title: Heterologous expression and genetic engineering of the tubulysin biosynthetic gene cluster using Red/ET recombineering and inactivation mutagenesis.
abstract: Although the tubulysin (tub) biosynthetic gene cluster has been located in two myxobacterial strains, it appears in both cases to be incomplete as obvious candidates for acyl transfer and oxidation functions are lacking. Here, we report the engineering of a heterologous expression system for the tub biosynthetic pathway from strain Cystobacter sp. SBCb004. The entire tub core cluster was reconstituted from two cosmids using Red/ET recombineering and heterologous expression achieved in strains Pseudomonas putida and Myxococcus xanthus. Availability of the heterologous expression system and the natural producer strain SBCb004 provided a platform for the functional investigation of various biosynthetic genes by targeted inactivation. In addition, BLAST analysis of SBCb004 genome data was used to identify multiple candidate monooxygenases, whose involvement in tubulysin assembly was evaluated using a combination of knockout mutagenesis and heterologous expression.
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Department of Pharmaceutical Biotechnology, Saarland University, Saarbrücken 66041, Germany.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chai
authors.forename: Shiping
authors.initials: S
authors.lastname: Shan
authors.forename: Kira J
authors.initials: KJ
authors.lastname: Weissman
authors.forename: Shengbiao
authors.initials: S
authors.lastname: Hu
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D005818:Genetic Engineering; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D016296:Mutagenesis; D016941:Myxococcus xanthus / Q000201:enzymology / Q000235:genetics; D009842:Oligopeptides / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics
chemical_list: D009842:Oligopeptides; C510756:tubulysin A; D006899:Mixed Function Oxygenases