Extracellular Production of Glutathione by Recombinant <i>Escherichia coli</i> K-12
2023
Hideyuki Suzuki | Kazuki Nishida | Tatsuya Nakamura
The goal of this study was to produce a sufficient amount of glutathione in the fermentation medium without the addition of cysteine. This would simplify and reduce the cost of its purification. In addition to reducing the cost of cysteine, it also avoids the inhibition of bacterial growth by cysteine. The <i>gshA</i>, <i>gshB</i>, and <i>cysE</i> genes of <i>Escherichia coli</i> were cloned under the control of the strong T5 promoter of the pQE-80L plasmid and introduced into an <i>E. coli</i> strain knocked out for the genes encoding γ-glutamyltranspeptidase and the GsiABCD glutathione transporter, which are responsible for the recycling of excreted glutathione. The overexpression of the <i>gshA</i> and <i>gshB</i> genes, genes for γ-glutamylcysteine synthetase and glutathione synthetase, and the <i>cysE</i><sup>V95R D96P</sup> gene, a gene for serine acetyltransferase with the V95R D96P mutation that makes it insensitive to cysteine, were effective on glutathione production. Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> was a good sulfur source for glutathione production, while the addition of Na<sub>2</sub>SO<sub>4</sub> did not affect the glutathione production. With the addition of 50 mM glutamic acid and 75 mM glycine, but without the addition of cysteine, to the simplified SM1 medium, 4.6 mM and 0.56 mM of the reduced and oxidized glutathione, respectively, were accumulated in the extracellular space after 36 h of batch culture. This can eliminate the need to extract glutathione from the bacterial cells for purification.
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