title: Genetic engineering in Actinoplanes sp. SE50/110 - development of an intergeneric conjugation system for the introduction of actinophage-based integrative vectors.
abstract: The α-glucosidase inhibitor acarbose is used for treatment of diabetes mellitus type II, and is manufactured industrially with overproducing derivatives of Actinoplanes sp. SE50/110, reportedly obtained by conventional mutagenesis. Despite of high industrial significance, only limited information exists regarding acarbose metabolism, function and regulation of these processes, due to the absence of proper genetic engineering methods and tools developed for this strain. Here, a basic toolkit for genetic engineering of Actinoplanes sp. SE50/110 was developed, comprising a standardized protocol for a DNA transfer through Escherichia coli-Actinoplanes intergeneric conjugation and applied for the transfer of ϕC31, ϕBT1 and VWB actinophage-based integrative vectors. Integration sites, occurring once per genome for all vectors, were sequenced and characterized for the first time in Actinoplanes sp. SE50/110. Notably, in case of ϕC31 based vector pSET152, the integration site is highly conserved, while for ϕBT1 and the VWB based vectors pRT801 and pSOK804, respectively, no sequence similarities to those in other bacteria were detected. The studied plasmids were proven to be stable and neutral with respect to strain morphology and acarbose production, enabling future use for genetic manipulations of Actinoplanes sp. SE50/110. To further broaden the spectrum of available tools, a GUS reporter system, based on the pSET152 derived vector, was also established in Actinoplanes sp. SE50/110.
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Tetiana
authors.initials: T
authors.lastname: Gren
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Ortseifen
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wibberg
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Schneiker-Bekel
authors.affiliation: Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld University, Postfach 100131, D-33501 Bielefeld, Germany.
authors.forename: Hanna
authors.initials: H
authors.lastname: Bednarz
authors.affiliation: Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld University, Postfach 100131, D-33501 Bielefeld, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Niehaus
authors.affiliation: Product Supply, Bayer Pharma AG, Friedrich Ebert Str. 217-475, 42117 Wuppertal, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Zemke
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Persicke
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Alfred
authors.initials: A
authors.lastname: Pühler
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany. Electronic address: Joern.Kalinowski@Cebitec.Uni-Bielefeld.DE.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
mesh_terms: D020909:Acarbose / Q000378:metabolism; D000192:Actinomycetales / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*
chemical_list: D020909:Acarbose
keywords: Acarbose; Actinoplanes sp. SE50/110; Genetic engineering; Integrative vectors