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gshF and GSH Biosynthesis

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iGEM20_UNSW_Australia

Present in its reduced (thiol) form, Glutathione (GSH) is a ubiquitous primary antioxidant which prevents the oxidation of biological molecules, such as proteins and enzymes, by donating its electrons to reactive oxygen species (Wang et al. 2019). Enzymatic synthesis and fermentation are the two processes that GSH is currently produced from. The intracellular synthesis of GSH is mediated by two ATP-dependent reactions which are catalysed by 𝛾-glutamylcysteine Synthetase (𝛾-GCS) (reaction 1) and Glutathione Synthetase (GS) (reaction 2). 𝛾-GCS and and GS are encoded by two different genes in most cells, the gshA and gshB genes, respectively. Since the activity of 𝛾-GCS is regulated by the feedback inhibition of GSH, over-accumulation of this antioxidant in the cell is not possible, thus, glutathione production is limited (Li et al. 2011). To improve the situation, a number of methods have been studied in one of which the exposure of cells to low pH resulted in secretion of GSH out of the cell and hence, less inhibition feedback on 𝛾-GCS activity (Liang, Du & Chen, 2008). A number of microorganisms, including Streptococcus thermophilus, have been found to have both 𝛾-GCS and GS encoded by a single gene, gshF. This gene codes a bifunctional enzyme in which its N-terminal sequence resembles 𝛾-GCS whereas its C-terminal sequence is similar to GS’s function. As a result, the activity of 𝛾-GCS is not inhibited and more GSH is produced, allowing the cell to have a higher threshold for reactive oxygen species without damaging any of its components (Li et al. 2011).

Due to its antioxidant nature, recombinant GSH expression has been explored as a means to increase yeast robustness for ethanol manufacturing from lignocellulosic feedstock. For cost-effective production, it is therefore imperative for high-yield, high-rate fermentation microbes. Previously, three homologous genes involved with GSH metabolism, GSH1, CYS3 and GLR1, were overexpressed which saw an increase in the robustness of Saccharomyces cerevisiae (Ask et al. 2013). However, the GSH feedback inhibition system posed a problem. To circumvent this, one study integrated the ribosomal DNA of S. cerevisiae with the GshF gene at a high copy number via the Cre-LoxP system (Qiu et al. 2015). The resulting yeast observed a three-fold increase in accumulated GSH leading to a greater tolerance to H2O2 (3mM), high temperatures (40℃), furfural (10mM), hydroxymethylfurfural (HMF - 10mM) and Cd+2 (0.5mM) concentrations compared to reference strains. From this, a product of two-fold higher ethanol concentration was achieved. However, yeast with higher thermotolerance is still required for a simultaneous saccharification (which occurs at 55℃) and fermentation process. Although the robustness of yeast can be improved via gshF, further means of increasing ROS resilience is a necessity for industrial biofuel production.

Although lacking an evaluation for ethanol production, a study by Tang presents an interesting method of increasing GSH production in yeast (Tang et al. 2015). This involves combining all three known biosynthesis pathways of GSH into one system - the first and second as explained previously (gshA/gshB and gshF) and the third utilising a side reaction from the proline synthesis pathway. For this, glutamate is converted to γ-glutamyl phosphate via γ-glutamyl kinase (Pro1). The substrate then reacts with cysteine to become γ-glutamylcysteine which is further conjugated with glycine via gsh2 to produce GSH. In the experiment, GshF from Actinobacillus pleuropneumoniae and Pro1 was expressed in S. cerevisiae. Additionally, two fusion proteins were constructed to mimic gshF’s two-step coupling efficiency; gsh2-gsh1 and Pro1-gsh2. The resulting W303-1b/FGP strain observed the highest GSH concentration at 216.50mg/L - a 2.19 fold increase compared to two-pathway strains. When amino acid precursors were introduced to shake flask cultures, a further 61.37% increase of GSH levels was observed. As such, this combinatorial strategy of GSH biosynthesis shows promise however requires evaluation as a feasible solution for increasing yeast robustness in applications such as ethanol production.

