γ-Glutamyl Cysteine Ligase of Lactobacillus reuteri Synthesizes γ-Glutamyl Dipeptides in Sourdough

Kokumi-active gamma-glutamyl dipeptides (gamma-GPs) accumulate in fermented food. gamma-Glutamyl transferase, glutaminase, glutathione synthetase, and gamma-glutamyl cysteine ligase (GCL) may synthesize gamma-GPs. The genome of Lactobacillus reuteri encodes GCL but not glutathione synthetase or glutamyl transferase; therefore, this study investigated the role of GCL in gamma-GP synthesis by L. reuteri LTH5448. Phylogenomic analysis of gcl in lactobacilli demonstrated that three genes coding for GCL are present in L. reuteri; two of these are present in L. reuteri LTH5448. Two deletion mutants of L. reuteri LTH5448, L. reuteri LTH5448 Delta gcl1 and LTHS448 Delta gcl1 Delta gcl2, were constructed by double crossover mutagenesis. Growth and oxygen resistance of the mutants were comparable to the wild type. gamma-Glu-Glu, gamma-Glu-Leu, gamma-Glu-Ile, gamma-Glu-Val, and gamma-Glu-Cys were quantified in buffer and sourdough fermentations by liquid chromatography mass spectrometry. The wild type and L. reuteri Delta gcl1 but not Delta gcl1 Delta gcl2 converted amino acids to gamma-Glu-Cys. gamma-Glu-Ile accumulation was reduced in both mutants; however, the disruption of gcl did not alter the biosynthesis of the other gamma-GPs. In conclusion, gcl1 in L. reuteri mediates gamma-Glu-Ile synthesis, gcl2 mediates gamma-Glu-Cys synthesis, but neither gene affected synthesis of other gamma-GPs. This study facilitates selection of starter cultures that synthesize gamma-Glu peptides with kokumi activity and, thus, improve the taste of fermented foods.