caa a22 4500 605504857 CHVBK 20210128100618.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6871-z doi (NATIONALLICENCE)springer-10.1007/s00253-015-6871-z Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa [Elektronische Daten] [Jian Chen, Haiyang Xia, Fujun Dang, Qingyu Xu, Wenjun Li, Zhongjun Qin] Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA Ser gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa. Springer-Verlag Berlin Heidelberg, 2015 Saccharopolyspora nationallicence Plasmid nationallicence Integration nationallicence Spinosyn nationallicence Chen Jian Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut Xia Haiyang Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut Dang Fujun Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut Xu Qingyu Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut Li Wenjun Yunnan Institute of Microbiology, Yunnan University, 650091, Kunming, China aut Qin Zhongjun Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10141-10149 0175-7598 99:23<10141 2015 99 253 https://doi.org/10.1007/s00253-015-6871-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6871-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Chen Jian Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut NATIONALLICENCE 700 1- Xia Haiyang Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut NATIONALLICENCE 700 1- Dang Fujun Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut NATIONALLICENCE 700 1- Xu Qingyu Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut NATIONALLICENCE 700 1- Li Wenjun Yunnan Institute of Microbiology, Yunnan University, 650091, Kunming, China aut NATIONALLICENCE 700 1- Qin Zhongjun Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10141-10149 0175-7598 99:23<10141 2015 99 253