Applications of BBa_K2571005

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@@ Line 3: / Line 3: @@
 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 how you used this part and how it worked out.
+===gshF and GSH Biosynthesis===
+<b> Author </b>
+iGEM20_UNSW_Australia
+Present in its reduced (thiol) form, Glutathione (GSH) is a ubiquitous primary antioxidant which prevents the oxidation of biological molecules, such as proteins and enzymes, by donating its electrons to reactive oxygen species (Wang et al. 2019). Enzymatic synthesis and fermentation are the two processes that GSH is currently produced from. The intracellular synthesis of GSH is mediated by two ATP-dependent reactions which are catalysed by 𝛾-glutamylcysteine Synthetase (𝛾-GCS) (reaction 1) and Glutathione Synthetase (GS) (reaction 2). 𝛾-GCS and and GS are encoded by two different genes in most cells, the gshA and gshB genes, respectively. Since the activity of 𝛾-GCS is regulated by the feedback inhibition of GSH, over-accumulation of this antioxidant in the cell is not possible, thus, glutathione production is limited (Li et al. 2011). To improve the situation, a number of methods have been studied in one of which the exposure of cells to low pH resulted in secretion of GSH out of the cell and hence, less inhibition feedback on 𝛾-GCS activity (Liang, Du & Chen, 2008). A number of microorganisms, including Streptococcus thermophilus, have been found to have both 𝛾-GCS and GS encoded by a single gene, gshF. This gene codes a bifunctional enzyme in which its N-terminal sequence resembles 𝛾-GCS whereas its C-terminal sequence is similar to GS’s function. As a result, the activity of 𝛾-GCS is not inhibited and more GSH is produced, allowing the cell to have a higher threshold for reactive oxygen species without damaging any of its components (Li et al. 2011).
+Due to its antioxidant nature, recombinant GSH expression has been explored as a means to increase yeast robustness for ethanol manufacturing from lignocellulosic feedstock. For cost-effective production, it is therefore imperative for high-yield, high-rate fermentation microbes. Previously, three homologous genes involved with GSH metabolism, GSH1, CYS3 and GLR1, were overexpressed which saw an increase in the robustness of Saccharomyces cerevisiae (Ask et al. 2013). However, the GSH feedback inhibition system posed a problem. To circumvent this, one study integrated the ribosomal DNA of S. cerevisiae with the GshF gene at a high copy number via the Cre-LoxP system (Qiu et al. 2015). The resulting yeast observed a three-fold increase in accumulated GSH leading to a greater tolerance to H2O2 (3mM), high temperatures (40℃), furfural (10mM), hydroxymethylfurfural (HMF - 10mM) and Cd+2 (0.5mM) concentrations compared to reference strains. From this, a product of two-fold higher ethanol concentration was achieved. However, yeast with higher thermotolerance is still required for a simultaneous saccharification (which occurs at 55℃) and fermentation process. Although the robustness of yeast can be improved via gshF, further means of increasing ROS resilience is a necessity for industrial biofuel production.
+Although lacking an evaluation for ethanol production, a study by Tang presents an interesting method of increasing GSH production in yeast (Tang et al. 2015). This involves combining all three known biosynthesis pathways of GSH into one system - the first and second as explained previously (gshA/gshB and gshF) and the third utilising a side reaction from the proline synthesis pathway. For this, glutamate is converted to γ-glutamyl phosphate via γ-glutamyl kinase (Pro1). The substrate then reacts with cysteine to become γ-glutamylcysteine which is further conjugated with glycine via gsh2 to produce GSH. In the experiment, GshF from Actinobacillus pleuropneumoniae and Pro1 was expressed in S. cerevisiae. Additionally, two fusion proteins were constructed to mimic gshF’s two-step coupling efficiency; gsh2-gsh1 and Pro1-gsh2. The resulting W303-1b/FGP strain observed the highest GSH concentration at 216.50mg/L - a 2.19 fold increase compared to two-pathway strains. When amino acid precursors were introduced to shake flask cultures, a further 61.37% increase of GSH levels was observed. As such, this combinatorial strategy of GSH biosynthesis shows promise however requires evaluation as a feasible solution for increasing yeast robustness in applications such as ethanol production.
 ===Applications of BBa_K2571005===