Overexpression of the glucoamylase-encoding STA1 gene of Saccharomyces cerevisiae var. diastaticus in laboratory and industrial strains of Saccharomyces

Abstract

Production of glucoamylase encoded by the Saccharomyces cerevisiae (var. diastaticus) STA1 gene has been assayed in laboratory S. cerevisiae strains of different ploidy and in different industrial Saccharomyces strains, in which STA1 was expressed under control of an inducible promoter. Highest enzyme activity was achieved with a tetraploid strain constructed by crossing preselected parental strains. Maximal glucoamylase production correlated with heterogeneity in enzyme mass, likely due to incomplete glycosylation, suggesting that the secretionglycosylation process is the limiting step in the production of the STA-encoded glucoamylase by Saccharomyces. Industrial strains showed quite different capacity to produce glucoamylase. High production was achieved with a S. pastorianus brewer's strain. Overall, our results allowed the selection of strains capable of yielding a high level of glucoamylase and suggest specific approaches for further enhancing this capability.

Influence of Different Substrates on the Production of a Mutant Thermostable Glucoamylase in Submerged Fermentation

“…The mutation did not affect the protein secretion nor the production of GA studied in this research, although some researchers reported that mutations could affect the enzyme conformation secreted and thus influence its activity [12,[35][36][37][38]. The enzyme activity and secretion might also be affected by a high quantity of glycosylation [39][40]. Our results are significant when compared to others in the literature.…”

Probiotic Saccharomyces cerevisiae strains as biotherapeutic tools: is there room for improvement?

“…To date, several S. cerevisiae laboratory strains have been genetically engineered to express proteins of interest that have a wide range of biotechnological applications, including biofuels, pharmaceutical and food industry, and models for eukaryotic cell mechanisms and physiology research (Schneiter 2004;Latorre-García et al 2008;Ardiani et al 2010;Da Silva and Srikrishnan 2012;Reis et al 2012). Strains have been optimized and genetic tools have been developed and characterized with the ultimate goal of achieving efficient and low cost production of desired biomolecules.…”

“…Also, some genetic manipulations require auxotrophic mutants, i.e., strains lacking the ability to synthesize an organic molecule, which can be restored upon transformation with a plasmid encoding both the protein of interest to be expressed and the genetic sequence of such organic molecule. However, as suggested elsewhere, genetic engineering of polyploid industrial yeast strains presents the advantage of higher growth and metabolic rates (Latorre-García et al 2008). Although probiotic S. cerevisiae strains have been widely studied for their probiotic potential, very few works focused on their genetic transformation.…”

“…Since there are innumerous techniques, plasmids, and other DNA sequences optimized and used successfully in genetic manipulation of laboratorial non-probiotic S. cerevisiae strains (Da Silva and Srikrishnan 2012), the similarity among different strains at the genetic and protein level justifies the application of these genetic tools for probiotic S. cerevisiae transformation. In fact, most of the aforementioned works concerning genetic manipulation of S. boulardii used either genetic sequences fully (Latorre-García et al 2008;Pohlmann et al 2013;Michael et al 2013;Douradinha et al 2014;Hudson et al 2014) or partially (Wang et al 2014) derived from nonprobiotic S. cerevisiae strains. Only two works used S. boulardii homologous sequences (Hamedi et al 2013;Wang et al 2014).…”

“…cerevisiae genetic manipulations have helped us to unravel many of the physiological and metabolic processes of the eukaryotic cell, and have been routinely use to produce molecules of biotechnological relevance (Ardiani et al 2010;Da Silva and Srikrishnan 2012;Reis et al 2012). More recently, transformation of S. boulardii showed it is possible to use this probiotic yeast to also produce molecules with industrial (Latorre-García et al 2008) and biotherapeutic potential (Pohlmann et al 2013;Michael et al 2013;Wang et al 2014). For instance, probiotic strains could be engineered to express antigens for specific intestinal disorders, such as mucin MUC-1 for colon cancer (Byrd and Bresalier 2004) or adhesion protein intimin for pathogenic E. coli O157:H7 (Gedek 1999;Oliveira et al 2012).…”