Three-pathway combination for glutathione biosynthesis in Saccharomyces cerevisiae
- Liang Tang†1,
- Weiwei Wang†3,
- Wenlong Zhou2,
- Kai Cheng3,
- Yan Yang1,
- Minzhi Liu2,
- Kedi Cheng2 and
- Wei Wang1, 2Email author
© Tang et al. 2015
Received: 28 March 2015
Accepted: 26 August 2015
Published: 16 September 2015
Glutathione (GSH), a pivotal non-protein thiol, can be biosynthesized through three pathways in different organisms: (1) two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (Gsh1 or GshA) and glutathione synthetase (Gsh2 or GshB); (2) a bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (GshF); (3) an alternative condensation of γ-glutamyl phosphate synthesized by γ-glutamyl kinase (Pro1 or ProB) with cysteine to form γ-glutamylcysteine which was further conjugated to glycine by glutathione synthetase. The Gsh1 and Gsh2 of conventional GSH biosynthetic pathway or the bifunctional GshF reported previously have been independently modulated for GSH production. This study developed a novel three-pathway combination method to improve GSH production in Saccharomyces cerevisiae.
A bifunctional enzyme GshF of Actinobacillus pleuropneumoniae was functionally expressed in S. cerevisiae and Pro1 in proline biosynthetic pathway was exploited for improving GSH yield. Moreover, two fusion proteins Gsh2-Gsh1 and Pro1-GshB were constructed to increase the two-step coupling efficiency of GSH synthesis by mimicking the native domain fusion of GshF. The engineered strain W303-1b/FGP with three biosynthetic pathways presented the highest GSH concentration (216.50 mg/L) and GSH production of W303-1b/FGP was further improved by 61.37 % when amino acid precursors (5 mM glutamic acid, 5 mM cysteine and 5 mM glycine) were fed in shake flask cultures. In batch culture process, the recombinant strain W303-1b/FGP also kept high efficiency in GSH production and reached an intracellular GSH content of 2.27 % after 24-h fermentation.
The engineered strains harbouring three GSH pathways displayed higher GSH producing capacity than those with individually modulated pathways. Three-pathway combinatorial biosynthesis of GSH promises more effective industrial production of GSH using S. cerevisiae.
KeywordsGlutathione Three-pathway combination Combinatorial biosynthesis Saccharomyces cerevisiae
Glutathione (γ-l-glutamyl-l-cysteinylglycine, GSH) is an intracellular redox-active tripeptide thiol found in living organisms [1, 2]. The free sulfhydryl moiety of cysteine residue contributes to a wide variety of biological activities, such as anti-oxidization , detoxification [4, 5] and immune regulation , etc. GSH has been widely used in pharmaceutical, food and cosmetic industries.
An overview of literature on the production of GSH without addition of amino acid precursors by microorganisms
GSH content in mutant
GSH content in wild type
pGSR2518-x containing gshA of E. coli B fused with a S. cerevisiae promoter fragment P8 was used to transform S. cerevisiae YNN27
A recombinant plasmid pGMF with GSH1 from S. cerevisiae was introduced into S. cerevisiae YSF-31
Plasmids, pδAUR-GCS and pδAUR-GS, containing GSH1 and GSH2 from S. cerevisiae were linearized and integrated at δ-sites with high copy numbers into the ribosomal DNA of S. cerevisiae YPH499
Sulfate assimilation metabolism and GSH synthetic metabolism were combinatorially engineered in S. cerevisiae YPH499
gshF derived from Streptococcus thermophilus was integrated at a high copy number into the ribosomal DNA of S. cerevisiae BY4741
54.9 μM/g DCW (1.69 %)
11.7 μM/g DCW (0.36 %)
An integrative expression vector, pGAPZHGSH, containing GSH1 and GSH2 from S. cerevisiae regulated by GAP promoter was transformed into P. pastoris GS115
0.92 g (GSH)/L
94.98 g (DCW)/L (0.97 %)
An integrative expression vector, pGAPZHGSH, containing GSH1 and GSH2 from S. cerevisiae regulated by GAP promoter was transformed into P. pastoris GS115
<0.05 mM/g DCW (<1.54 %)
<0.02 mM/g DCW (<0.61 %)
An integrative expression vector, pGAPZH-Lmgsh, containing gshF from Listeria monocytogenes regulated by GAP promoter was transformed into P. pastoris GS115
<0.04 mM/g DCW (<1.23 %)
Although genetic manipulations of a single pathway have increased GSH production of engineered strains, discovering how to further improve GSH production is still a real challenge for us. In this study, we focused on direct modulation of the GSH biosynthetic pathway via a method of three-pathway combination. Two artificially recombinant enzymes glutathione synthetase-linker-γ-glutamylcysteine synthetase fusion protein (Gsh2-Gsh1) from S. cerevisiae, γ-glutamyl kinase-linker-glutathione synthetase hybrid (Pro1-GshB) from S. cerevisiae and E. coli and a resynthesized GshF of Actinobacillus pleuropneumoniae were introduced into S. cerevisiae genome via independent or combinatorial genetic integration. The overexpression of genes from different pathways and synthetic capacities of GSH of different engineered strains were investigated. The data demonstrated that three-pathway combination was superior to single-pathway for synthesis of GSH.
