BAT2缺失酿酒酵母基因工程安全菌株的构建及其杂交育种

为了获得低产高级醇基因工程安全菌株,首先将带有Cre重组酶编码基因的pGAPZA质粒转入BAT2缺失单倍体突变株A8-B和C22-B中,利用Cre/loxp系统去除G418抗性基因(KanMX),获得酿酒酵母单倍体突变株ΔBAT2a和ΔBAT2α。然后将这2株单倍体杂交成双倍体菌株ΔBAT2,该菌株具有良好的遗传稳定性。以野生菌株AY15和2株单倍体出发菌株为对照,对基因工程安全菌株进行白酒发酵试验,实验结果显示,基因工程安全菌株ΔBAT2与出发菌株相比,CO2失重高,残糖低,乙醇含量高;与野生菌株AY15相比,异丁醇、异戊醇、总高级醇生成量分别降低50.00%、33.40%和30.57%。

Construction and Crossing Trial of BAT2 Deletion Mutants Lacking Antibiotic Resistant Gene in Saccharomyces cerevisiae

In order to obtain an engineering strain lacking antibiotic resistant gene with low-yield of higher alcohols,the plasmid pGAPZA with the gene encoding the Cre recombinase was transformed into the BAT2 deletion haploid mutants A8-B and C22-B.Then the G418 resistance gene(KanMX) was wiped off by Cre/loxp system from the mutants A8-B and C22-B,and Saccharomyces cerevisiae haploid mutants ΔBAT2a and ΔBAT2α were obtained.These two haploids were mated into a diploid ΔBAT2,which was verified to be genetically stable.Moreover,at the end of the liquor fermentation,the main fermentation profiles of the wild-type strain AY15,the parental strains A8-B and C22-B and the engineering strain ΔBAT2 were determined.The experiment results showed that the engineering strain ΔBAT2 was compared with the parental strains,weight loss of CO2 was higher,the residual total sugar was lower,ethanol was higher,yet the engineering strain was compared with the wild-type strain,the amount of isobutyl alcohol,isoamyl alcohol and the total higher alcohols were respectively reduced by 50.00%,33.40% and 30.57%.The engineering strain lacking antibiotic resistant genes with low-yield of higher alcohols was used as an excellent strain in the liquor industry.