Function Of Gene ZmaS In The Zwittermicin A Biosynthetic Gene Cluster

Zwittermicin A (ZwA) is a linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis strains, and it has antimicrobial activity to many Oomycetes, algal protists, gram-positive and negative bacteria strains. In the ZwA biosynthetic gene cluster from Bacillus cereus UW85, the encoding product of zmaS is a putative phosphopantetheinyl transferase (PPTase). Usually, PPTase can transfer the 4’-phosphopantetheine moiety from CoA to the conserved Ser residue of the apo-carrier protein to produce the holo-carrier protein, resulting in the mass of carrier protein increase of 340 Da. ZmaS in the ZwA biosynthetic gene cluster from UW85 was disrupted by gene homologous double-crossover. Results showed that the mutant UW86 still produced ZwA and showed more powerfully antimicrobial activity(Erwinia herbicola LS005 as indicative bacterium) compared with strain UW85.The carrier protein domains from Bacillus thuringiensis strain YBT-1520, ACP111and PCP103, are equivalent to ACP domain of ZmaK and PCP domain of ZmaC from UW85. The PPTase from strains YBT-1520 is equivalent to ZmaS of strain UW85. The ACP111 and PCP 103 were expressed respectively or coexpressed respectively with PPTase in Escherichia coli BL21 (DE3). The molecular mass of carrier protein domains which were expressed in the presense or absense of PPTase were identified by MALDI-TOF analysis. Results showed that the masses of the two carrier protein domains (ACP111 and PCP 103) co-expressed with PPTase kept the same as the solely expressed carrier protein domains in Escherichia coli BL21 (DE3). These results indicated that PPTase (ZmaS) could not modify the carrier protein domains-ACP111 and PCP103.In the ZwA biosynthetic gene cluster from UW85, the sequence covering about 6.1 kb between zmaS and zmaT,with five genes being named kabR and kabA through kabD, is not involved in the biosynthesis of ZwA. The 6.1 kb DNA fragment was heterologously expressed in Bacillus thuringiensis strain BMB171. Mass spectrum analysis and antimicrobial activity assay showed that the sequence between kabA-kabD was the minimal biosynthesis gene cluster of Kanosamine, another antibiotic produced by strain UW85. Furthermore, the kabR was verified to up-regulate the biosynthesis of Kanosamine.The biosynthesis gene cluster of Kanosamine were solely expressed or coexpressed with zmaS in host-BMB171. The result of antimicrobial activity experiment showed that the biosynthesis of Kanosamine was down-regulated by ZwaS. Therefore, the yield of Kanosamine was increased, resulting in the antimicrobial activity was enhanced in the mutant-UW86 compared to strain UW85.