Cloning of inulinase gene from Aspergillus kawachii in Saccharomyces cerevisiae

FRATEBIANCHI DE LA PARRA, D; CHESINI, M; ROJAS, N. L; CAVALITTO, S. F

Mar del Plata

Congreso; VIII Encuentro Latinoamericano y del Caribe de Biotecnología - REDBIO 2013; 2013

Fundación REDBIO

Inulinases comprise an important group of enzymes that hidrolyze the [2→1] β-fructan linkages of the inulin molecule and are thus used for the production of fructose and fructo-oligosaccharides. These products are extensively used as sweeteners and functional food additives, among other uses. Although Aspergillus kawachii produces inulinases its low expression levels makes cloning and over-expression of its gene a need for its industrial application. The aim of this work was to generate two alternative constructs of the inulinase gene (inu) ?with (inuSI) and without its signal peptide (inuSISPS)- cloned into a S. cerevisiae expression vector under the regulation of the inducible Gal1 promoter. Previous studies showed that the primary transcript of the inu gene presents an intron which S. cerevisiae was unable to remove during maturation; as a consequence the intron was in-vitro deleted, generating the inuSI construction from which the present work began. The inu gene without its signal peptide (inuSISPS) was successfully cloned into the pYES2 expression vector in the correct reading frame (according to sequencing results). However, no inulinase activity was obtained and the SDS-PAGE showed no difference between intracelullar protein patterns of the induced and non-induced clones, perhaps due to an expression level of the protein not high enought to be detected by SDS-PAGE. In any case, a new cloning strategy is under development based on bioinformatic tools, towards the analysis of intron and signal peptide sequences and/or potential post transductional modifications in order to consider a more appropriate expression system.