rDNA介导的菊粉酶基因整合载体构建及在K.marxianus中应用

Construction of INU gene integration vector at rDNA-targeting locus and its application to K.marxianus

为提高Kluyveromyces marxianus(K.marxianus)发酵菊芋粉生产乙醇过程中的菊粉酶活性,以酿酒酵母rDNA为整合位点,构建菊粉酶基因的整合表达载体pFA6a-rDNA-pgk-inu.经Sph I线性化后,采用电击法转化K.marxianus,使表达载体整合到K.marxianus的染色体上.经PCR鉴定的阳性转化子能够分泌菊粉酶且能在无筛选压力下稳定遗传50代.重组菌K/r-2分泌的菊粉酶活力为140U/mL,比出发菌株提高了80%.以粗菊芋粉生料为底物进行了补料发酵,重组菌株的发酵终点乙醇浓度为76.5g/L,比出发菌株提高了5g/L,达到理论转化率的96%.这些研究工作为非粮作物菊芋生产燃料乙醇奠定了基础.

In order to improve inulinase activity of Kluyveromyces marxianus （K. marxianus） in the process of producing ethanol with Jerusalem artichoke meal, using rDNA of S. cerevisiae as integration sites, the integration vector pFA6a-rDNA-pgk-inu of INU gene is constructed. Digested by Sph I, the linear vector pFA6a-rDNA-pgk-inu is integrated into genome of K. marxianus by electroporation. The positive integrants identified by PCR show the inulinase activity and are mitotically stable for 50 generations under the non-selective pressure conditions. The inulinase activity of recombinant strain K/r-2 gets the highest 140 U/mL, 80% higher than that of the wild-type. In the experiments of fed-batch fermentation using the uncooked Jerusalem artichoke meal, the ethanol concentration in broth of integrants is 76.5 g/L, 5 g/L higher than that of the wild-type strain with 96% of its theoretical value. Such research work lays a good foundation for producing fuel ethanol from Jerusalem artichoke.