title: Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa.
abstract: 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 14,611 bp 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.
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: Jian
authors.initials: J
authors.lastname: Chen
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. hyxia@sibs.ac.cn.|Shanghai Institute of Plant Physiology and Ecology, 300 Fenglin Road, Shanghai, 200032, China. hyxia@sibs.ac.cn.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Xia
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: Fujun
authors.initials: F
authors.lastname: Dang
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: Qingyu
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Yunnan Institute of Microbiology, Yunnan University, Kunming, 650091, China.
authors.forename: Wenjun
authors.initials: W
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. qin@sibs.ac.cn.|Shanghai Institute of Plant Physiology and Ecology, 300 Fenglin Road, Shanghai, 200032, China. qin@sibs.ac.cn.
authors.forename: Zhongjun
authors.initials: Z
authors.lastname: Qin
mesh_terms: D001287:Attachment Sites, Microbiological; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D018942:Macrolides / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D016366:Open Reading Frames; D010575:Pesticides / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016930:Saccharopolyspora / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D018942:Macrolides; D010575:Pesticides; D011994:Recombinant Proteins
keywords: Integration; Plasmid; Saccharopolyspora; Spinosyn