title: Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes.
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 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.
authors.affiliation: 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.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Guosong
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Shanghai Laiyi Center for Biopharmaceuticals R&D, Shanghai 200240, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Ge
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Jiangsu National Synergetic Innovation Center for Advanced Materials, SICAM, Nanjing 210009, China. Electronic address: whjiang@sibs.ac.cn.
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, China. Electronic address: yhlu@sibs.ac.cn.
authors.forename: Yinhua
authors.initials: Y
authors.lastname: Lu
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics; D002701:Chloramphenicol / Q000096:biosynthesis* / Q000302:isolation & purification; D024861:Genetic Enhancement / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics; D005810:Multigene Family / Q000235:genetics*; D010080:Oxazoles / Q000302:isolation & purification; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D064210:Secondary Metabolism / Q000235:genetics*; D013302:Streptomyces / Q000502:physiology*; D015854:Up-Regulation / Q000235:genetics
chemical_list: D010080:Oxazoles; D010456:Peptides, Cyclic; C499972:YM 216391; D002701:Chloramphenicol
keywords: Actinomycetes; Gene cluster amplification; Genome mining; Secondary metabolite; Site-specific recombination