Activity analysis of GshF from A. pleuropneumoniae expressed under the control of different promoters in S. cerevisiae
Strains and plasmids used in this study
Strains or plasmids
Source or references
E. coli Trans1-T1
F− φ80(lacZ) ΔM15 ΔlacX74 hsdR(rK− mK+) ΔrecA1398 endA1 tonA
S. cerevisiae W303-1b
MATα ade2-1 leu2-3,112 his3-11,15 ura3-1 trp1-1
W303-1b derivative with pΔgsh1h, GSH1::HygB
W303-1b derivative with pΔgsh1h-pro1, GSH1::P gap -PRO1-T pgk1
W303-1b derivative with pΔgsh1h-proB, GSH1::P gap -proB-T pgk1
W303-1b derivative with pδGAPg-gshF
W303-1b derivative with pδGAP′g-gshF
W303-1b derivative with pδPGK1g-gshF
W303-1b derivative with pδGAL1g-gshF
W303-1b/F derivative with pΔgsh1h, GSH1::HygB
W303-1b derivative with pδGAPh-gsh2gsh1
W303-1b derivative with pδGAPg-pro1gshB
W303-1b derivative with pδGAPg-proBgshB
W303-1b/F derivative with pδGAPg-gshF
W303-1b/F derivative with pδGAPh-gsh2gsh1
W303-1b/F derivative with pδGAPg-pro1gshB
W303-1b/FG derivative with pδGAPg-pro1gshB
pBluescript II KS(+) derivative with homologous region of GSH1
pBluescript II KS(+) derivative with homologous region of GSH1, HygBr
pG derivative with P gap , PRO1, T pgk1 , HygBr
pG derivative with P gap , proB, T pgk1 , HygBr
pBluescript II KS(+) derivative with homologous δ region, P gap , T pgk1 , G418r
pBluescript II KS(+) derivative with homologous δ region, P gap′, T pgk1 , G418r
pBluescript II KS(+) derivative with homologous δ region, P pgk1 , T pgk1 , G418r
pBluescript II KS(+) derivative with homologous δ region, P gal1 , T pgk1 , G418r
pBluescript II KS(+) derivative with homologous δ region, P gap , T pgk1 , HygBr
pδGAPg derivative with A. pleuropneumoniae gshF, G418r
pδGAP′g derivative with A. pleuropneumoniae gshF, G418r
pδPGK1 g derivative with A. pleuropneumoniae gshF, G418r
pδGAL1 g derivative with A. pleuropneumoniae gshF, G418r
pδGAPh derivative with GSH2-GSH1 from S. cerevisiae, HygBr
pδGAPg derivative with PRO1-gshB from S. cerevisiae and E. coli, respectively, G418r
pδGAPg derivative with proB-gshB from E. coli, G418r
Active GK for GSH biosynthesis in S. cerevisiae W303-1b
In the cells lacking Gsh1-dependent biosynthetic pathway of GSH, it was found that γ-glutamyl phosphate is a common precursor shared by proline and GSH biosynthetic pathways. Thus, biosynthesis of proline could be partially diverted toward GSH production via γ-glutamyl phosphate (Fig. 1) and GK may play a central role in the alternative pathway.
