scholarly journals ATP and NADPH engineering of Escherichia coli to improve the production of 4-hydroxyphenylacetic acid using CRISPRi

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yu-Ping Shen ◽  
Yu-Ling Liao ◽  
Qian Lu ◽  
Xin He ◽  
Zhi-Bo Yan ◽  
...  
Keyword(s):  
Raw Material ◽  
E Coli ◽  
Hydroxyphenylacetic Acid ◽  
Short Palindromic Repeat ◽  
Final Strain ◽  
Encoding Gene ◽  
Novel Strategy ◽  
High Level ◽  
Encoding Genes ◽  
Level Production

Abstract Background 4-Hydroxyphenylacetic acid (4HPAA) is an important raw material for the synthesis of drugs, pesticides and biochemicals. Microbial biotechnology would be an attractive approach for 4HPAA production, and cofactors play an important role in biosynthesis. Results We developed a novel strategy called cofactor engineering based on clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening (CECRiS) for improving NADPH and/or ATP availability, enhancing the production of 4HPAA. All NADPH-consuming and ATP-consuming enzyme-encoding genes of E. coli were repressed through CRISPRi. After CRISPRi screening, 6 NADPH-consuming and 19 ATP-consuming enzyme-encoding genes were identified. The deletion of the NADPH-consuming enzyme-encoding gene yahK and the ATP-consuming enzyme-encoding gene fecE increased the production of 4HPAA from 6.32 to 7.76 g/L. Automatically downregulating the expression of the pabA gene using the Esa-PesaS quorum-sensing-repressing system further improved the production of 4HPAA. The final strain E. coli 4HPAA-∆yfp produced 28.57 g/L of 4HPAA with a yield of 27.64% (mol/mol) in 2-L bioreactor fed-batch fermentations. The titer and yield are the highest values to date. Conclusion This CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.

Biosensor-assisted evolution for high-level production of 4-hydroxyphenylacetic acid in Escherichia coli

2021 ◽  
Author(s):  
Yu-Ping Shen ◽  
Yuyang Pan ◽  
Fu-Xing Niu ◽  
Yu-Ling Liao ◽  
Mingtao Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydroxyphenylacetic Acid ◽  
High Level ◽  
Level Production

scholarly journals High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG

2015 ◽  
Vol 14 (1) ◽  
Author(s):  
Zhe Zhang ◽  
Grietje Kuipers ◽  
Łukasz Niemiec ◽  
Thomas Baumgarten ◽  
Dirk Jan Slotboom ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
E Coli ◽  
High Level ◽  
Level Production

High-level production of soluble adenine nucleotide translocator from Schistosoma japonicum in E. coli cell-free system

2012 ◽  
Vol 47 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Zhong Yang ◽  
Rongrong Ma ◽  
Lei Huang ◽  
Xiangcheng Zhu ◽  
Jiayuan Sheng ◽  
...  
Keyword(s):  
Schistosoma Japonicum ◽  
Adenine Nucleotide ◽  
Coli Cell ◽  
Adenine Nucleotide Translocator ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
High Level ◽  
Level Production

Expression, Enzymatic Characterization, and High-Level Production of Glucose Isomerase from Actinoplanes missouriensis CICIM B0118(A) in Escherichia coli

2011 ◽  
Vol 66 (11-12) ◽  
pp. 605-613 ◽  
Author(s):  
He Wang ◽  
Ruijin Yang ◽  
Xiao Hua ◽  
Zhong Zhang ◽  
Wei Zhao ◽  
...  
Keyword(s):  
Ph Value ◽  
Glucose Isomerase ◽  
Medium Composition ◽  
Gene Encoding ◽  
E Coli ◽  
Optimum Ph ◽  
One Step ◽  
Ph Range ◽  
High Level ◽  
Level Production

High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0 - 9.0. Its optimum temperature was around 85 °C, and it exhibited good thermostability when the temperature was lower than 90 °C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 μM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool

scholarly journals Constitutive High Level Expression of an Endoxylanase Gene from the Newly IsolatedBacillus subtilisAQ1 inEscherichia coli

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Is Helianti ◽  
Niknik Nurhayati ◽  
Maria Ulfah ◽  
Budiasih Wahyuntari ◽  
Siswa Setyahadi
Keyword(s):  
Specific Activity ◽  
Biochemical Properties ◽  
Extracellular Expression ◽  
High Level Expression ◽  
Periplasmic Fraction ◽  
16S Rdna Sequence ◽  
E Coli ◽  
Encoding Gene ◽  
High Level ◽  
Very High

A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted asBacillus subtilis(B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed inE. coli. InE. colithe recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely,2173.6 ± 51.4and2745.3 ± 11 U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the nativeB. subtilisAQ1 endoxylanase and that of 95% homologous recombinant one fromB. subtilisDB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression inE. coliand hence made the production inE. colifeasible.

