scholarly journals Efficient Biosynthesis of R-(-)-linalool through Adjusting Expression Strategy and Increasing GPP Supply in Escherichia coli

2019 ◽  
Author(s):  
Xun Wang ◽  
Jing Wu ◽  
Jiaming Chen ◽  
Longjie Xiao ◽  
Xun Li
Keyword(s):  
Shake Flask ◽  
Streptomyces Clavuligerus ◽  
Batch Cultivation ◽  
Market Demand ◽  
Protein Fusion ◽  
Fed Batch ◽  
Geranyl Diphosphate ◽  
E Coli ◽  
Key Enzyme ◽  
Linalool Synthase

Abstract Background: R-(-)-linalool is a versatile acyclic monoterpene alcohol with applications in the flavor and fragrance, pharmaceutical, and agrochemical industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method of R-(-)-linalool synthesis is urgently needed. Results: To induce the R-(-)-linalool biosynthesis pathway in E. coli , we expressed heterologous (3R)-linalool synthase (LIS) from Lavandula angustifolia (laLIS). We then enhanced R-(-)-linalool production in the cells using a suitable LIS from Streptomyces clavuligerus (bLIS). The bLIS expression was markedly elevated by using optimized ribosomal binding sites (RBSs) and protein fusion tags. R-(-)-linalool output rose from 4.8 mg L -1 to 33.4 mg L -1 . To increase the geranyl diphosphate (GPP) content in E. coli , we tested various alterations in geranyl diphosphate synthases (GPPSs) and their mutants. The final E. coli strain harboring GPPS from Abies grandis ( Ag GPPS) accumulated ≤ 100.1 mg L -1 R-(-)-linalool after 72 h shake-flask fermentation. This yield gain constitutes a 60.7-fold improvement in R-(-)-linalool biosynthesis over the parent strain. Fed-batch cultivation of the engineered strain in a 1.3-L fermenter yielded 1,027.3 mg L -1 R-(-)-linalool. Conclusions: In this study, an efficient R-(-)-linalool production pathway was induced in E. coli via the heterologous MVA pathway, AgGPPS, and (3R)-linalool synthase (bLIS). By overexpressing the key enzyme in the engineered E. coli strain, R-(-)-linalool production reached 100.1 mg L -1 and 1,027.3 mg L -1 under shake flask- and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date using an engineered E. coli strain. The strategies of key enzyme overexpression and mutation could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.

Identification and Optimization of Recombinant E. coli Fed-Batch Fermentation Producing γ-Interferon Protein

2013 ◽  
Vol 11 (1) ◽  
pp. 123-134
Author(s):  
Mahdi Feyzdar ◽  
Ahmad Reza Vali ◽  
Valiollah Babaeipour
Keyword(s):  
Neural Network ◽  
Batch Cultivation ◽  
Model Complexity ◽  
Fed Batch ◽  
E Coli ◽  
Novel Approach ◽  
Fed Batch Fermentation ◽  
Optimal Brain Surgeon ◽  
Fed Batch Cultivation ◽  
Γ Interferon

Abstract A novel approach to identification of fed-batch cultivation of E. coli BL21 (DE3) has been presented. The process has been identified in the system that is designed for maximum production of γ-interferon protein. Dynamic order of the process has been determined by Lipschitz test. Multilayer Perceptron neural network has been used to process identification by experimental data. The optimal brain surgeon method is used to reduce the model complexity that can be easily implemented. Validation results base on autocorrelation function of the residuals, show good performance of neural network and make it possible to use of it in process analyses.

scholarly journals Fed-Batch Cultivation and Adding Supplements to Increase Yield of β-1,3-1,4-Glucanase by Genetically Engineered Escherichia coli

Catalysts ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 269
Author(s):  
Lijuan Zhong ◽  
Zheng Liu ◽  
Yinghua Lu
Keyword(s):  
Escherichia Coli ◽  
Feeding Strategy ◽  
Batch Cultivation ◽  
Genetically Engineered ◽  
Fed Batch ◽  
Glucanase Activity ◽  
E Coli ◽  
Constant Rate ◽  
Engineered Escherichia Coli ◽  
Fed Batch Cultivation

