ◽  
Wide Range ◽  
Glycolysis Pathway

1AbstractQuiescent (Q-Cell) Escherichia coli cultures can be created by using the signalling molecule indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks. However, the reason for the increased productivity of cells in this state was previously unknown. We show here that Q-cells can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater per-cell productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Metabolomic data show that by disrupting the proton-motive force, indole interrupts the tricarboxylic acid cycle, leading to the accumulation of metabolites in the glycolysis pathway that are excellent starting points for high-value chemical production. By comparing protein expression patterns between wild-type and Q-cell cultures we show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g.L-1 of 3HB compared to 18.6 g.L-1 in the control. Therefore, Q-cells have high potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals.

scholarly journals Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative

2020 ◽  
Vol 8 ◽  
Author(s):  
Zhijian Ni ◽  
Zhongkui Li ◽  
Jinyong Wu ◽  
Yuanfei Ge ◽  
Yingxue Liao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Batch Fermentation ◽  
Infant Nutrition ◽  
High Titer ◽  
Production Performance ◽  
Cell Factory ◽  
Efficient Production ◽  
Healthy Development ◽  
Fucosyltransferase Gene ◽  
Fed Batch Fermentation

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

scholarly journals Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

2020 ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
Chemical Production ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Shock Proteins

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter were powerful tools to mediate the protein expression. Moreover, Escherichia coli W3110 strain possesses more advantages than the B strain due to more heat shock proteins and higher tolerance to chemicals. Therefore, implementation of T7-based system in W3110 strain is a conceivable strategy to develop the cell factory. Results Three novel W3110 strains with chromosome-equipped T7RNAP (i.e W3110:IL5, W3110::L5 and W3110::pI) were engineered to demonstrate the feasibility on protein expression and chemical production. At first, the LacZ and T7RNAP with IPTG induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), Cas9, carbonic anhydrase (CA) and lysine decarboxylase (CadA). All the proteins were expressed higher and enzymatic functions were better in W3110::L5 and W3110::pI. Moreover, the highest cadaverine production, lysine consumption and the yield were obtained in W3110::L5(+) strain with pET28a(+)-CadA which reached 32.2 g/L, 45 g/L and 91.7% at 24 h, while the W3110::pI(-) strain with pSU-T7-CadA achieved 36.9 g/L, 43.8 g/L and 103.4% at 12 h which is unnecessary of inducer. Conclusion Inducer and lacI/lacO regulators on chromosome and plasmid have been investigated in W3110 strains with T7RNAP. The newly engineered W3110::L5 and W3110:pI both possessed similar protein expression compared to commercial BL21(DE3). Furthermore, among all strains, W3110::pI displayed the greatest potential as cell factory in the future.

scholarly journals Novel Mode Engineering for β-Alanine Production in Escherichia coli with the Guide of Adaptive Laboratory Evolution

2021 ◽  
Vol 9 (3) ◽  
pp. 600
Author(s):  
Jian Xu ◽  
Li Zhou ◽  
Meng Yin ◽  
Zhemin Zhou
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Condition ◽  
Energy Production ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Production Efficiency ◽  
Genetic Mutations ◽  
Cell Factory ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
E Coli

The strategy of anaerobic biosynthesis of β-alanine by Escherichia coli (E. coli) has been reported. However, the low energy production under anaerobic condition limited cell growth and then affected the production efficiency of β-alanine. Here, the adaptive laboratory evolution was carried out to improve energy production of E. coli lacking phosphoenolpyruvate carboxylase under anaerobic condition. Five mutants were isolated and analyzed. Sequence analysis showed that most of the consistent genetic mutations among the mutants were related with pyruvate accumulation, indicating that pyruvate accumulation enabled the growth of the lethal parent. It is possible that the accumulated pyruvate provides sufficient precursors for energy generation and CO2 fixing reaction catalyzed by phosphoenolpyruvate carboxykinase. B0016-100BB (B0016-090BB, recE::FRT, mhpF::FRT, ykgF::FRT, mhpB:: mhpB *, mhpD:: mhpD *, rcsA:: rcsA *) was engineered based on the analysis of the genetic mutations among the mutants for the biosynthesis of β-alanine. Along with the recruitment of glycerol as the sole carbon source, 1.07 g/L β-alanine was generated by B0016-200BB (B0016-100BB, aspA::FRT) harboring pET24a-panD-AspDH, which was used for overexpression of two key enzymes in β-alanine fermentation process. Compared with the starting strain, which can hardly generate β-alanine under anaerobic condition, the production efficiency of β-alanine of the engineered cell factory was significantly improved.

scholarly journals Metabolic engineering Escherichia coli for efficient production of icariside D2

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xue Liu ◽  
Lingling Li ◽  
Jincong Liu ◽  
Jianjun Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Carbon Sources ◽  
Gene Expression Pattern ◽  
Fine Tuning ◽  
Cell Factory ◽  
Efficient Production ◽  
Uridine Diphosphate ◽  
Microbial Cell Factory ◽  
E Coli

