Phenolic acid decarboxylase (PADC) gene, encoding phenolic acid decarboxylase, was cloned from Bacillus subtilis and ligated with a shuttle vector YEp352 to generate a novel plasmid YPADC. By analysis of sequencing and the restriction endonuclease digestion, the validity of construction was proved. Subsequently, the new vector was successfully transformed into wild-type top-fermenting yeast strain W303-1A; the mutant yeast strain W303+padc was obtained, which was tested on the laboratory-scale mashing and fermentation experiments. At the end of fermentation, the results showed an obvious increase of 4-vinylguaiacol content in top-fermented beers brewed with mutant yeasts. The final 4-vinylguaiacol concentration obtained with wild-type and mutant yeasts was 1.20 and 1.70mg/l, respectively. Additionally, the level of esters produced by the mutant strain was higher than that of the wild-type; there were therefore a marked clove-like and ester aroma in top-fermented beers brewed with the former. However, no evident differences were found in brewing characteristic between wild-type and mutant strains, especially the ability of utilizing fermentable sugar and reducing diacetyl. Taken together, these approaches indicated the possibility of cloning PADC gene and enhancing the concentration of 4-vinylguaiacol in top-fermented beers.
Key words: Clone, phenolic acid decarboxylase, top-fermenting yeast, 4-vinylguaiacol.
4VG, Vinylguaiacol; 4VP, 4-vinylphenol; 4EG, 4-ethylguaiacol; 4EP,4-ethylphenol; POF, phenolic off-flavor; PADC, phenolic acid decarboxylase gene;HPLC, high performance liquid chromatography; YPD, yeast peptone dextrose; LB,Luria Bertani; PCR, polymerase chain reaction; PET, polyethylene terephthalate.
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