Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.
Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.
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DOI：
10.1371/journal.pone.0158682
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Luzhetskyy A
中科院分区：
文献类型：
--
作者：
Bilyk O;Sekurova ON;Zotchev SB;Luzhetskyy A
酵母中的转化相关重组（TAR）是一种快速且廉价的克隆和组装大DNA片段的方法，其依赖于天然同源重组。基于p15 a和F因子复制子的两个载体，可以在酵母中维持，E.大肠杆菌和链霉菌。这些载体已成功地用于装配链霉菌Acta 1362的grecocycline生物合成基因簇。通过PCR获得簇的片段，并与“捕获”载体一起转化到酵母细胞中，产生携带整个基因簇的构建体。获得的构建体在S. albus J1074，产生几种grecocycline同系物。Grecocyclines具有独特的结构部分，如二糖侧链、C-5位的额外氨基糖和巯基。来自该途径的酶可用于已知活性angucyclines的衍生化，以改善其所需的生物学性质。
Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the “capture” vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties.