Heterologous expression as a tool to increase GABA and riboflavin production in fermented vegetable beverages

Gamma-aminobutyric acid (GABA) and riboflavin are bioactive compounds with health-promoting properties that can be produced by particular lactic acid bacteria (LAB) strains. GABA synthesis from glutamate involves the enzyme glutamate decarboxylase and the glutamate/GABA antiporter encoded by gadB and gadC, respectively, albeit yields are not usually high. In this study, different strategies were applied for obtaining fermented vegetable beverages enriched with high yields of both riboflavin and GABA. First, it was explored the construction and selection of LAB engineered to overexpress the gadB and/or gadC genes, and their effects on fermented plant-based beverages to enhance GABA production. For this, we designed the plasmids pNZ:TuR.gadB, pNZ:TuR.gadC and pNZ:TuR.gadCB to heterologously overexpress gadB, gadC or both genes, respectively, for increasing GABA production in different GABA- and/or riboflavin-producing LAB strains and this production was confirmed in broth media and vegetable beverages supplemented with glutamate. Subsequently, selected engineered strains producing high GABA yields were co-cultured in plant-based beverages with a riboflavin-overproducing Lactococcus lactis strain, obtaining increased GABA production in some combinations, but with reduced riboflavin yields. Thus, Lactiplantibacillus plantarum Al03R3, a riboflavin-overproducing spontaneous mutant harboring pNZ:TuR.gadB, was finally selected as the best starter for vegetable beverages due to its double ability to improve GABA (11 g/L) and riboflavin (6 mg/L) yields in fermented oat beverages with increasing monosodium glutamate supplementation between 80 and 150 mM. In conclusion, the implementation of the designed heterologous expression systems in a riboflavin-overproducing strain gave rise a fermented plant-based beverage with high concentrations of both GABA and riboflavin.