title: [Prediction, overexpression and activity confirmation of adenylation domain in Zwittermicin A biosynthesis gene cluster].
abstract: OBJECTIVE
  The adenylation domain is required for the substrate activation of non-ribosomal peptide synthesis. The objective of this research was to prove that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis.
  
  
  METHODS
  We cloned the adenylation domain in the Zwittermicin A synthesis cluster of Bacillus thuringiensis strain YBT-1520 with PCR amplification. After a series of enzyme digestions and subclonings, new expression vectors pBMB1312 was obtained. In order to detect the proper condition for overexpression, we tried different Isopropyl beta-D-1-thiogalactopyranoside (IPTG) concentration and temperature during overepression.
  
  
  RESULTS
  The overexpression protein of this domain could be purified under 20 degrees C, 0.1 mmol/L Isopropyl beta-D-1-thiogalactopyranoside (IPTG), BL21 codon plus RP (DE3) as the host strain. Then, PPi release assay indicated that 2, 3-diaminopropionate, the presume precursor of Zwittermicin A, could be adenylated by the adenylation domain.
  
  
  CONCLUSIONS
  This research confirmed that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Song
authors.initials: S
authors.lastname: Chunxu
authors.forename: Zhao
authors.initials: Z
authors.lastname: Changming
authors.forename: Yu
authors.initials: Y
authors.lastname: Ziniu
authors.forename: Sun
authors.initials: S
authors.lastname: Ming
mesh_terms: D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D010455:Peptides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; C088290:zwittermicin A