Construction Of S. Carlsbergensis With Low Protease A Activity For Brewing

Protease A[EC3.4.23.6], an aspartic proteolytic enzyme, is encoded by PEP4 gene in S. cerevisiae. It can be excreted under stress conditions or leaked by autolysis into fermented wort during Lager beer brewing process using S. carlsbergensis, Lager beer yeast, which detriments foam stability of final draft beer. With an aim to improve foam stability of draft beer by managing to get a commercial strain with low-protease A level, following efforts were taken in this study: identify proteins profile of the draft beer foam, find out the difference of foam stability between draft beer and pasteurized beer, trace the change of protease A in larger scale production, construct low or non protease A strain by gene knock-out, select out low protease A upgraded strain within resultant transformants, and investigate fermentation performance of upgraded strains by 100L pilot trial.Composition and concentrations of foam proteins play a key role in foam stability of draft beer, while no unequivocal view on key foam proteins profile has been achieved by far. Traditional measurements such as SDS PAGE, chromatography, amino acids analysis, were employed to clarify foam proteins profile in previous investigations, which possibly hid the truth of it. To better understand foam proteins, advanced modern measurements were adopted including two dimensional electrophoresis,MALDI-TOF MS,LC/MS/MS in this study, besides the traditional SDS PAGE and amino acids analysis. Results of SDS PAGE and amino acids analysis suggested primarily that proteins were complex, not only confined to protein Z and LTP1. Results of two dimensional electrophoresis further clarified the complexity of foam proteins and uncovered the similar distributions of proteins between foam and beer with different content and characterizations. Glycosylatin forms of LTP1 were detected by MALDI -TOF MS in beer foam though no other valuable information was gained. Totally, there were 23 proteins detected in draft beer foam by LC/MS/MS, most of them were not reported by other before, which enlarges and updates the list of proteins related to foam.Foam stability of draft beer and pasteurized beer were compared which indicated that protease A is direct negative factor of foam stability of draft beer. Karyotypes of S. carlsbergensis 04-1 and B, used by different breweries in Tsingtao Brewery Group, were similar with a little difference. The relation between cropped generations and protease A activity of them was parallel. The protease A activity of drafted beer brewed with 1&2 cropped generation S. carlsbergensis 04-1 and B was low, while higher brewed with higher generation yeast. The mimic PU treatment was adopted to verify the effect of pasteurization units on protease A and invertase activity. There was no activity of protease A in beer treated with PU 7, while invertase activity still could be detected. The foam stability increased relatively as the treated PU value increased in the range of 0-7.It is effective and direct means to increase foam stability of draft beer by constructing low or no protease A activity brewing strain through encoding gene PEP4 deletion. S. carlsbergensis is a tetraploid whose detailed copy number and detailed sequence of PEP4 are uncertain. To achieve a PEP4 absent strain in tetraploid, multiple gene disruptions should be taken. In this study, dominant selective marker Sh ble and KanMX were applied by construction plasmid pSH-Zeo and PCR with pUG6 as template. Combined Cre-LoxP and PCR meditated gene disruption, mutants with pep4::LoxP/PEP4 were gained. As KanMX was deleted ultimately from transformant pep4::KanMX/PEP4 in this study, gene security of modified strain was improved without heterogenous gene remained.To avoid reverse mutation caused by repeated deleting allelic genes in allopliod with same PCR product, retractive primer design strategy was suggested and applied in this study. By this means, a pep4::LoxP/pep4::KanMX strain was gained. But it did not survive by its special expressive and regulative regime after second PEP4 deletion. It is indicated that there are only two PEP4 allelic genes with the same sequence to S. cerevisiae in S. carlsbergensis used by Tsingtao Brewery Group, which play a key role in S. carlsbergensis metabolism and survival.The low protease A upgraded strain, S. carlsbergensis 04-1-M and B-M, was gained by successive selection within pep4::LoxP/PEP4 transformants, mainly taking fermentation degree and protease A activity of fermented wort of laboratory brewing trials in account. PCR verified that the mutant gene pep4::LoxP remained stable in offspring strains which ensured the inheritance of corresponding low protease A characteristic of the upgraded strain. The fermentation degrees of upgraded strains were lower than their wild strains, while flocculation values are higher. Lethal temperature of S. carlsbergensis 04-1-M was 51℃, which was different from all the others.In 100 L pilot brewing trail, all the two upgraded strains perform similar to their counterparts, such as assimilation of amino acids & fermentable saccharides, propagation of yeast, ect. Relatively slow attenuation rate of upgraded strains resulted in a little more maltotriose left in theirs final fermented worts. The concentration of diacetyl, pentanedion, acetoin in final fermented worts brewed by upgraded strains were higher, while ratio of higher alcohol to ester, fermentation degree, protease A activity were lower, than those of their counterparts. The flavor and taste of final fermented worts brewed by upgraded ones were evaluated by experienced taste panel of Tsingtao Brewery Group, no defect were detected by them. More trails are suggested for industrial application though its promising prospective is implied by our 100 L pilot brew.