Induction of production and secretion β(1→4) glucanase withSaccharomyces cerevesiaeby replacing theMET10gene withegl1gene fromTrichoderma reesei

Abstract

Aims: To construct novel brewer’s yeast strains with the ability to degrade β‐glucan and increase sulfite levels in beer brewing by genetic manipulation. Methods and Results: The recombinant plasmid pA15ME containing Pmet10‐egl1‐Tmet10 expression cassette was constructed. BamHI‐linearized target plasmid pA15ME was transformed into the industrial brewer’s yeast strain Z0103 to replace the MET10 locus through one‐step gene replacement. The recombinants Z8, Z7 and Z3 with the ability to secrete active endo‐β‐1,4‐glucanase I into the culture medium were isolated by Congo red dyeing. The enzymatic activities of EG I of Z8, Z7 and Z3 were 3·3, 1·5, 1·3 U l−1, and the hydrolysing degrees of β‐glucans in wort were increased 11·9%, 8·6% and 6·9%, respectively, than that of original strain Z0103. The MET10 gene deletions were confirmed by real‐time PCR, and the sulfite levels of the culture mediums inoculated with Z8, Z7 and Z3 were increased 26%, 16% and 17%, respectively, compared to that of Z0103. Conclusions: The novel endoglucanase‐producing brewer’s yeast strains with inserted endoglucanase gene and deficient MET10 gene led to reduced content of barley β‐glucans, enhanced filterability and increased sulfur dioxide in fermenting wort. Thus, the cost for addition of microbial β‐glucanase enzyme and sulfite preparations in normal beer brewing processes could be reduced. Significance and Impact of the Study: These results suggested that genetic engineering approach is a powerful tool to construct the novel recombinant brewer’s yeast strains with different properties to reduce the cost of beer brewing and improve the flavour of a beer, and the strains obtained have potential application value in beer brewing.

Synthetic enzyme mixtures for biomass deconstruction: Production and optimization of a core set

“…1). The same protein expressed in Saccharomyces cerevisiae had a MW of ∼97 kDa due to hyperglycosylation (Lu et al, 2009), and hyperglycosylation might also account for the higher MW of EG1 in P. pastoris . EX3 expressed in P. pastoris ran as a tight doublet with apparent MW's of 30–32 kDa (Fig.…”

Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae

“…Cellulase-expressing recombinant Saccharomyces cerevisiae strains have potential applications in bioethanol production from cellulosic substrates, as part of a consolidated bio-processing strategy [16, 33-35, 62, 64], in wine production, as a way to improve the efficiency of the maceration process [31,61], and in beer brewing, by reducing the content of barley b-glucans in fermenting wort and enhancing filterability as a result [4,21,23,32]. The aim of the work we present was to evaluate the possibility of expressing the celA (GenBank Access No.…”

“…The expression of an endo-b-1,4-glucanase, among other enzymes, in a recombinant wine yeast strain, decreased turbidity after maceration and increased free-flow wine, showing the potential of recombinant cellulase-secreting wine yeast strains in the commercial-scale processing and clarification, color extraction and stabilization and aroma enhancement of wine [31,61]. Similarly, the expression of endo-b-1,4-glucanase egl1 gene from Trichoderma reesei in a brewer's yeast strain led to a reduction of the content of barley b-glucans in the beer wort, enhancing filterability as a result [32].…”

“…Other potential, and more traditional, fields of application of S. cerevisiae strains expressing recombinant cellulases are those of wine-making [12,31,49,61] and beer brewing [4,21,23,32]. The expression of an endo-b-1,4-glucanase, among other enzymes, in a recombinant wine yeast strain, decreased turbidity after maceration and increased free-flow wine, showing the potential of recombinant cellulase-secreting wine yeast strains in the commercial-scale processing and clarification, color extraction and stabilization and aroma enhancement of wine [31,61].…”

Trichoderma

“…As an alternative, genetic engineering of brewer's yeast strains with genes encoding β-glucanases offers a tool to improve beer quality and to reduce brewing costs. Strains of S. cerevisiae have been constructed harboring the gene egl1 from T. reesei which encodes an endo-β-1,4-glucanase, with application resulting in an improvement not only in hydrolysis of β-glucans but also with regard to beer flavor (Penttilä et al, 1987(Penttilä et al, , 1988Zurbriggen et al, 1991;Lu et al, 2009).…”