Overexpression of enzymes involved in GSH biosynthesis
In earlier studies, Gsh1 and Gsh2 (GshA and GshB) derived from yeast or E. coli were independently applied to various model microorganisms to generate genetically engineered strains with higher GSH productive capacities [15–19]. GshF from bacteria was also expressed in E. coli and yeast [20–22]. Here, A. pleuropneumoniae GshF was successfully expressed in S. cerevisiae as mentioned above. However, the recombinant constructs of Gsh2-Gsh1 and Pro1-GshB with a flexible linker have not been exploited for improving intracellular GSH synthesis until now.
To evaluate the potential of different pathways geared toward improving GSH synthesis, several δ DNA-mediated integrative expression vectors pδGAPh-gsh2gsh1, pδGAPg-pro1gshB or pδGAPg-proBgshB harbouring different pathway genes were constructed. Recombinant S. cerevisiae strains W303-1b/G, W303-1b/P or W303-1b/P′ (Table 2) were then generated by transforming different linearized plasmids into S. cerevisiae W303-1b as constructed W303-1b/F. The pathway genes with strong constitutive GAP promoters were introduced into the genome of W303-1b via homologous integration at δ-sites. To further compare GSH synthetic capacities of different pathways, GSH1 gene of engineered strain W303-1b/F was deleted to eliminate synthesis of GSH by endogenous enzymes. The resulting strain W303-1b/Δgsh1/F (F pathway) together with W303-1b/G (G pathway) and W303-1b/Δgsh1/pro1 (P pathway) represent different pathway strains (Table 2). No dramatic differences in biomass between W303-1b/G, W303-1b/F, W303-1b/Δgsh1/F, W303-1b/P or W303-1b/P′ and original strain W303-1b were observed during shake flask cultures (Fig. 4). Three engineered strains W303-1b/G, W303-1b/F and W303-1b/P displayed higher activities for synthesizing GSH than the parental strain. The highest intracellular concentration of GSH was 186.77 mg/L produced by strain W303-1b/F, and most of the synthesized GSH could be attributed to the function of the introduced GshF as strain W303-1b/Δgsh1/F yielded a similar amount (about 90 %) GSH to W303-1b/F. Other strains W303-1b/G, W303-1b/P and W303-1b produced 145.72, 111.82 and 98.75 mg/L of GSH, respectively. Results of the synthetic capacities of different pathways (ranked in descending order) are as follows: F pathway, G pathway and P pathway.
Since W303-1b/F synthesized the highest GSH concentration among tested strains, it was chosen to examine the effect of gene dosage on GSH production. The antibiotic-resistant gene was removed from the genomic DNA of W303-1b/F using the Cre-loxP system, then W303-1b/F cells were further transformed with the linearized recombinant plasmid pδGAPg-gshF to obtain engineered strain W303-1b/FF (Table 2). Three recombinant strains harbouring two copies of gshF gene relative to that of W303-1b/F (Additional file 1: Table S8) were obtained and compared for their GSH production with W303-1b/F. However, no further improvement was observed (Fig. 4). This result suggests that increasing copy number was shown to be effective up to a certain limit of gene copy number.
Three biosynthetic pathways combined to increase GSH production
To obtain a higher GSH concentration, three biosynthetic pathways of GSH were combinatorially expressed in S. cerevisiae W303-1b. Strain W303-1b/F with higher synthetic capacity of GSH was chosen as the starting strain for further introduction of other biosynthetic pathways. The two-pathway strains W303-1b/FG and W303-1b/FP were obtained by the integration of linearized pδGAPh-gsh2gsh1 and pδGAPg-pro1gshB, respectively. Further integration of linearized pδGAPg-pro1gshB into the engineered W303-1b/FG generated the three-pathway strain W303-1b/FGP (Table 2).
