Ma Sijia, Wang Yanan, Jiao Xiang, Zhang Sufang, Zhao Zongbao K
Wei Sheng Wu Xue Bao. 2015 Nov 4;55(11):1505-11.
To construct a phosphate starvation derepressed expression vector for functional integration and expression of exogenous genes in Rhodosporidium toruloides.
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.
pPHO89 was regulated by phosphate and activated when the recombinant R. toruloides strains were grown under phosphate-limited condition.
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.
构建一种用于外源基因在球拟酵母中进行功能整合与表达的磷酸盐饥饿去阻遏表达载体。
通过生物信息学分析预测钠：无机磷酸盐共转运体启动子pPHO89和热休克蛋白终止子tHSP。从产油酵母球拟酵母NP11的cDNA中扩增这两个元件的DNA片段。然后，分别使用无限制（RF）克隆方法，将载体pZPK-pPGK-hyg-tNOS中的甘油酸激酶启动子pPGK和终止子tNOS替换为pPHO89和tHSP。保留潮霉素磷酸转移酶基因hyg作为报告基因。使用农杆菌介导的转化（ATMT）将所得载体pZPK-pPHO89-hyg-tHSP转化到球拟酵母中。具有潮霉素抗性的转化体可证实pPHO89启动子和tHSP终止子的功能。此外，我们开发了新的诱导型载体，其包含由组成型pPGK启动子和诱导型pPHO89启动子驱动的两个表达盒。
pPHO89受磷酸盐调节，当重组球拟酵母菌株在磷酸盐限制条件下生长时被激活。
就磷酸盐调节、相对响应强度、简便性和成本效益而言，pPHO89是一个理想的启动子。这些ATMT载体可为产油酵母的合理工程改造提供有用工具，以生产脂肪酸衍生的生物燃料和生物化学品。
