A general method for selection of alpha-acetolactate decarboxylase-deficient Lactococcus lactis mutants to improve diacetyl formation

The enzyme acetalactate decarhoxylase (Ald) plays a key role in the regulat ion of the a-acetolactate pool in both pyruvate catabolism and the biosynth esis of the branched-chain amino acids, isoleucine, leucine, and valine (IL V) This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus la ctis subsp, lactis biovar diacetylactis. Such mutants can be selected as le ucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lact ococcus strains are auxotrophic for the three amino acids. Therefore, the p lasmid pMC004 containing the ilv genes (encoding the enzymes involved in th e biosynthesis of IV) of L. lactis NCDO2118 was constructed, introduction o f pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-proto trophic phenotype. By plating the strains on a chemically defined medium su pplemented with leucine but not valine and isoleucine, spontaneous leucine- resistant mutants were obtained. These mutants were screened by Western blo tting with Aid-specific antibodies for the presence of slid. Selected mutan ts lacking Aid were subsequently cured of pMC004, Except for a defect in th e expression of Aid, the. resulting strain, MC010, was identical to the wil d-type strain, as shown by Southern blotting and DNA fingerprinting. The mu tation resulting in the lack of Aid in MC010 occurred spontaneously, and th e strain does not contain foreign DNA; thus, it can be regarded as food gra de. Nevertheless, its application in dairy products depends on the regulati on of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis stra ins.