Bioscience Laboratories, Meiji Seika Kaisha, Ltd., 788 Kayama, Odawara-shi, Kanagawa 250-0852, Japan.
J Bacteriol. 2011 Apr;193(8):1815-22. doi: 10.1128/JB.01325-10. Epub 2011 Feb 4.
Previously, we showed that a 145-kb DNA region, including the entire kanamycin biosynthetic gene cluster (with two kanamycin resistance genes), was tandemly amplified up to 36-fold in an industrial strain of Streptomyces kanamyceticus. Strain improvement had included the use of increased kanamycin resistance as an initial potential indicator of higher kanamycin productivity. We were able to recapitulate the DNA amplification by cultivating S. kanamyceticus under selection for kanamycin resistance. To identify the genes required for amplification, various chromosome deletions were constructed, and the DNA amplification was shown to depend on orf1082 (zouA), present in a putative mobile genetic element. ZouA consists of 1,481 amino acids and is homologous to the products of traA-like genes of some conjugative plasmids. These genes encode relaxases that initiate DNA transfer during conjugation by single-strand nicking at oriT. As in the original high-producing strain, DNA amplification occurred between 16-nucleotide (nt) sites (RsA and RsB) containing 14 identical nucleotides. Interestingly, RsA lies just 80 bp upstream of the initiation codon of zouA and is partially contained in an inverted repeat structure similar to those found in plasmid oriT sequences, suggesting that it might function in a manner similar to that of oriT. We therefore propose that DNA amplification in S. kanamyceticus is initiated by relaxase-mediated recombination between oriT-related sequences.
先前，我们表明，在链霉菌（Streptomyces kanamyceticus）的工业菌株中，包括整个卡那霉素生物合成基因簇（带有两个卡那霉素抗性基因）在内的 145kb DNA 区域被串联扩增了 36 倍。菌株改良包括增加卡那霉素抗性作为提高卡那霉素生产力的潜在指标。我们通过在卡那霉素抗性选择下培养 S. kanamyceticus 能够重现 DNA 扩增。为了鉴定扩增所需的基因，构建了各种染色体缺失，结果表明扩增依赖于或f1082（zouA），其存在于一个假定的可移动遗传元件中。ZouA 由 1481 个氨基酸组成，与一些可接合质粒的 traA 样基因的产物同源。这些基因编码松弛酶，在接合过程中通过 oriT 处的单链切口启动 DNA 转移。与原始高产菌株一样，DNA 扩增发生在含有 14 个相同核苷酸的 16 个核苷酸（nt）位点（RsA 和 RsB）之间。有趣的是，RsA 刚好位于 zouA 的起始密码子上游 80bp 处，并且部分包含类似于在质粒 oriT 序列中发现的反向重复结构，这表明它可能以类似于 oriT 的方式发挥作用。因此，我们提出链霉菌（Streptomyces kanamyceticus）中的 DNA 扩增是由松弛酶介导的 oriT 相关序列之间的重组引发的。
