| Zwittermicin A(ZwA) is a novel,broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis,whose chemical formula is C13H28N6O8.Zwittermicin A is highly active against the Oomycetes and their relatives, the algal,protists,and has moderate activity against diverse Gram-negative bacteria and certain Gram-positive bacteria as well as against a wide range of plant pathogenic fungi. Meanwhile,ZwA could acilitates the insecticidal activity of the protein toxin produced by B.thuringiensis.Jo Handelsman group pedicted that ZwA could be synthesised by five proposed precursors:L-serine,malonyl CoA,Amino-malonyl-ACP, Hydroxy-malonyl-ACP and 2,3-diamino-propionate.Its biosynthesis pathway is different to both polyketides synthases(PKS) and NRPS(nonribosomal peptide synthetases) way, but a hybrid pathway.Since the whole biosynthesis gene cluster is not complete and the biosynthesis pathway is unknown,we carried on this work.The reported 16 kb sequence includes the relative genes of Amino-malonyl-ACP and Hydroxy-malonyl-ACP,but there is no evidence on the relative biosynthesis gene of 2,3-diamino-propionate.This work is to verify 2,3-diamino-propionate is the direct precursor of Zwittermicin A and completes the whloe gene biosynthesis pathway.This research is based on the 67 Kb biosynthesis gene cluster of Bacillus thuringiensis strain YBT-1520 in our lab.After bioinformatics analysis,we found three genes ZwA5A,ZwA5B and orf3 are relative with the precursor 2,3-diaminopropionate. ZwA5A and ZwA5B are homologs of cysteine synthetase and ornithine cyclodeaminase respectively.The ZwA5A-mutant could not produce ZwA when ZwA5A,the 2, 3-diaminopropionate biosynthesis related gene,was interrupted by homologous exchange, while the ZwA5A-mutant resumed to produce ZwA when 2,3-diaminopropionate was fed.It indicates that ZwA5A gene is necessary for the biosynthesis of ZwA and 2, 3-diaminopropionate can replace ZwA5A in the biosynthesis of ZwA.The adenylation domain ZWAA in the ZwA biosynthesis cluster ORF3 was over expressed in E.coli by pGEX-6P-1.ATP-dependent PPi exchange experiment result showed that ZwAA2 can active 2,3-diaminopropionate.Taken together,we have demonstrated that 2, 3-diaminopropionate is one of the precursors during the biosynthesis of ZwA. |
