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首页> 外文期刊>Applied Microbiology >β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes
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β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes

机译：β-葡萄糖醛酸苷酶作为放线菌的灵敏而多功能的报告基因

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摘要

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA , which encodes the β-glucuronidase enzyme. A series of gusA- containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1 , plaR2 , and plaR3 , involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N -epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter.
机译：在这里，我们描述了一种基于gusA的放线菌通用且敏感的报告系统，该系统编码β-葡萄糖醛酸苷酶。构建了一系列包含gusA的转录和翻译融合载体，并用于研究苯乙内酯生物合成基因簇的调控级联。此外，这些载体用于研究ATG，GTG，TTG和CTG起始密码子的翻译起始效率。出人意料的是，使用TTG起始密码子的构建体显示出最佳的活性，而使用ATG或GTG的构建体的活性分别约为二分之一或三分之一。 CTG融合仅显示了TTG融合活性的5％。自杀载体pKGLP2在其主链上带有gusA，可用于肉眼观察类金属倍体的形成和分解，从而使放线菌中的基因靶向变得更快，更容易。使用该系统将参与苯乙内酯生物合成的三个调节基因plaR1，plaR2和plaR3有效地替换为阿普霉素抗性标记。最后，我们以GusA蛋白为报告基因，通过引入非蛋白氨基酸N-ε-环戊氧基羰基-1-赖氨酸，扩大了放线菌的遗传密码。

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