Biomass, GSH concentration, GSH content and molar ratio of γ-GC to GSH of various strains in flask cultures with or without addition of amino acid precursors (AA)
GSH concentration (mg/L)
GSH content (%)
Molar ratio of γ-GC to GSH (%)
9.14 ± 0.48
98.75 ± 7.30
1.09 ± 0.14
6.74 ± 0.24
9.09 ± 0.13
186.77 ± 3.19
2.05 ± 0.06
10.29 ± 0.87
9.56 ± 0.26
145.72 ± 14.87
1.52 ± 0.14
9.72 ± 0.93
9.23 ± 0.32
111.82 ± 11.34
1.21 ± 0.09
8.58 ± 2.53
8.74 ± 0.44
104.24 ± 9.86
1.19 ± 0.08
7.34 ± 0.42
9.35 ± 0.38
206.70 ± 7.23
2.21 ± 0.02
11.26 ± 0.67
9.04 ± 0.20
190.57 ± 1.56
2.11 ± 0.05
10.92 ± 0.93
9.26 ± 0.07
216.50 ± 11.46
2.34 ± 0.13
9.97 ± 0.40
8.58 ± 0.09
252.33 ± 28.62
2.94 ± 0.36
9.65 ± 0.51
8.38 ± 0.37
316.77 ± 45.69
3.77 ± 0.43
9.50 ± 1.43
Shake flask culture with addition of amino acid precursors
Batch culture of engineered strain with three biosynthetic pathways
Recently, a novel bifunctional enzyme GshF has aroused concerns about GSH productive capacity as most reported GshFs are insensitive to feedback inhibition of GSH except that of P. multocida and L. monocytogenes [8–11, 20]. In this study, the putative gshF from A. pleuropneumoniae resynthesized according to codon usage of S. cerevisiae was firstly functionally expressed in S. cerevisiae, and then the relationship between GSH production and the strengths of three different glucose-based promoters, including GAP and PGK1 promoters of S. cerevisiae and GAP promoter from P. pastoris (GAP′), and a galactose-based GAL1 promoter of S. cerevisiae was analyzed. Figure 2 showed that the expressed GshF under the control of constitutive GAP promoter of S. cerevisiae produced the highest concentration of GSH. Thus it is presumed that the expression of GshF driven by GAP promoter of S. cerevisiae functioned well for GSH synthesis. Through the comparison of the productive capacity of GSH of engineered strain W303-1b/F with that of the reported GS115/LmgshF with L. monocytogenes GshF, an observed variance was that F pathway presented higher synthetic capacity of GSH than G pathway (Fig. 4) . This conflicting result may be attributed to the different sensitivities of GshFs from L. monocytogenes and A. pleuropneumoniae to feedback inhibition of GSH [8, 11]. Since single F pathway could produce higher amounts of GSH compared with other pathways, we tried to augment the copies of F pathway by further integration of gshF to improve the productive capacity of GSH of engineered strain W303-1b/F. However, the gshF copy number increase didn’t further improve GSH concentration in W303-1b/F, indicating that a reasonable rather than high copy number of gshF gene driven by GAP promoter is sufficient for the highest expression levels and synthetic capacity of GSH through one pathway is limited.
It has been reported that proline biosynthesis could contribute to GSH synthesis. γ-glutamyl phosphate is required as the common intermediate for the crosstalk between proline biosynthetic pathway and conventional Gsh1/Gsh2 pathway. Veeravalli et al. reported that, different from in vitro assay, as l-cysteine is present in micromolar concentrations in the intracellular environment, the activity of γ-glutamyl phosphate reductase (GPR) encoded by PRO2 in eukaryotes or proA in prokaryotes must be shut off to allow γ-GC formation from γ-glutamyl phosphate . In this study, as amino acid precursors could be fed to improve concentrations of intracellular free l-cysteine, we resorted to overexpression of GK instead of loss-of-function mutations in GPR for accumulation of γ-glutamyl phosphate to strengthen the redirection of γ-glutamyl phosphate to GSH synthesis. It was indirectly confirmed by the result of patch assay that W303-1b/Δgsh1/pro1 with Pro1 overexpression construct performed a relatively stronger H2O2 tolerance than W303-1b/Δgsh1/proB and W303-1b/Δgsh1 (Fig. 3). Overexpressed Pro1 mediated accumulation of γ-glutamyl phosphate which was partially diverted toward GSH production, while heterologous ProB (optimized according to codon usage of S. cerevisiae) did not work well. Consistent with the result of patch assay, W303-1b/P provided a slightly higher GSH production than W303-1b/P′ and strain W303-1b in shake flask cultures (Fig. 4). When P pathway was upregulated, the accumulated γ-glutamyl phosphate could improve utilization of extra l-cysteine by addition of amino acid precursors for improvement of GSH synthesis. Thus, P pathway could be modulated for improving intracellular GSH synthesis.
