Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

Abstract

The first step of amino acid degradation in lactococci is a transamination, which requires an ␣-keto acid as the amino group acceptor. We have previously shown that the level of available ␣-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding ␣-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce ␣-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added ␣-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added ␣-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding ␣-ketoglutarate to improve aroma development in cheese.

CodY-Regulated Aminotransferases AraT and BcaT Play a Major Role in the Growth of Lactococcus lactis in Milk by Regulating the Intracellular Pool of Amino Acids

“…Growth experiments in buffered 75 mM ␤-glycerophosphate milk were done at 30°C and monitored as previously described (26). Milk was reconstituted from NILAC low-heat spray powder (NIZO, Ede, The Netherlands) at a concentration of 10% (wt/vol) in distilled sterilized water at 30°C.…”

“…The catabolism of amino acids by whole cells of L. lactis subsp. cremoris TIL46 and mutants was studied by using radiolabeled amino acids as tracers according to a previously described protocol (26,38). Briefly, each reaction mixture contained 100 mM Tris-HCl buffer (pH 8), unlabeled amino acid at a concentration of 2 mM, 0.05 M tritiated amino acid, and 10 mM ␣-ketoglutarate.…”

Sequence analysis of the mobilizable lactococcal plasmid pGdh442 encoding glutamate dehydrogenase activity

“…The first property is a glutamate dehydrogenase (GDH) activity which catalyses the reversible oxidative deamination of glutamate to 2-oxoglutarate and ammonia, mainly using NADP as the co-factor. This activity stimulates amino acid catabolism in LAB by supplying the 2-oxoglutarate required for amino acid transamination, which is the first step of amino acid conversion to aroma compounds (Rijnen et al, 2000;Tanous et al, 2002). Indeed, inactivation of the gdh gene in L. lactis has been shown to totally prevent the strain from degrading amino acids (Tanous et al, 2006).…”

Safe use of genetically modified lactic acid bacteria in food. Bridging the gap between consumers, green groups, and industry

“…GM-LAB with increased proteolytic propertied are generated by the over expression of the genes encoding PepN, PepX, PepC and PepI peptidases from a highly proteolytic L. helveticus strain (Joutsjoki et al 2002) or PepI, PepL, PepW, and PepG from L. delbrueckii (Wegmann et al 1999) into L. lactis. Moreover, the expression of gdh from Peptoniphilus asaccharolyticus, encoding glutamate dehydrogenase, into L. lactis increases the production of α-ketoglutarate (Rijnen et al 2000). This enhances the degradation of amino acids, which also benefits the cheese ripening process.…”