[Prediction, overexpression and activity confirmation of adenylation domain in Zwittermicin A biosynthesis gene cluster].
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.
Chunxu
Song
S
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Changming
Zhao
Z
Ziniu
Yu
Y
Ming
Sun
S
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
D001426:Bacterial Proteins; D010455:Peptides; C088290:zwittermicin A