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

Abstract

Background: L-glutathione (GSH) is a non-protein thiol compound with important biological properties and is widely used in pharmaceutical, food, cosmetic and health products. The cellular GSH is determined by the activity and characteristic of GSH-synthesizing enzymes, energy and precursor supply, and degradation of formed GSH.

Results: In this study, genes encoding enzymes related to the precursor amino acid degradation and glycogen formation as well as GSH degradation were systematically manipulated in Escherichia coli strains over-expressing gshF from Actinobacillus succinogenes. The manipulation included disrupting the precursor degradation pathways (tnaA and sdaA), eliminating L-glutathione degradation (ggt and pepT), and manipulating the intracellular ATP level (disruption of glgB). However the constructed mutants showed lower levels of GshF expression. 2-D electrophoresis was performed to elucidate the reasons for this discrepancy, and the results indicated obvious changes in central metabolism and amino acid metabolism in the penta-mutant. Fed-batch culture of the penta-mutant ZJ12345 was performed where the GshF expression level was enhanced, and both the GSH production (19.10 mM) and the yield based on added L-cysteine (0.76 mmol/mmol) were significantly increased.

Conclusion: By interrupting the degradation pathways of L-cysteine, serine and GSH and blocking glycogen formation, the GSH production efficiency was significantly improved.

Fig. 1

Metabolic pathways of Escherichia coli for GSH production, including the pathways related to central carbon metabolism, precursor amino acid production, and GSH degradation. glgB glycogen-branching enzyme, serA D-3-phosphoglycerate dehydrogenase, cysE serine acyltransferase, sdaA l-serine deaminase, tnaA cysteine desulfhydrase, gdh glutamate dehydrogenase, ggt γ-glutamyltranspeptidase, pept tripeptidase, gshF glutathione synthetase

Fig. 2

The residual l-cysteine concentrations in the mutant and wild type strains after 2 h of incubation

Fig. 3

The residual GSH concentrations in the mutants and wild type strains after 2 h of incubation

Fig. 4

Effect of the glgB knockout on the concentration of intracellular ATP in E. coli during cultivation

Fig. 5

Expression of the GSH synthase GshF in different hosts with different genetic backgrounds. The concentration of IPTG was 0.5 mM and the induction time was 4 h. 1, ZJ012; 2, ZJ123; 3, ZJ1234; 4, ZJ12345, 5, ZJ000

Fig. 6

The production of GSH by whole-cell biocatalyst in the engineered strains using ZJ000 as the control. a The concentration of GSH during the process; b the highest yield of GSH

Fig. 7

Profiles of fed-batch fermentation by E. coli strains ZJ000 (a), ZJ1234 (b) and ZJ12345 (c). (filled square) DCW; (filled triangle) GSH; (filled star) Glucose; (filled circle) Acetic acid