title: Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids.
abstract: Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving "scar" sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression.
authors.affiliation: Research Center for Molecular Medicine, Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Su
authors.affiliation: Research Center for Molecular Medicine, Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China; School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China.
authors.forename: Xin-Qing
authors.initials: XQ
authors.lastname: Zhao
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China.
authors.forename: Hai-Na
authors.initials: HN
authors.lastname: Wang
authors.affiliation: Research Center for Molecular Medicine, Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China; Beijing Biostar Technologies, Ltd., Beijing 101111, China.
authors.forename: Rong-Guo
authors.initials: RG
authors.lastname: Qiu
authors.affiliation: Research Center for Molecular Medicine, Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China; Beijing Biostar Technologies, Ltd., Beijing 101111, China. Electronic address: tangli63b@yahoo.com.
authors.forename: Li
authors.initials: L
authors.lastname: Tang
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005822:Genetic Vectors; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics*; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C506766:RpsL protein, E coli; D048630:Polyketide Synthases
keywords: Co-existing; Heterologous expression; Polyketide synthases (PKS); Precise seamless stitching; Red/ET recombination