圆红冬孢酵母磷酸盐饥饿诱导表达载体的构建

Phosphate starvation derepressed expression vector for engineering oleaginous yeast Ｒhodosporidium toruloides

摘要:

摘要:【目的】建立一种适用于圆红冬孢酵母代谢工程的磷酸盐饥饿诱导表达系统。【方法】对圆红冬孢酵母pho89基因5'侧翼序列进行生物信息学分析，设计相应引物，PCR扩增pho89基因启动子(pPHO89)和hsp70基因终止子(tHSP)，利用RF克隆方法置换出发载体上的pPGK组成型启动子和tNOS终止子，以潮霉素磷酸转移酶基因hyg为报告基因，得到响应磷酸盐饥饿诱导的单表达盒载体pZPK-pPHO89-hyg-tHSP，利用ATMT方法转化圆红冬孢酵母，通过转化子潮霉素抗性表型鉴定pPHO89和tHSP的启动子和终止子活 性。在此基础上，构建了适合外源基因表达的双表达盒诱导表达载体pZPK-HYG-pPHO89-MCS-tHSP，并利用该载体构建了苹果酸酶重组表达菌株。【结果】成功构建了响应磷酸盐饥饿的圆红冬孢酵母诱导性表达载体，该载体在圆红冬孢酵母中可表现出启动子和终止子活性。【结论】该启动子受磷酸盐浓度的严谨调节，响应度高，操作简单，无需额外诱导剂，经济便捷，为后续圆红冬孢酵母代谢工程研究提供了基本材料。

Abstract:

Abstract:［Objective］To construct a phosphate starvation derepressed expression vector for functional integration and expression of exogenous genes in Rhodosporidium toruloides.［Methods］Sodium: inorganic phosphate symporter promoter pPHO89 and heat shock protein terminator tHSP were predicted by bioinformatics assay．DNA fragments of the two elements were amplified from the cDNA of the oleaginous yeast R. toruloides NP11． Then the lyceric acid kinase promoter pPGK and terminator tNOS in vector pZPK-pPGK-hyg-tNOS were replaced by pPHO89 and tHSP，respectively，using restriction free (RF) cloning method． Hygromycin phosphotransferase gene hyg was retained and as a report gene． The resulting vector pZPK-pPHO89-hyg-tHSP was transformed into R． toruloides using agrobacterium-mediated transformation (ATMT). The function of the pPHO89 promoter and the tHSP terminator can be confirmed by the transformant harboring the hygromycin resistance． Furthermore，we developed new inducible vectors containing two expression cassettes driven by the constitutive pPGK promoter and inducible pPHO89 promoter． ［Results］pPHO89 was regulated by phosphate and activated when the recombinant R. toruloides strains were grown under phosphate-limited condition.［Conclusion］pPHO89 is an ideal promoter in terms of regulation by phosphate，comparative response strength，simplicity，and cost effectiveness． These ATMT vectors can provide useful tools for rational engineering of oleaginous yeast to produce fatty acid derived biofuels and biochemicals.