The aim of this study was to analyze the major influence factors of culture medium on the expression level of β-1,3-1,4-glucanase, and to further develop an optimized process for the extracellular production of β-glucanase at a bioreactor scale (7 L) with a genetically engineered Escherichia coli (E. coli) JM109-pLF3. In this study, batch cultivation and fed-batch cultivation including the constant rate feeding strategy and the DO-stat (DO: Dissolved Oxygen) feeding strategy were conducted. At a 7 L bioreactor scale for batch cultivation, biomass reached 3.14 g/L and the maximum β-glucanase activity was 506.94 U/mL. Compared with batch cultivation, the addition of glycerol, complex nitrogen and complete medium during fed-batch cultivation increased the production of biomass and β-1,3-1,4-glucanase. The maximum biomass and β-glucanase activity, which were 7.67 g/L and 1680 U/mL, respectively, that is, 2.45 and 3.31 times higher than those obtained with batch cultivation, were obtained by feeding a complex nitrogen source at a constant rate of 1.11 mL/min. Therefore, these nutritional supplements and strategies can be used as a reference to enhance the production of other bioproducts from E. coli.

scholarly journals Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
P. Philip ◽  
D. Kern ◽  
J. Goldmanns ◽  
F. Seiler ◽  
A. Schulte ◽  
...  
Keyword(s):  
Shake Flask ◽  
Fed Batch ◽  
E Coli ◽  
Substrate Supply ◽  
Optimal Screening

scholarly journals Improved Mannanase Production from Penicillium occitanis by Fed-Batch Fermentation Using Acacia Seeds

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Monia Blibech ◽  
Raoudha Ellouz Ghorbel ◽  
Fatma Chaari ◽  
Ilyes Dammak ◽  
Fatma Bhiri ◽  
...  
Keyword(s):  
Batch Fermentation ◽  
Shake Flask ◽  
Fold Increase ◽  
Batch Cultivation ◽  
Fed Batch ◽  
Fed Batch Fermentation ◽  
Culture Process ◽  
Coconut Meal ◽  
Penicillium Occitanis ◽  
Shake Flask Fermentation

By applying a fed-batch strategy, production of Penicillium occitanis mannanases could be almost doubled as compared to a batch cultivation on acacia seeds (76 versus 41 U/mL). Also, a 10-fold increase of enzyme activities was observed from shake flask fermentation to the fed-batch fermentation. These production levels were 3-fold higher than those obtained on coconut meal. The high mannanase production using acacia seeds powder as inducer substrate showed the suitability of this culture process for industrial-scale development.

Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

2019 ◽  
Vol 20 (6) ◽  
pp. 497-505 ◽  
Author(s):  
Abeer M. Abd El-Aziz ◽  
Mohamed A. Shaker ◽  
Mona I. Shaaban
Keyword(s):  
Gold Nanoparticles ◽  
Lipolytic Activity ◽  
Market Demand ◽  
Biotechnological Applications ◽  
Lipase Gene ◽  
Expression Plasmid ◽  
E Coli ◽  
Nickel Affinity Chromatography ◽  
Recombinant Production ◽  
Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

scholarly journals Dynamic consolidated bioprocessing for innovative lab-scale production of bacterial alkaline phosphatase from Bacillus paralicheniformis strain APSO

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Nadia A. Soliman ◽  
Gaber A. Abo-Zaid ◽  
Yasser R. Abdel-Fattah
Keyword(s):  
Alkaline Phosphatase ◽  
Consolidated Bioprocessing ◽  
Black Liquor ◽  
Batch Cultivation ◽  
Efficient Production ◽  
Scale Production ◽  
Market Demand ◽  
Stirred Tank Bioreactor ◽  
Bacterial Alkaline Phosphatase ◽  
Bacillus Paralicheniformis

AbstractTo meet the present and forecasted market demand, bacterial alkaline phosphatase (ALP) production must be increased through innovative and efficient production strategies. Using sugarcane molasses and biogenic apatite as low-cost and easily available raw materials, this work demonstrates the scalability of ALP production from a newfound Bacillus paralicheniformis strain APSO isolated from a black liquor sample. Mathematical experimental designs including sequential Plackett–Burman followed by rotatable central composite designs were employed to select and optimize the concentrations of the statistically significant media components, which were determined to be molasses, (NH4)2NO3, and KCl. Batch cultivation in a 7-L stirred-tank bioreactor under uncontrolled pH conditions using the optimized medium resulted in a significant increase in both the volumetric and specific productivities of ALP; the alkaline phosphatase throughput 6650.9 U L−1, and µ = 0.0943 h−1; respectively, were obtained after 8 h that, ameliorated more than 20.96, 70.12 and 94 folds compared to basal media, PBD, and RCCD; respectively. However, neither the increased cell growth nor enhanced productivity of ALP was present under the pH-controlled batch cultivation. Overall, this work presents novel strategies for the statistical optimization and scaling up of bacterial ALP production using biogenic apatite.

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