Construction Of Self-cloning Industrial Brewing Yeast With High-SOD And Low-diacetyl Production

Diacetyl is one of the main flavor mass which influence beer,whose leak point is very low in beer, so the content of diacetyl in beer is the important signal of the quality's fit and unfit.Ageing-resistance also is a problem to beer brewing people for a long time.The oxyradical in beer is one of the major reasons which induce beer's flavor to ageing.If we can research the tow things together,it must bring to good result in improving stability of beefs flavor,perverting beer's ageing,and even being good for drinker's health.This experimentation uses TSINGTAO brewers yeast industry strains YSF31 as materials,deploying self-cloning technique to construct a new brewers yeast industry strains,whose content of diacetyl and capability of aging-resistant comparing with YSF31 is lower and higher.Used the general DNA of TSINGTAO brewers yeast industry strains YSF31 as a template to amplify superoxide dismutase gene SOD1 and a-factor gene by PCR.A 0.47kb fragment flanged with SphI-SalI recognition site sequence containing SOD1 gene from PCR was inserted into the SphI and SalI sites of plasmid pYCUP(containing CUP1 gene, conserved by China Academic of Since,microorganism institute,yeast genetic breeding laboratory) to form a new recombination plasmid pMC2.A 0.31kb RcoRI-SphI fragment containingα-factor gene from PCR was inserted into the EcoRI and SamI sites of plasmid pMP1(containing 3-phosphoglyceric kinase gene PGK1,conserved by laboratory) to form another recombined plasmid pMC1.A about 1.5kb Bglâ…¡-SphI fragment containing SOD1 and CUP1 from plasmid pMC2 and the about 1.7kb KpnI-SphI fragment containingα-factor and PGK1 from plasmid pMC1 were inserted into the Kpnâ… and Bglâ…¡sites of plasmid pPILV4(containing ILV2 gene,conserved by laboratory) to generate the recombinant plasmid pMC572.In pMC572 internal fragment ofα-acetohydroxy acid synthase(AHAS) gene(ILV2) was replaced with a copper resistant gene(CUP1) and a fused gene.The fused gene was constructed with Saccharomyces cerevisiae genes,PGK1 promoter,α-factor sequence and open reading frame of SOD gene.YSF31 was transformed with fragment linear from pMC572.The major results as follows:1.According to the resistance to CuSO₄,transformant is higher than recipient. Recipient could only grow on YPD with 5mM CuSO₄,but transformant could grow on YPD with 8mM CuSO₄.2.Using different assemblies of PCR amplification,find transformant can get all destination strap,but recipient can't.3.Detecting AHAS activity,the activities of transformant is half of that of recipient. Certificating the fragments of SOD1,CUP1,PGK1,a-factor all have been integrated in chromosome,a copy of allelomorphic gene of ILV2 has been destroyed.The transformant was designated as Z-2-8.4.SOD in Saccharomyces cerevisiae can't secrete by itself.Because ofα-factor gene integration,SOD was successfully secreted.But there is no activity of SOD in recipient's fermentation liquor.Meanwhile acetoin was produced at concentrations of 50%lower than that in the control,which must be induced in depressing the content of diacetyl.5.In CO₂ weight relief experimentation,detecting biomass,and others fermentation indexes and flavor substances experimentation,the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type.There are not any exogenous genes involved in this experimentation.They are all come from Saccharomyces cerevisiae itself.It may be more acceptable to the public.