Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis

The gene encoding the major intracellular tributyrin esterase of Lactococcu s lactis was cloned using degenerate DNA probes based on 19 known N-termina l amino acid residues of the purified enzyme. The gene, named estA was sequ enced and found to encode a protein of 258 amino acid residues. The transcr iption start site was mapped 233 nucleotides upstream of the start codon, a nd a canonical promoter sequence was identified. The deduced amino acid seq uence of the estA product contained the typical GXSXG motif found in most l ipases and esterases. The protein was overproduced up to 170-fold in L. lac tis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integra tion of a temperature-sensitive integration vector. This resulted in the co mplete loss of esterase activity; which could then be recovered after compl ementation of the constructed esterase-deficient strain with the wild-type est gene. This confirms that EstA is the main enzyme responsible for estera se activity in L. lactis. Purified recombinant enzyme showed a preference f or short-chain acyl eaters, surprisingly also including phospholipids. Medi um- and long-acyl-chain lipids were also hydrolyzed, albeit less efficientl y., Intermediate characteristics between esterases and lipases make intrace llular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved i n (phospho)lipid metabolism or cellular detoxification or both, as its sequ ence show ed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a un iversal formaldehyde detoxification pathway.