scholarly journals IS1R-Mediated Plasticity of IncL/M Plasmids Leads to the Insertion ofblaOXA-48into the Escherichia coli Chromosome

2014 ◽  
Vol 58 (7) ◽  
pp. 3785-3790 ◽  
Author(s):  
R. Beyrouthy ◽  
F. Robin ◽  
J. Delmas ◽  
L. Gibold ◽  
G. Dalmasso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Insertion Sequences ◽  
Content Type ◽  
Genetic Environment ◽  
E Coli ◽  
The Family ◽  
Insertion Sites ◽  
Low Levels ◽  
Encoding Gene ◽  
Encoding Genes

ABSTRACTThe OXA-48 carbapenemase is mainly encoded by ∼62-kb IncL/M plasmids. However, chromosome-mediated genes have been observed inEscherichia coliisolates. In this work, we investigated the genetic environment of OXA-48 in members of the familyEnterobacteriaceae(n= 22) to understand how the OXA-48-encoding gene is transferred into theE. colichromosome. The OXA-48-encoding gene was located within intact Tn1999.2transposons in the ∼62-kb plasmids or within a truncated variant of Tn1999.2for the OXA-48-encoding genes located in the chromosomes ofE. colibacteria. The analysis of the Tn1999.2genetic environment revealed an inverted orientation of the transposon in five ∼62-kb plasmids (5/14 [35%]) and in all chromosome inserts (n= 8). The sequencing of pRA35 plasmid showed that this orientation of Tn1999.2and the acquisition of an IS1Rinsertion sequence generated a 21.9-kb IS1R-based composite transposon encoding OXA-48 and designated Tn6237. The sequencing of a chromosomal insert encoding OXA-48 also revealed this new transposon in theE. colichromosome. PCR mapping showed the presence of this element in all strains harboring an OXA-48-encoding chromosomal insert. However, different insertion sites of this transposon were observed in theE. colichromosome. Overall, these findings indicate a plasticity of the OXA-48 genetic environment mediated by IS1Rinsertion sequences. The insertion sequences can induce the transfer of the OXA-encoding gene intoE. colichromosomes and thereby promote its persistence and expression at low levels.

High level production of bovine angiogenin in E. coli by an efficient refolding procedure

2004 ◽  
Vol 26 (19) ◽  
pp. 1501-1504 ◽  
Author(s):  
Seung-Hwan Jang ◽  
Dong-Ku Kang ◽  
Soo-Ik Chang ◽  
Harold A. Scheraga ◽  
Hang-Cheol Shin
Keyword(s):  
E Coli ◽  
High Level ◽  
Level Production

scholarly journals Resistance to aztreonam, in combination with a bicyclic boronate β-lactamase inhibitor in Escherichia coli identified following mixed culture selection

2019 ◽  
Author(s):  
Ching Hei Phoebe Cheung ◽  
Jacqueline Findlay ◽  
Kate J. Heesom ◽  
Matthew B. Avison
Keyword(s):  
Escherichia Coli ◽  
Mixed Culture ◽  
Clinical Analysis ◽  
Bacterial Populations ◽  
E Coli ◽  
High Level ◽  
Inhibitor Resistance ◽  
Level Production ◽  
Inhibitor Dose ◽  
Lactamase Inhibitor

AbstractBackgroundBicyclic boronates are a new and potentially important class of β-lactamase inhibitor, with the ability to inhibit β-lactamases from all molecular classes, including mobile metallo-β-lactamases.ObjectiveOur objective was to identify mutants resistant to the actions of the bicyclic boronate inhibitor 2, when being used in combination with aztreonam.MethodsOvernight cultures were plated on to agar containing increasing concentrations of aztreonam with a fixed 10 mg/L concentration of the inhibitor. Resistant derivatives and parent strains were analysed by whole genome sequencing and LC-MS/MS proteomics to identify mechanism of resistance.ResultsWhen using a mixed overnight culture containing one Escherichia coli (TEM-1, CTX-M-15, CMY-4 producer) and one Klebsiella pneumoniae (SHV-12, CTX-M-15, NDM-1 producer) mobilisation of an IncX3 plasmid carrying blaSHV-12 from the K. pneumoniae into the E. coli generated an aztreonam/boronate resistant derivative.ConclusionsHigh-level production of three bicyclic boronate susceptible enzymes (CMY-4, CTX-M-15, SHV-12) capable of hydrolysing aztreonam plus TEM-1, which binds the inhibitor, overcomes the fixed inhibitor dose used. This was only identified when using a mixed culture for selection. It would seem prudent that to allow for coalescence of the myriad β-lactamase genes commonly found in bacterial populations colonising humans, this mixed culture approach should be the norm when testing the potential for generating β-lactamase inhibitor resistance in pre-clinical analysis.

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