Regulation of the pleiotropic transcriptional regulator CodY on the conversion of branched-chain amino acids into branched-chain aldehydes in Lactococcus lactis

Abstract

ABSTRACT Lactococcus lactis is a key strain in the production of branched-chain aldehydes that contribute to the nutty flavor in cheese. It has been hypothesized that the conversion of branched-chain amino acids (BCAAs) into branched-chain aldehydes is under the regulation of a global transcriptional regulator, CodY; however, the functions remain unclear. This study aimed to elucidate the regulatory effect of CodY on the key genes for the BCAAs conversion into branched-chain aldehydes in L. lactis , namely branched-chain aminotransferase (encoded by bcaT ) α-keto acid decarboxylase (encoded by kdc A) and α-keto acid dehydrogenase (encoded by pdh ABCD). The deletion of codY was associated with a significant increase in the production of 3-methylbutanal and 2-methylbutanal, indicating the inhibitory effect of CodY on the BCAAs conversion into branched-chain aldehydes. Real-time quantitative reverse transcription-PCR demonstrated the negative regulatory effect of CodY on bcaT and kdc A, whereas pdh ABCD was activated by CodY. Potential CodY binding sites in the promoter regions of bcaT , kdc A, and pdh ABCD were predicted, and four CodY-boxes within the regulatory regions were verified by electrophoretic mobility shift assay and DNase I footprinting assay. The sites located within the promoter of bcaT and kdc A led to repression by CodY. When one site was located upstream and the other was located within the promoter of pdh ABCD, the ultimate effect was activation. Overall, the study revealed that CodY, together with isoleucine, acts as a pleiotropic transcriptional regulator of the conversion of BCAAs into branched-chain aldehydes in L. lactis via binding to various regulatory regions of key genes. IMPORTANCE Branched-chain aldehydes are the primary compounds that contribute to the nutty flavor in cheddar cheese. Lactococcus lactis , which is often applied as primary starter culture, is a significant contributor to the nutty flavor of cheddar cheese due to its ability of conversion of BCAAs into branched-chain aldehydes. In the present study, we found that the regulatory role of CodY is crucial for the conversion. CodY acts as a pleiotropic transcriptional regulator via binding to various regulatory regions of key genes. The results presented valuable knowledge into the role of CodY on the regulation and biosynthetic pathway of branched-chain amino acids and the related aldehydes. Furthermore, it provided new insight for increasing the nutty flavor produced during the manufacture and ripening of cheese.