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.
Conclusion: This research confirmed that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis.