title: Translocation of the thioesterase domain for the redesign of plipastatin synthetase.
abstract: Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation betweenL-Try3with the last few residuesL-Pro7 andL-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T-TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Ling
authors.initials: L
authors.lastname: Gao
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Liu
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Ma
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Jinzhi
authors.initials: J
authors.lastname: Han
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
authors.affiliation: College of Life Science, Nanjing Agricultural University, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Chen
authors.initials: C
authors.lastname: Dai
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lv
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang Nanjing 210095, P.R. China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Bie
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D002853:Chromatography, Liquid; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D055666:Lipopeptides / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D009876:Operon / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D000072417:Protein Domains; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013869:Thiolester Hydrolases / Q000737:chemistry*
chemical_list: D005227:Fatty Acids; D055666:Lipopeptides; D009842:Oligopeptides; D010456:Peptides, Cyclic; D021122:Protein Subunits; C049901:plipastatin; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase