Engineering a Lactococcus lactis strain to produce gamma-aminobutyric-acid-enriched, putrescine-free cheese

The neuroactive compound γ-aminobutyric acid (GABA) is widely recognized for its potential health-promoting effects, which are driving increasing industrial interest in the development of GABA-enriched fermented foods. Microorganisms such as GABA-producing Lactococcus lactis strains are key to this approach. As the primary starter culture in the dairy industry, L. lactis is essential for the manufacture of fermented products such as cheese. While this species is generally recognized as safe, certain strains can produce putrescine, a toxic biogenic amine whose accumulation in food products must be minimized or avoided. The main objective of this study was to delete the AGDI cluster-responsible for putrescine biosynthesis-in a GABA-producing L. lactis strain to obtain a variant suitable for use as a starter culture in the production of GABA-enriched, putrescine-free cheeses. The mutant was successfully generated via double homologous recombination. When cultivated in a medium supplemented with agmatine, the knockout strain did not produce putrescine; however, it retained its ability to synthesize GABA in a glutamate-supplemented medium. Furthermore, when employed as a starter culture for Cabrales-like mini-cheeses, the mutant strain effectively produced cheeses with elevated GABA concentrations (ranging from 545.50 to 865.86 mg kg^-1) and no detectable putrescine accumulation. Eliminating putrescine accumulation in cheese resolves the major food-safety concern associated with the wild-type strain, thereby enabling its safe use as a starter culture for the production of GABA-enriched cheese, while also preventing sensory defects linked to this biogenic amine.