MXR1基因对毕赤酵母工程菌代谢调控的影响

The Effect of MXR1 Gene on Metabolic Regulation in Pichia pastoris

为了研究毕赤酵母中转录因子Mxr1p在毕赤酵母代谢调控中所起的作用,构建一株以南极假丝酵母脂肪酶B基因(CALB)作为报告基因,MXR1基因完全缺失的毕赤酵母基因工程菌株。将重组质粒pPIC9K-CALB转化毕赤酵母GS115,利用三丁酸甘油酯平板筛选得到分泌表达CALB的重组菌GS115/pPIC9K-CALB。通过重叠延伸PCR方法获得一段中间含有博来霉素抗性基因sh b le,两翼大约各有1 200 bp与毕赤酵母MXR1基因上下游同源的基因片段,将此片段用氯化锂法转化毕赤酵母细胞GS115/pPIC9K-CALB后,利用博来霉素抗性及CALB酶活力丧失双重筛选的方法得到一株MXR1基因完全缺失的毕赤酵母基因工程菌株。该菌株在以甲醇为唯一碳源的培养基中不生长,在以乙醇、葡萄糖或者甘油为唯一碳源的培养基中生长缓慢。结果表明转录Mxr1p因子在毕赤酵母中的多条代谢途径中起着关键性的作用,主要涉及甲醇、乙醇、甘油和葡萄糖等代谢途径。

To investigate the effect of transcriptional factor Mxr1p on metabolism regulation in P.pastoris,we constructed a recombinant strain which was knocked out the gene of MXR1 with Candida antarctica Lipase B（CALB）as a report gene,then transformed P.pastoris GS115 using plasmid pPIC9K-CALB,obtained recombinant strain GS115/pPIC9K-CALB by screening with Butanoic acid,1,2,3-propanetriyl ester as substrate.A large fragment was obtained by connecting the gene sequence of the upper stream and lower stream of MXR1,and the gene sequence of sh ble gene using overlap extension PCR was transformed into P.pastoris GS115/pPIC9K-CALB by LiAc method,then the transformants were selected through the resistance of zeocin and the enzyme activity of CALB.A transformant with a characteristic of litter growth using methanol as sole carbon source and low growth using glycerol,glucose,alcohol as sole carbon source was obtained.The study indicated that Mxr1p may play a key role in many metabolism pathways,including methanol metabolism,glycerol metabolism,glucose metabolism and ethanol metabolism.