Bacterial β-etherases and glutathione lyases are glutathione-dependent enzymes that catalyze the selective cleavage of β-O-4 aryl ether bonds found in lignin. Their glutathione (GSH) dependence is regarded as major limitation for their application in the production of aromatics from lignin polymer and oligomers, as stoichiometric glutathione amounts are required. Thus, recycling of the GSH cofactor by a NAD(P)H-dependent glutathione reductase was proposed previously. Herein, the use of a whole-cell catalyst was studied for efficient β-O-4 aryl ether bond cleavage with intracellular GSH supply and recycling. After optimization of the whole-cell catalyst as well as reaction conditions, up to 5 mM lignin model substrate 2,6-methoxyphenoxy-α-veratrylglycerone (2,6-MP-VG) were efficiently converted into 2,6-methoxyphenol (2,6-MP) and veratryl glycerone (VG) without addition of external GSH. Unexpectedly, no glucose supply was required for glutathione recycling within the cells up to this substrate concentration. To demonstrate the applicability of this whole-cell approach, a whole-cell cascade combining a stereoselective β-etherase (either LigE from Sphingobium sp. SYK-6 or LigF-NA from Novosphingobium aromaticivorans) and a glutathione lyase (LigG-TD from Thiobacillus denitrificans) was employed in the kinetic resolution of racemic 2,6-MP-VG. This way, enantiopure (S)- and (R)-2,6-MP-VG were obtained on semi-preparative scale without the need for external GSH supply.
Keywords: Escherichia coli; Glutathione lyase; Glutathione recycling; Whole-cell catalysis; β-etherase.
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