Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of Saccharomyces cerevisiae

Abstract

Glutathione (GSH) is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. In this study, we demonstrated that engineering in sulfate assimilation metabolism can significantly improve GSH production. The intracellular GSH content of MET14 and MET16 over-expressing strains increased up to 1.2 and 1.4-fold higher than that of the parental strain, respectively, whereas those of APA1 and MET3 overexpressing strains decreased. Especially, in the MET16 overexpressing strain, the volumetric GSH concentration was up to 1.7-fold higher than that of the parental strain as a result of the synergetic effect of the increases in the cell concentration and the intracellular GSH content. Additionally, combinatorial mutant strains that had been engineered to contain both the sulfur and the GSH synthetic metabolism synergistically increased the GSH production. External addition of cysteine to S. cerevisiae is well known as a way to increase the intracellular GSH content; however, it results a decrease in cell growth. This study showed that the engineering of sulfur metabolism in S. cerevisiae proves more valuable than addition of cysteine as a way to boost GSH production due to the increases in both the intracellular GSH content and the cell growth.

Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in Saccharomyces cerevisiae

“…Sample preparation was carried out according to a previous report (Hara et al 2012). GSH and GSSG concentrations were determined using high-performance liquid chromatography (Shimadzu, Kyoto, Japan) equipped with the YMC-Pack ODS-A column (YMC, Kyoto, Japan).…”

“…The control vector pGK406 and the constructed plasmids, pGK406-ERV1, pGK406-ERV2, and pGK406-ERO1, were digested with NcoI or EcoRV and transformed into the S. cerevisiae YPH499 strain or GSH1/GSH2 cocktail δ-integrated YPH499 strain (GCI strain) harboring pGK405. The integrated numbers of GSH1 genes and GSH2 genes in the GCI strain were 7 and 14, respectively (Hara et al 2012). A GLR1 gene encoding glutathione reductase Glr1 was deleted from the S. cerevisiae genomic DNA of the GCI strain by homologous replacement (Kiriyama et al 2013).…”

“…Many studies have been carried out to increase glutathione production using S. cerevisiae (Li et al 2004;Hara et al 2014). Overexpression of GSH1 and GSH2 increased the intracellular glutathione content in S. cerevisiae (Hara et al 2012). From an industrial viewpoint, production of glutathione as the GSSG form is more valuable, particularly for storage after the cellular extraction process, because GSSG has higher stability compared with GSH.…”

Development of bio-based fine chemical production through synthetic bioengineering

“…Overexpression of GCS is critical for enhancing glutathione fermentation [92,93]. Enhancement of cysteine synthesis by engineering of sulfate metabolism also improved glutathione production [93]. In contrast, the overexpression of the transcription factors YAP1 and MET4 increased glutathione production [104][105][106].…”

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

“…Glutathione Overexpression of YAP1 [58] Manipulation of the sulphate assimilation pathway by overexpressing MET14 and MET16 [59] Improved oxidized glutathione production by overexpression of GSH1, GSH2, and ERV1 and the deletion of GLR1 [60] Adaptive laboratory evolution in the presence of increasing levels of acrolein and screening for enhanced glutathione production [61] Whole-genome engineering via genome shuling and screening for enhanced glutathione production [62] Artemisinin/artemisinic acid Reconstruction of the complete biosynthetic pathway of artemisinic acid, including the three-step oxidation of amorphadiene to artemisinic acid by expression of CYP71AV1, CPR1, CYB5, ADH1 and ALDH1 from Artemisia annua [48] Taxol/taxadiene Expression of a truncated version of the endogenous tHMG1 and GGPPS from Taxus chinensis or Sulfolobus acidocaldarius together with TDC1 from T. chinensis [66] Prediction of the eiciency of diferent GGPPS enzymes via computer aided protein modelling [67] Forskolin Expression of a promiscuous cytochrome P450 from Salvia pomifera [68] Polyketides Heterologous expression of 6-MSA synthase gene from Penicillium patulum together with PPTases from either Bacillus subtilis or Aspergillus nidulans [69] Construction of polyketide precursor pathways by expressing prpE from Salmonella typhimurium and PCC pathway from Streptomyces coelicolor [70] Enhanced cofactor supply by expressing 2-PS from Gerbera hybrida [71] Resveratrol Reconstruction of a de novo pathway by expressing TAL from Herpetosiphon aurantiacus, 4-CL1 from Arabidopsis thaliana and VST1 from Vitis vinifera [49] Expression of 4CL1 from A. thaliana and STS from Arachis hypogaea [73] Expression of PAL from Rhodosporidium toruloides, C4H and 4-CL1 from A. thaliana, and STS from A. hypogaea [74] Expression of 4-coumaroyl-coenzyme A ligase (4CL1) from A. thaliana and stilbene synthase (STS) from V. vinifera [75] Overexpression of the resveratrol biosynthesis pathway, enhancement of P450 activity, increasing the precursor supply for resveratrol synthesis via phenylalanine pathway [76] Dihydrochalcones Expression of the heterologous pathway genes in a TSC13-overexpressing S. cerevisiae strain [78] Alkaloids Expression of 14 monoterpene indole alkaloid pathway genes from Catharanthus roseus and enhanced secondary metabolism to produce strictosidine de novo [79] Construction of the complete de novo biosynthetic pathway to norcoclaurine by expressing a mammalian TyrH enzyme and DODC from Pseudomonas putida, along with four genes required for biosynthesis of its electron carrier cosubstrate …”