Department of Biological Sciences and Bioengineering, Inha University, Incheon 22212, Republic of Korea.
Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea.
J Microbiol Biotechnol. 2022 Jul 28;32(7):911-917. doi: 10.4014/jmb.2205.05036. Epub 2022 Jun 13.
As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by , contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in . The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.
作为有价值的抗生素，微生物天然产物在各个领域已经使用了几十年。其中包括多烯化合物，如制霉菌素、两性霉素和制霉菌素样多烯（NPP）。多烯大环内酯类化合物具有多种生物活性，如抗真菌和抗病毒活性。由 产生的 NPP A1 在四烯大环内酯骨架中含有独特的二糖部分。通过对 NPP A1 生物合成基因簇（BGC）的遗传操作，开发出了具有庚烷结构和提高抗真菌活性的 NPP B1。在这里，我们生成了一个人工染色体 DNA 文库，以分离出一个大型 NPP B1 BGC。通过构建和筛选细菌人工染色体（BAC）文库，成功地从 染色体中分离出 NPP B1 BGC，尽管分离出的 140-kb BAC 克隆（命名为 pNPPB1s）缺少 NPP B1 BGC 的右末端约 8 kb 部分。通过引入额外的 pNPPB1s 以及共同表达包含缺失 8 kb 的 32-kb 部分，导致 NPP B1 在 中的产量增加了 7.3 倍。qRT-PCR 证实了含有两个 NPP B1 BGC 的 菌株中 NPP B1 BGC 的转录水平显著增加。有趣的是，NPP B1 在体外表现出先前未知的 SARS-CoV-2 RNA 依赖性 RNA 聚合酶（RdRp）抑制活性。这些结果表明，在放线菌中对大型天然产物 BGC 进行 BAC 克隆是提高天然产物效价和筛选生物活性的一种有价值的方法。
