Prediction, overexpression and activity confirmation of adenylationdomain 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 thuringien-sis 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 β-D-1-thiogalactopyranoside (IPTG) concentration and temperature during overepression. [Results]The overexpression protein of this domain could be purified under 20℃, 0.1 mmol/L Isopropyl b-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.