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, GIULIANA; EBERHARDT, MARÍA F.; MORTERA, PABLO; MAGNI, CHRISTIAN; ESPARIZ, MARTIN

Congreso; XII Congreso Argentino de SAMIGE.; 2017

SAMIGE

Enterococcus strains usually dominatethe non-starter flora of traditional cheeses such as Mozzarella and Fontina. Theycontribute positively on the development of flavor compounds during ripening.The most important enzymatic activities of non-starter lactic acid bacteria involvedin flavor production are proteolysis and lipolysis. As the applications ofenzymes in the food industry are expanding in this project we have performed ascreening of E. faecalis esterases inorder to produce flavor-enhancing esterases in a GRAS host. First, twenty-threehydrolases with possible esterase activity were identified in the availablegenome of E. faecalis JH2-2 using in silico tools. In an attempt toidentify wall-anchored or secreted enzymes the program SignalP 4.0 was subsequentlyused. Hence, nine out of twenty-three hydrolases showed to have a signalpeptide indicating possible secretion. Four of these hypothetical esterases codinggenes, named estA, estB, estC and estD, werecloned in pET 28b and expressed in Escherichiacoli DH5α. Then, cell fractions of IPTG-induced recombinant strains wereobtained and analyzed. The presence of EstB and EstC was only observed in thecytoplasmic fractions which suggests that neither of the putative enzymes couldbe recognized or transported by E. coli secretionsystem. On the other hand, EstD wasnot detected in soluble form under tested conditions. Interestingly, EstAputative esterase could be recovered in the periplasmic fraction whichindicates that the hypothetical signal peptide is being recognized and theprotein secreted by the E. coli Secsystem. In an attempt to corroborate the hydrolytic capability of the putativeenzymes, the esterolysis of p-nitrophenyl (pNP) monoesters of fatty acids wereevaluated. EstA showed to hydrolyze only short chain acyl pNP derivatives (C4),while EstC over C4, C16 and C18 acyl pNP derivative. Noteworthy, short-chainfree fatty acids are indicators of quality and source of flavor in cheese. Inorder to broaden the knowledge of EstA and EstC regarding its origin, aphylogenetic analysis was performed. As a result, EstA and EstC were identifiedin all E. faecalis strains analyzed andin lesser extent within the genus.Finally, in order to study thecontribution of 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 wassynthetized and cloned in pNZ8048, a NICE (nisin-controlled expression) systemvector, which derives from the nisoperon (nisABTCIPRKEFG). As hosts, L. lactis NZ9000 and a derivate straindeficient in ClpP and HtrA major proteases were employed. As an alternativeexpression tool, the promotor of NICE system was also exchanged by the promotorof P170 expression system which is up-regulated as lactate accumulates in thegrowth medium. Currently, the best combination of host, promotor type, andexpression conditions are under evaluation.