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.
Aims: To construct a yeast one-hybrid system and isolate transcriptional activators. Methods and Results: A 1AE1-kb promoter region of xylanase2 from Trichoderma reesei was cloned by PCR and sequenced (GenBank accession number: AY263380). Sequence analysis revealed that typical binding sites for several transcription factors in filamentous fungi, such as CREI, XLNR, ALCR, AREA and CCAAT enhancer, are located in the promoter. To isolate xyn2 transcription factors, the reporter plasmid of a yeast one-hybrid system was constructed on the backbone of the plasmid pRS415 containing the leu2 selective marker, with the xyn2 promoter region and Saccharomyces cerevisiae his4 as a reporter gene. The reporter gene contained 123-bp minimal promoter region. The S. cerevisiae H158 strain containing the reporter plasmid was transformed with a T. reesei expression cDNA library, and 34 transformants were collected from SC-Leu-His-Ura plates. The isolation of the gene ace2 from several transformants showed that the one-hybrid system approach was successful. Then, approx. 59 mg l )1 of ace2 was overexpressed in Escherichia coli BL21. Significance and Impact of the Study: The yeast one-hybrid system is suitable for isolating transcription factors of filamentous fungi. ACE II is a main and universal transcriptional activator that controls cellulase and hemicellulase transcription regulation in T. reesei.
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