Construction Of Recombinant Industrial S. Cerevisiae Strain With Barley LTP1 Secretion Capability And PrA Low Expression By Homologous Recombination

Yeast proteinase A(PrA)〔EC3.4.23.25〕, is a kind of acidic proteolytic enzyme found in vacuole of Saccharomyces cerevisiae. Membrane filtration has no effect on the activity of proteinase A in green draft beer, so the proteins and polypeptides in green draft beer could be degraded by proteinase A which derived from yeast cells before the consumption. The fact that barley lipid transfer protein 1 being a kind of crucial protein associated with beer foam formation and stability has been proved by previous studies. The degradation of this foaming protein in unpasteurized beer by proteinase A can lead to the decline of beer foam stability.In order to improve the foam properties of green draft beer, the brewing yeast gene expression regulatory elements of ADH1 promoter, MFal signal peptide sequence and CYC1 terminator were amplified by PCR using Saccharomyces cerevisiae genome as template. Barley non-specific lipid transfer protein 1 coding gene was also ampilified by PCR using the extracted genome DNA of Hordeum vulgare as template. The Barley lipid transfer protein 1(LTP1) expression cassette was constructed with ADH1 promoter, MFal signal peptide sequence and CYC1 terminator of Saccharomyces cerevisiae and LTP1 coding sequence. The Geneticin-G 418 resistance coding sequence KanMX was selected as screening marker and was cloned into the MCS of shuttle vector YEplacl81 together with the Ltpl expression cassette. The Saccharomyces cerevisiae WZ65 cells were transformed with the yeast expression plasmid YEpl81-KAMLC. The LTP1 secreted by yeast in fermentation liquor fermented with the transformant strains was assayed using HPLC method. The concentration of LTP1 in fermentation liquor was increased slowly within 24h during fermentation, then a faster incrase appeared between 24h and 72h. With the fermentation continued, the yield of LTP1 increased. The LTP1 with the concentration of 29.45mg/L was gained through 132h fermentation using YEPD liquid medium. For the stable expression of barley lipid transfer protein 1 and lower the activity of yeast proteinase A, the constructed DNA fragment containing barley Ltp1 expression cassette and KanMX was integrated into one allele of PEP4 gene. The recombinant strains with genotype of PEP4/pep4::KanMX-Ltp1 were attained by screening and verifications. The comparison performed between the two electrophoresis methods for analyzing LTP1 secreted by yeast cells indicated that Tricine-SDS-PAGE was more suitable than SDS-PAGE for separating LTP1 from the other components in fermentation liquor. The band corresponding to molecular mass of lOKDa was proved being barley lipid transfer protein 1 by further study of western-blotting.The G418 resistance marker integrated into the chromosome of yeast recombinants need to be rescued for the considering safety of the recombinant strains for industrial applications. In this study, we found that the TEF1 promoter in plasmid pTEF1/ZEO could not initiate the expression of coding gene downstream. So TEF1 promoter of Sh ble expression cassette was replaced with the TEF promoter. Plasmid pSH47/ZEO for Cre-recombinase expression was constructed by cloning the Sh ble expression cassette into plasmid pSH47. The KanMX between the two loxP sites in chromosome of the recombinant Saccharomyces cerevisiae strains was rescued using Cre/loxP system. The curing of the plasmid pSH47/ZEO in recombinant strains was carried out by subculturing. The experimental results showed that the recombinant strains with barley LTP1 secretion capability and KanMX rescued were constructed.The physiological characteristics of the recombinant strains S.c-Ltp1αand S.c-Ltp1βwere tested including genetic stability, growth performance, fermenting force, flocculability, etc. The testing results of LTP1 concentrations in fermentation liquor showed that the LTP1 concentrations secreted by recombinant strains S.c-Ltpla and S.c-Ltp1βat 108h reached 18.76 and 18.28mg/L in 12°P wort, respectively. And the LTP1 concentrations secreted by recombinant strains S.c-Ltp1αand S.c-Ltp1βat 108h reached 12.07 and 12.13mg/L in 8°P wort, respectively. After 30 generations subculturing, the genetic performance of the recombinant strains were stable. The activity of proteinase A of recombinant strains decreased compared with the control strain WZ65. The growth speed during logarithmic phase is lower than that of the control strain, but the cell-concentrations of the recombinant strains and the control strains were similarly. There are obvious differences of the other physiological characteristics between the recombinant strains and the control strain WZ65.The experimental data from the beer-brewing trial showed that the flavor of the beer brewed with the recombinant strains was similar to that of current dry-beer. Every physical-chemical indexes were reached superior grade according to the national standard, especially the head retention time was improved from 202sec to 326sec.