Previous studies have reported individual modulation of G pathway and F pathway for GSH production [15–22]. In this study, P pathway was exploited for GSH production for the first time. Combining G, F and P three pathways is a novel method for GSH production. Genes of three pathways were uniformly regulated and expressed by exactly the same strategy to evaluate synthetic capacities of engineered strains with different combinations of biosynthetic pathways of GSH. As expected, the engineered strain W303-1b/FGP presented the highest yield of GSH. Moreover, the result of shake flask cultures with addition of amino acid precursors (Fig. 6) further proved the advantages of W303-1b/FGP. In batch culture process, the recombinant strain W303-1b/FGP also afforded high efficiency in GSH production and reached an intracellular GSH content of 2.27 % after 24-h fermentation. Generally, a GSH content of 3–5 % could be obtained using strains selected via classical selection strategy, but the exact mechanisms underlying higher GSH accumulation in these mutants has remained rather obscure. Nisamedtinov et al. reported that GSH over-accumulation in S. cerevisiae mutants selected via random mutagenesis is caused by the combined effect of higher cysteine synthesis and increased flux through Gsh1 reaction . These genetic manipulations could confer a GSH content of 1–2 % on the engineered strains. The GSH synthetic capacity of engineered strain W303-1b/FGP constructed in this study was comparable to that of reported genetically engineered strains and higher than that of engineered strains with single modulation of G pathway or F pathway (Table 1). Increase in GSH content of W303-1b/FGP may be attributed to the fact that the three pathways form a complex network of GSH biosynthesis producing comprehensive effects. Here, it should be noted that the balance of two consecutive enzymatic reactions especially in the complex network of GSH biosynthesis is very important. Previous studies have reported that overexpression of conventional Gsh1/Gsh2 pathway improved production of intracellular GSH along with accumulation of intermediate γ-GC which increased significantly when amino acid precursors were added . In our study, as the intracellular γ-GC level was only increased slightly compared with that of original strain W303-1b when F pathway was introduced, we artificially introduced a flexible six-glycine linker between the two enzymes of G pathway as well as P pathway to improve the coupling efficiency by mimicking the native domain fusion of GshF. As expected, intracellular γ-GC amounts of W303-1b/FGP were still maintained at low levels even when amino acid precursors were added (Table 3).
In summary, a three-pathway combinatorial method of GSH biosynthesis presents higher GSH yield and utilization efficiency of amino acid precursors than that of a single pathway. This strategy is an important development toward improving industrial production of GSH using S. cerevisiae.
E. coli Trans1-T1 (TransGen, Beijing, China) was used for propagation and manipulation of the recombinant DNA. S. cerevisiae W303-1b (MATα ade2-1 leu2-3,112 his3-11,15 ura3-1 trp1-1) was used as host strain for DNA transformation and expression of recombinant genes. Yeast transformants were grown at 30 °C in YPD medium (10 g/L yeast extract, 20 g/L tryptone and 20 g/L glucose).
