rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用

根据酵母整合质粒的设计要求，PCR扩增特定的2．2kb rDNA片段，并以此替换酿酒酵母(Saccharomyces cereristae)整合载体YIp5的URA3片段；在此基础上，引入G418抗性基因KanMX和酵母磷酸甘油激酶(phosphoglycerale kinase，PGK)组成型强启动子和终止子序列(PGKp-t)，构建适合酿酒酵母工业菌株高拷贝整合表达载体pYMIKP，以细菌木糖异构酶(xylose isomerase，XI)基因xy/A为目标基因，通过载体pYMIKP引入到酵母工业菌株NAN-27中，酵母转化子在非选择培养条件下，连续生长50世代质粒稳定性为99．72％，目标基因高拷贝重组菌的木糖异构酶比酶活是对照菌株的67．2倍，达到0．672U／mg蛋白，实现了外源基因在酿酒酵母工业菌株中的稳定高效表达。

Construction of a ribosomal DNA multi-copy integration vector and application in the industrial Saccharomyces cerevisiae strain

Based on the YIp5 (yeast integration vector), construction of the multi-copy integration vector pYMIKP was investigated by using a 2.2 kb designed rDNA fragment from Saccharomyces cerevisiae to replace the original URA3 gene for targeted homologous recombination. The G418 resistance gene KanMX was used as the dominant selection marker, and yeast phosphoglyceric kinase (PGK) constitutive promoter and terminator (PGKp-t) were used for expression control element in the pYMIKP respectively. The plasmid carrying xylA gene was transformed and the xylA gene was integrated into genome rDNA locus of S. cerevisiae NAN-27 by using the pYMIKP. The xylose isomerase XI specific activity of recombinant strain XH34 is 0.672 U/mg protein, which is 67.2 times as much as the parent strain. The integrants are mitotically stable for 50 generations under the non-selective pressure condition.