Induction of production and secretion beta(1-->4) glucanase with Saccharomyces cerevesiae by replacing the MET10 gene with egl1 gene from Trichoderma reesei.
OBJECTIVE
To construct novel brewer's yeast strains with the ability to degrade beta-glucan and increase sulfite levels in beer brewing by genetic manipulation.


RESULTS
The recombinant plasmid pA15ME containing P(met10)-egl1-T(met10) 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-beta-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 beta-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 beta-glucans, enhanced filterability and increased sulfur dioxide in fermenting wort. Thus, the cost for addition of microbial beta-glucanase enzyme and sulfite preparations in normal beer brewing processes could be reduced.


CONCLUSIONS
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.
Shandong University, Jinan, China.
Y
Y
Lu
T-H
TH
Wang
X-L
XL
Ding
D001515:Beer / Q000382:microbiology; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005285:Fermentation; D005516:Food Microbiology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D050864:Sulfite Reductase (NADPH) / Q000235:genetics; D013447:Sulfites / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics; D047071:beta-Glucans / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D013447:Sulfites; D047071:beta-Glucans; D050864:Sulfite Reductase (NADPH); C494326:sulfite reductase (NADPH), S cerevisiae; D002480:Cellulase; C090774:endoglucanase EGL1