Optimization and evaluation of heterologous lysozyme production in saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzymepresent in high concentrations in chicken (Gallus gallus) egg whites. It hydrolysesthe link between N-acetylmuramic acid and N-acetylglucosamine in Gram positivebacterial cell walls, resulting in cell death. It is thus active against lactic acidbacteria (LAB), which may be present in grape juices and musts. These bacteriaare responsible for malolactic fermentation of wines although many species, particularlyof the genera Lactobacillus and Pediococcus, are considered spoilage organisms.The growth of LAB is therefore closely monitored and controlled duringwinemaking. The most common means of control is growth inhibition by chemicaltreatment (usually with SO2). Lysozyme is a commonly used wine processing aid,complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2to be used, thus improving the wholesomeness of wine. The OIV (OrganisationInternationale de la Vigne et du Vin) approved its use in quantities up to 500 mg perliter of wine in 1997.This study evaluated the effect of different secretion signals on the secretion oflysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretionby an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglyceratekinase 1) prompter and terminator was also evaluated. In the case of FY23 foursecretion signals were used, namely the native lysozyme signal and the S. cerevisiaemating factor-a signal as well as mutants of these signals. These mutants incorporatedtwo additional arginines at the N-terminus of the signals immediately downstreamof the terminal methionine. The effect of these mutations was to increase thepositive charge of the secretion signal N-terminals. The secretion signal-lysozymefusions were placed under the regulation of the S. cerevisae PGK1 gene's promoterand terminator. The resulting expression cassettes were cloned into integrating vectorsYIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used totransform FY23 and VIN13.FY23 as well as VIN13 transformants were evaluated in an artificial mediumdesigned to reflect the nutrient content of grape juice, with particular attention beingpaid to assiminable nitrogen. Three hexose concentrations were tested in order todetermine the effect thereof on lysozyme secretion titer.Lysozyme secreted under all tested growth conditions was found to be too lowfor detection by either enzymatic assay or HPLC-FLD. For this reason secretedlysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently,lysozyme concentrations in the concentrates was determined by means ofthe aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates wasalso performed.No significant differences were found between native or MF-a secretion signalsand their mutated counterparts in terms of secretion titer or proteolytic maturation.Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases,with both an authentically processed and a larger form, in which the secretion signalwas not completely removed, being present. Lysozyme secreted with the nativesignal appeared to be correctly processed in all cases. Secretion titer from highcopy number episomal FY23 tranformants was similar to that of integrants containinga single copy of the gene. Sugar concentration affected lysozyme production,with higher quantities of the enzyme being secreted when higher initial sugar concentrationswere used. Lysozyme titers were extremely low (< 0:25 mg/L) withall expression cassettes under all the tested conditions with both FY23 and VIN13.In the case of the VIN13's a lower final biomass was found for the secretor straintested in comparrison to the VIN13 wild-type.

[发布机构] Stellenbosch University