Systematic manipulation of glutathione metabolism in Escherichia coli for improved glutathione production

Zhang, Jing; Quan, Cong; Wang, Cheng; Wu, Hui; Li, Zhimin; Ye, Qin

doi:10.1186/s12934-016-0439-1

Cited by 30 publications

(25 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With the aim of increasing the production of OA, we sought to identify and disrupt the enzymes responsible for OA degradation in E. coli. Previous studies have demonstrated that both GGT and tripeptidase (PepT) play important roles in GSH degradation and that GGT contributes to GSH degradation to a greater extent than PepT in E. coli (20,21).…”

Section: Resultsmentioning

confidence: 99%

Production of Ophthalmic Acid Using Engineered Escherichia coli

Ito

Tokoro

Hori

et al. 2018

Appl Environ Microbiol

View full text Add to dashboard Cite

Ophthalmic acid (OA; l-γ-glutamyl-l-2-aminobutyryl-glycine) is an analog of glutathione (GSH; l-γ-glutamyl-l-cysteinyl-glycine) in which the cysteine moiety is replaced by l-2-aminobutyrate. OA is a useful peptide for the pharmaceutical and/or food industries. Herein, we report a method for the production of OA using engineered cells.-deficient , which lacks the highly conserved pyridoxal 5'-phosphate-binding protein YggS and naturally accumulates OA, was selected as the starting strain. To increase the production of OA, we overexpressed the OA biosynthetic enzymes glutamate-cysteine ligase (GshA) and glutathione synthase (GshB), desensitized the product inhibition of GshA, and eliminated the OA catabolic enzyme γ-glutamyltranspeptidase. The production of OA was further enhanced by the deletion of and with the aim of increasing the availability of ATP and attenuating the unwanted degradation of amino acids, respectively. The final strain developed in this study successfully produced 277 μmol/liter of OA in 24 h without the formation of by-products in a minimal synthetic medium containing 1 mM each glutamate, 2-aminobutyrate, and glycine. Ophthalmic acid (OA) is a peptide that has the potential for use in the pharmaceutical and/or food industries. An efficient method for the production of OA would allow us to expand our knowledge about its physiological functions and enable the industrial/pharmaceutical application of this compound. We demonstrated the production of OA using cells in which OA biosynthetic enzymes and degradation enymes were engineered. We also showed that unique approaches, including the use of a Δ mutant as a starting strain, the establishment of an S495F mutation in GshA, and the deletion of or, facilitated the efficient production of OA in .

show abstract

Section: Resultsmentioning

confidence: 99%

Production of Ophthalmic Acid Using Engineered Escherichia coli

Ito

Tokoro

Hori

et al. 2018

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…In many cases, the optimization of the fermentation process to enhance the ATP level is an option [ 11 ]. ATP accumulation could also be elevated by gene knock-out strategies or small RNA regulation [ 13 , 23 , 24 ]. As CRISPRi system is advantageous over traditional gene-knockout strategies because of its ability to manipulate genes essential for growth [ 14 ], the development of CRISPRi system has offered an alternative approach to control the intracellular ATP level.…”

Section: Discussionmentioning

confidence: 99%

Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production

et al. 2018

View full text Add to dashboard Cite

BackgroundMicrobial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural flavonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modification of ATP concentration in Escherichia coli was applied in efficient pinocembrin production.ResultsIn the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efficiency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a final pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains.ConclusionsOur results introduce a strategy to approach the efficient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condition have been optimized for pinocembrin production. The results and engineering strategies demonstrated here would hold promise for the ATP level improvement of other flavonoids by CRISPRi system, thereby facilitating other flavonoids production.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0995-7) contains supplementary material, which is available to authorized users.

show abstract

“…A high titer of 10 mM GSH was achieved. Second, serious degradation of L-cysteine and GSH existing in whole-cell catalysis (Zhang et al, 2016) could be overcome. Although the cells overexpressing PPK were used, the heat-treatment could deactivate enzymes responsible for these degradation pathways and decrease the side reactions.…”

Section: One-pot Gsh Synthesis With Atp Regeneration From Polypmentioning

confidence: 99%

“…However, despite of using complicated process controls, the improvement of the conversion of L-cysteine was limited and the economy of system was generally less concerned. While considering these factors, a high yield of 76 % based on added L-cysteine was achieved by manipulating pathways of L-cysteine degradation, energy supply and GSH degradation, but the GSH titer was only 19.1 mM in a process lasted 24 h (Zhang et al, 2016). When GSH synthesis was performed by using whole-cell catalysis in S. cerevisiae, intracellular ATP generation system was generally used.…”

Section: Gsh Synthesis With the Fed-batch Modementioning

confidence: 99%

One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system

Xing

Huang

et al. 2017

Journal of Biotechnology

Self Cite

View full text Add to dashboard Cite

In vitro cascade catalysis using enzyme-based system is becoming a promising biomanufacturing platform for biofuels and biochemicals production. Glutathione is a pivotal non-protein thiol compound and widely applied in food and pharmaceutical industries. In this study, glutathione was synthesized by a bifunctional glutathione synthetase together with a thermophilic ATP regeneration system through a two-enzyme cascade in vitro. Four bifunctional glutathione synthetases from Streptococcus sanguinis, S. gordonii, S. uberis and Bacillus cereus were applied for glutathione synthesis. The bifunctional glutathione synthetase from S. sanguinis was selected and coupled with the polyphosphate kinase from Thermosynechococcus elongatus BP-1 for regenerating ATP to produce glutathione in one pot. In the optimized system, 28.5mM glutathione was produced within 5h due to efficient ATP regeneration from low-cost polyphosphate. The yield based on added l-cysteine reached 81.4% and the productivity of glutathione achieved 5.7mM/h. The one-pot system indicated a potential biotransformation platform for industrial production of glutathione.

show abstract

Systematic manipulation of glutathione metabolism in Escherichia coli for improved glutathione production

Cited by 30 publications

References 43 publications

Production of Ophthalmic Acid Using Engineered Escherichia coli

Production of Ophthalmic Acid Using Engineered Escherichia coli

Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production

One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system

Contact Info

Product

Resources

About