【摘要】 渗透压调控启动子在高底物浓度或盐类物质诱导下可实现目标基因的高表达和可控表达,因此可用于构建新的代谢工程菌株.利用来源于可耐高渗的产甘油假丝酵母(Candida glycerinogenes)的渗透压调控启动子PCgSTL3、PCgZWF和PCgGPD,在酿酒酵母(Saccharomyces cerevisiae)中控制木糖醇脱氢酶基因XYL2的表达水平,并研究其在NaCl诱导下对木糖醇脱氢酶活性及重组酿酒酵母木糖代谢流的影响.结果显示,以组成型强启动子PTPI为对照,添加0.4 mol/L NaCl作为渗透压诱导剂后,渗透压调控启动子控制下的木糖醇脱氢酶活性均得到增强,其中在PCgGPD控制下其活性变化最为明显,酶活提高了2.8倍.同时,以PCgGPD控制XYL2基因表达的重组菌木糖消耗量、乙醇产量和产率均为最高,与无诱导剂相比分别提高了62.3%、30.7%和9.7%,副产物木糖醇产率最低,下降了53.3%.本研究表明,渗透压调控启动子PCgGPD可显著提高木糖醇脱氢酶活性,通过添加NaCl作为渗透压诱导剂可实现对XYL2基因的可控表达,显著降低木糖醇积累,提高乙醇产率.更多还原

【Abstract】 This research aimed to investigate the xylose metabolism in Saccharomyces cerevisiae with the xyltiol dehydrogenase gene XYL2 overexpressed at different levels by osmo-responsive promoters. Xylose-metabolizing strains were constructed by expressing the xylitol dehydrogenase gene XYL2 using osmo-responsive promoters PCg STL3, PCg ZWF and PCg GPD fromCandida glycerinogenes in the multicopy plasmid p YX212. The activity of xylitol dehydrogenase and xylose-fermenting abilities of the recombination strains were measured. The recombinants with XYL2 gene expressed under osmo-responsive promoters were cultured with 0.4 mol/L Na Cl as inducers. Increased activities of XDH were exhibited. The XDH activity of the recombinant SPG-xyl2 whose XYL2 was controlled by PCg GPD was approximately 2.8 times that of the recombinant whose XYL2 gene was expressed under PTPI. The strain SPG-xyl2 showed higher ethanol production(30.7%), ethanol yield(9.7%) and lower xylitol(53.3%) yield when cultivated in YPX with 0.4 mol/L NaCl. The results indicated that the XDH activity can be significantly improved by the overexpression of XYL2 gene using an osmo-responsive promoter PCg GPD. When the additional NaCl was added as osmo-inducer, the utilization of xylose was promoted with an increased yield of ethanol as well as reduced accumulation of xylitol.更多还原