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Abstract：
Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).
摘要：
解脂耶氏酵母是用于异源蛋白质生产的替代酵母。基于自克隆载体，开发了一套18个显色克隆载体，每个都包含一个可切除的营养缺陷型选择标记URA3ex，LYS5ex和LEU2ex,六个不同的启动子之一：组成型pTEF，相位依赖性混合pHp4d，和来自pEYK1和pEYL1衍生物的赤藓糖醇诱导型启动子。这些载体允许增加目的基因的克隆速度。并行,一种改进的新rProt受体菌株JMY8647是通过消除丝线和引入赖氨酸营养缺陷型（Lys-）而开发的，为基因工程提供了额外的标记。使用这种克隆策略，确定了米根霉ROL脂肪酶分泌的最佳靶向序列。在八个靶向序列中，与野生型ROL信号序列相比,SP6信号序列导致脂肪酶活性提高23%。使用杂合赤藓糖醇诱导型启动子pHU8EYK和pEYL1-5AB获得了更高的比脂肪酶活性，分别为1.9倍和2.2倍，与组成型pTEF启动子相比。与pTEF单拷贝菌株相比，两个拷贝菌株的脂肪酶活性增加了3.3倍（266.7mU/mg对79.7mU/mg）。