Plasmid construction and yeast transformation
All plasmids were constructed using conventional restriction enzyme-mediated cloning methods. Based on the nucleotide sequences of the target genes, development of primer sets were designed and used to amplify gene fragments by PCR (Additional file 1: Tables S1–S6). δ DNA-mediated integrative expression vectors as described in Ref. , and plasmids for deletion of GSH1 gene were constructed as shown in Additional file 1: Figures S1–S4. The resulting plasmids were linearized by digestion with restriction enzyme NotI and transformed into S. cerevisiae using lithium acetate method. The positive transformants were selected on YPD plates with addition of antibiotics geneticin (G418, 4 mg/mL) or hygromycin B (HygB, 1 mg/mL) and verified by DNA sequencing after the integrated gene fragments were amplified by PCR using the corresponding primers and the genomic DNA templates. The copy numbers of integrated genes were verified using real-time PCR (primers GshF-RT1/GshF-RT2, Additional file 1: Table S7) and calculated by a comparative C T method . For each transformation, three positive colonies were picked randomly for further investigation. The repeated use of the marker genes were performed via a loxP-marker-loxP gene disruption cassette according to Ref. .
Single colonies of strains W303-1b, W303-1b/Δgsh1, W303-1b/Δgsh1/pro1 and W303-1b/Δgsh1/proB were diluted in distilled H2O and patched (about 500 cells/μL) on YPD plates containing various concentrations of H2O2. Plates were incubated for about 72 h at 30 °C to compare H2O2 tolerances by watching their growth.
GSH production of the engineered S. cerevisiae strains in shake flasks
Three positive colonies of each engineered S. cerevisiae strain were cultured at 30 °C in shake flasks containing 20 mL of liquid YPD medium with agitation at 250 rpm for 18–24 h for primary culture. An adequate volume of fresh cultures were then inoculated into a set of 250-mL flasks containing 50 mL of liquid YPD medium to keep an initial optical density at 600 nm (OD600) value of 0.2. The cultures were incubated at 30 °C with agitation at 250 rpm for additional 48 h. In order to further increase the production of GSH, the addition of amino acid precursors (5 mM glutamic acid, 5 mM cysteine and 5 mM glycine) was single shot after 36-h cultivation for feeding assay.
Batch culture of the engineered strain W303-1b/FGP for GSH production
The batch fermentation was operated in a 10-L fermentor (China Beauty). Liquid YPD medium for batch culture was sterilized by autoclave at 121 °C for 20 min. Engineered strain W303-1b/FGP grown on YPD agar plate was inoculated into liquid YPD medium and cultured at 30 °C with agitation at 250 rpm for 18–24 h. 10 % (v/v) seed cultures were transferred into the fermentor. The dissolved oxygen (DO) was maintained at 25 % through adjustment of stirrer speed (100–500 rpm) and the aeration rate (1–5 L/h). The temperature was set to 30 °C and the pH was uncontrolled.
Grown cultures were removed from the incubator at a given time and biomass was measured as optical density at 600 nm (OD600) and/or the dry cell weight (DCW). OD600 was determined after the sample was appropriately diluted. DCW (g/L) was determined after yeast cells were centrifuged at 4000 rpm for 10 min and washed twice with distilled water before drying at 105 °C to constant weight. OD600 was converted to DCW using calibration coefficient (OD600 = 10, equivalent to 1.19 g/L DCW in this study). To monitor the intracellular GSH and γ-GC amounts, cells were harvested and washed with distilled water. Intracellular GSH and γ-GC were extracted from the cells by 40 % ethanol for 2 h at 30 °C. The GSH and γ-GC concentrations (mg/L) of the sample were measured using the 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) derivatization method (Additional file 1: Figure S5) according to a previous report  with the reduced GSH as the standard. The GSH content (%) was calculated from the intracellular GSH concentration (mg/L) divided by DCW.
LT, WW, WZ and KC performed the experiments. LT, WW, YY and ML analyzed the data, and LT drafted the manuscript. KC commented on the manuscript. WW conceived and coordinated the study and revised the manuscript. All authors read and approved the final manuscript.
This work was financially supported by the National Science Foundation of China (Grant No. 30772677, 81072562), the Fundamental Research Funds for the Central Universities (Grant No. 2012N06), and the National Science and Technology Project of China (Grant No. 2012ZX09301002-001).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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