Rerouting citrate metabolism in Lactococcus lactis to citrate-driven transamination.
Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.
Top Institute Food and Nutrition, Wageningen, The Netherlands.
Agata M
AM
Pudlik
Juke S
JS
Lolkema
D000596:Amino Acids / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism*; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D000637:Transaminases / Q000378:metabolism
D000596:Amino Acids; D002264:Carboxylic Acids; D019343:Citric Acid; D000637:Transaminases