Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; University of Chinese Academy of Sciences, Beijing 100039, China.
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Metab Eng. 2017 Mar;40:80-92. doi: 10.1016/j.ymben.2017.01.004. Epub 2017 Jan 11.
Actinomycetes produce a large variety of pharmaceutically active compounds, yet production titers often require to be improved for discovery, development and large-scale manufacturing. Here, we describe a new technique, multiplexed site-specific genome engineering (MSGE) via the 'one integrase-multiple attB sites' concept, for the stable integration of secondary metabolite biosynthetic gene clusters (BGCs). Using MSGE, we achieved five-copy chromosomal integration of the pristinamycin II (PII) BGC in Streptomyces pristinaespiralis, resulting in the highest reported PII titers in flask and batch fermentations (2.2 and 2g/L, respectively). Furthermore, MSGE was successfully extended to develop a panel of powerful Streptomyces coelicolor heterologous hosts, in which up to four copies of the BGCs for chloramphenicol or anti-tumour compound YM-216391 were efficiently integrated in a single step, leading to significantly elevated productivity (2-23 times). Our multiplexed approach holds great potential for robust genome engineering of industrial actinomycetes and novel drug discovery by genome mining.
放线菌产生了大量具有药用活性的化合物，但为了发现、开发和大规模生产，其产量往往需要提高。在这里，我们描述了一种新的技术，即通过“一个整合酶-多个 attB 位点”的概念进行多重靶向特异性基因组工程（MSGE），用于稳定整合次级代谢产物生物合成基因簇（BGC）。使用 MSGE，我们在吸水链霉菌中实现了五拷贝染色体整合普那霉素 II（PII） BGC，在摇瓶和分批发酵中分别达到了报道的最高 PII 效价（分别为 2.2 和 2g/L）。此外，MSGE 成功扩展到开发了一系列强大的变铅青链霉菌异源宿主，其中氯霉素或抗肿瘤化合物 YM-216391 的 BGC 可以在一步中高效整合多达四个拷贝，从而显著提高了生产力（2-23 倍）。我们的多重方法为工业放线菌的稳健基因组工程和通过基因组挖掘进行新型药物发现提供了巨大的潜力。
