Construction of a Single PEP4 Allele Deletion in Saccharomyces carlsbergensis and a Preliminary Evaluation of its Brewing Performance

Junguang Hao^1,2, Jianjun Dong², R. Alex. Speers³, Wei Shen¹, Lianju Shan², Wei Fan², Qi Li^1,4, Guoxian Gu¹ and Jian Chen¹
¹ State Key Lab of Food Science and Technology, Ministry of Education, Jiangnan University, Wuxi, 214122, P. R. China.
² Research Center of Tsingtao Brewery Group, Qingdao, 266061, P. R. China.
³ Food Science Program, Dalhousie University, Halifax, NS, Canada.
⁴ Corresponding author. E-mail: liqi@jiangnan.edu.cn

J. Inst. Brew. 114(4), 322–328, 2008 | VIEW ARTICLE

ABSTRACT
The secretion of proteinase A (encoded by PEP4) from brewer’s yeast is detrimental to the foam stability of unpasteurized beer. The aim of this study was to construct mutants of the allopolyploid Saccharomyces carlsbergensis strain TT, which were partially or completely deficient in proteinase A activity. Allelic PEP4 genes were consecutively disrupted by using the Cre-loxP recombination system combined with PCR-mediated gene disruption. A single PEP4 deletion mutant TT-M was successfully constructed. However, no viable mutant could be obtained when the second allelic PEP4 gene was deleted. The brewing performances of the parent strain and the modified strain were compared on a 100 L pilot fermenter scale. Proteinase A activity in fermented wort brewed with mutant strain TT-M was significantly lower (p<0.05) than that of the parent strain TT, whereas no significant difference on either maltose or maltotriose assimilation (p>0.05) was found. The mutant TT-M remained genetically stable, as shown by diagnostic PCR, after re-streaking for 20 generations. The flavor and taste of the final fermented wort, brewed with the mutant strain TT-M, was evaluated by the Tsingtao expert sensory panel, and found to be comparable to that of the parent strain and exhibited no distinct defects. The flavor component profiles of these two finished products were also comparable. The study demonstrated allelic genes in polyploidy industrial yeasts could be efficiently and consecutively deleted by the retractive primer disruption strategy, and the mutant of Saccharomyces carlsbergensis partially deficient in proteinase A contributed to an improvement in foam stability.

Key words:
Beer foam stability, gene disruption, PEP4, Proteinase A.