Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes |
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| Affiliation: | 1. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;2. University of Chinese Academy of Sciences, Beijing 100039, China;3. Shanghai Laiyi Center for Biopharmaceuticals R&D, Shanghai 200240, China;4. Jiangsu National Synergetic Innovation Center for Advanced Materials, SICAM, Nanjing 210009, China;5. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, China;1. Helmholtz Institute for Pharmaceutical Research, 66123 Saarbrücken, Germany;2. University of Saarland, Pharmaceutical Biotechnology, 66123 Saarbrücken, Germany;1. Department of Pharmacy, Pharmaceutical Biotechnology, Saarland University, Campus C2.3, 66123, Saarbrücken, Germany;2. Department of Pharmacy, Pharmaceutical Biology, Saarland University, Campus C2.3, 66123, Saarbrücken, Germany;3. Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus E8.1, 66123, Saarbrücken, Germany;1. Helmholtz-Institute for Pharmaceutical Research Saarland, Saarland University, Campus C2.3, Saarbrücken 66123, Germany;2. PharmBioTec GmbH, Science Park 1, 66123 Saarbrücken, Germany;3. Universität Siegen, Fakultät IV, Organische Chemie II, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany;4. University of Freiburg, Institute for Pharmaceutical Sciences, Stefan-Meier str.14, 79104 Freiburg, Germany;5. BASF SE, 67056 Ludwigshafen, Germany;6. Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany;7. Department of Pharmaceuticla Biotechnology, Universität des Saarlandes, Campus Geb. C2.3, 66123 Saarbrücken, Germany;1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, PR China;2. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, PR China;3. Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, PR China;1. Molecular Engineering Lab, Institute of Bioengineering and Nanotechnology, A*STAR, 138669, Singapore;2. Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, 35053, Taiwan;3. Molecular Engineering Lab, Institute of Molecular and Cell Biology, A*STAR, 138669, Singapore;4. Singapore Institute of Food and Biotechology Innovation, A*STAR, 138669, Singapore |
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| Abstract: | 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 2 g/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. |
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| Keywords: | Secondary metabolite Actinomycetes Site-specific recombination Gene cluster amplification Genome mining |
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