IDENTIFICATION AND HETEROLOGOUS EXPRESSION OF Enterococcus faecalis ESTERASES FOR THE PRODUCTION OF SHORT CHAIN FATTY ACIDS COMPOUNDS THAT CONTRIBUTE TO FLAVOR GENERATION IN CHEESES

ACCIARRI, GUILIANA; EBERHARDT, MARIA FLORENCIA; MORTERA PABLO; ESPARIZ, MARTIN; MAGNI, CHRISTIAN

Congreso; XII Congreso Argentino de Microbiologia General; 2017

Enterococcus strains usually dominate the non-starter flora of traditional cheeses such as Mozzarella and Fontina. They contribute positively on the development of flavor compounds during ripening. The most important enzymatic activities of non-starter lactic acid bacteria involved in flavor productionare proteolysis and lipolysis. As the applications of enzymes in the food Industry are expanding in this project we have performed a screening of E. faecalis esterases in order to produce flavor-enhancing esterases in a GRAS host. First, twenty-three hydrolases with possible esterase activity were identified inthe available genome of E. faecalis JH2-2 using in silico tools. In an attempt to identify wall-anchored or secreted enzymes the program SignalP 4.0 was Subsequently used. Hence, nine out of twenty-three hydrolases showed to have a signal peptide indicating possible secretion. Four of these hypotheticalesterases coding genes, named estA, estB, estC and estD, were cloned in pET 28b and expressed in Escherichia coli DH5a. Then, cell fractions of IPTG-induced recombinant strains were obtained and analyzed.The presence of EstB and EstC was only observed in the cytoplasmic fractions which suggests that neither of the putative enzymes could be recognized or transported by E. coli secretion system. On theother hand, EstD was not detected in soluble form under tested conditions. Interestingly, EstA putative esterase could be recovered in the periplasmic fraction which indicates that the hypothetical signal peptide is being recognized and the protein secreted by the E. coli Sec system. In an attempt to corroboratethe hydrolytic capability of the putative enzymes, the esterolysis of p-nitrophenyl (pNP) monoesters of fatty acids were evaluated. EstA showed to hydrolyze only short chain acyl pNP derivatives (C4), while EstC over C4, C16 and C18 acyl pNP derivative. Noteworthy, short-chain free fatty acids are indicatorsof quality and source of flavor in cheese. In order to broaden the knowledge of EstA and EstC regarding its origin, a phylogenetic analysis was performed. As a result, EstA and EstC were identified in all E. faecalis strains analyzed and in lesser extent within the genus. Finally, in order to study the contributionof EstA in the production of cheese sensorial relevant compounds, constructions of GRAS EstA?producing strains were conducted. In order to do so, a codon optimized version of estA was synthetized and cloned in pNZ8048, a NICE (nisin-controlled expression) system vector, which derives from the nis operon (nisABTCIPRKEFG). As hosts, L. lactis NZ9000 and a derivate strain deficient in ClpP and HtrA major proteases were employed. As an alternative expression tool, the promotor of NICE system was also exchanged by the promotor of P170 expression system which is up-regulated as lactate accumulates in the growth medium. Currently, the best combination of host, promotor type, and expression conditions are under evaluation.