Abstract Background Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. Results We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. Conclusions We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

scholarly journals Long Non-coding Antisense RNA DDIT4-AS1 Regulates Meningitic Escherichia coli-Induced Neuroinflammation by Promoting DDIT4 mRNA Stability

2022 ◽  
Author(s):  
Bo Yang ◽  
Bojie Xu ◽  
Ruicheng Yang ◽  
Jiyang Fu ◽  
Liang Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inflammatory Responses ◽  
Expression Patterns ◽  
Therapeutic Interventions ◽  
E Coli ◽  
Protein Levels ◽  
Wide Range ◽  
The Stability ◽  
Duplex Formation ◽  
The Brain

AbstractOur previous studies have shown that meningitic Escherichia coli can colonize the brain and cause neuroinflammation. Controlling the balance of inflammatory responses in the host central nervous system is particularly vital. Emerging evidence has shown the important regulatory roles of long non-coding RNAs (lncRNAs) in a wide range of biological and pathological processes. However, whether lncRNAs participate in the regulation of meningitic E. coli-mediated neuroinflammation remains unknown. In the present study, we characterized a cytoplasm-enriched antisense lncRNA DDIT4-AS1, which showed similar concordant expression patterns with its parental mRNA DDIT4 upon E. coli infection. DDIT4-AS1 modulated DDIT4 expression at both mRNA and protein levels. Mechanistically, DDIT4-AS1 promoted the stability of DDIT4 mRNA through RNA duplex formation. DDIT4-AS1 knockdown and DDIT4 knockout both attenuated E. coli-induced NF-κB signaling as well as pro-inflammatory cytokines expression, and DDIT4-AS1 regulated the inflammatory response by targeting DDIT4. In summary, our results show that DDIT4-AS1 promotes E. coli-induced neuroinflammatory responses by enhancing the stability of DDIT4 mRNA through RNA duplex formation, providing potential nucleic acid targets for new therapeutic interventions in the treatment of bacterial meningitis.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

scholarly journals Defects and uncertainties of adhesively bonded composite joints

2021 ◽  
Vol 3 (9) ◽  
Author(s):  
Sadik Omairey ◽  
Nithin Jayasree ◽  
Mihalis Kazilas
Keyword(s):  
Composite Materials ◽  
Composite Structures ◽  
Production Efficiency ◽  
Adhesive Bonding ◽  
Detection Methods ◽  
Bonded Joints ◽  
Adhesively Bonded ◽  
Adhesively Bonded Joints ◽  
Wide Range ◽  
Bonded Composite

AbstractThe increasing use of fibre reinforced polymer composite materials in a wide range of applications increases the use of similar and dissimilar joints. Traditional joining methods such as welding, mechanical fastening and riveting are challenging in composites due to their material properties, heterogeneous nature, and layup configuration. Adhesive bonding allows flexibility in materials selection and offers improved production efficiency from product design and manufacture to final assembly, enabling cost reduction. However, the performance of adhesively bonded composite structures cannot be fully verified by inspection and testing due to the unforeseen nature of defects and manufacturing uncertainties presented in this joining method. These uncertainties can manifest as kissing bonds, porosity and voids in the adhesive. As a result, the use of adhesively bonded joints is often constrained by conservative certification requirements, limiting the potential of composite materials in weight reduction, cost-saving, and performance. There is a need to identify these uncertainties and understand their effect when designing these adhesively bonded joints. This article aims to report and categorise these uncertainties, offering the reader a reliable and inclusive source to conduct further research, such as the development of probabilistic reliability-based design optimisation, sensitivity analysis, defect detection methods and process development.

scholarly journals UV Nanoimprint Lithography: Geometrical Impact on Filling Properties of Nanoscale Patterns

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 822
Author(s):  
Christine Thanner ◽  
Martin Eibelhuber
Keyword(s):  
Layer Thickness ◽  
Process Parameters ◽  
Nanoimprint Lithography ◽  
Failure Modes ◽  
Production Method ◽  
Efficient Production ◽  
Comprehensive Overview ◽  
Replication Method ◽  
Wide Range ◽  
Pattern Size

Ultraviolet (UV) Nanoimprint Lithography (NIL) is a replication method that is well known for its capability to address a wide range of pattern sizes and shapes. It has proven to be an efficient production method for patterning resist layers with features ranging from a few hundred micrometers and down to the nanometer range. Best results can be achieved if the fundamental behavior of the imprint resist and the pattern filling are considered by the equipment and process parameters. In particular, the material properties and pattern size and shape play a crucial role. For capillary force-driven filling behavior it is important to understand the influencing parameters and respective failure modes in order to optimize the processes for reliable full wafer manufacturing. In this work, the nanoimprint results obtained for different pattern geometries are compared with respect to pattern quality and residual layer thickness: The comprehensive overview of the relevant process parameters is helpful for setting up NIL processes for different nanostructures with minimum layer thickness.

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