Characterization Of TzwM And TzwO Genes Involved In Zwittermicin A Biosynthesis

Zwittermicin A is a linear aminopolyol antibiotic produced by Bacillus cereus (Bc) andBacillus thuringiensis (Bt) with broad spectrums. Zwittermicin A inhibits many eukaryotesand prokaryotes, and interacts synergistically with Bt toxin to enhance the insecticidalactivity of Bt. So, the study of Zwittermicn A biosynthetic cluster is one of the hotstops. In2004, Emmert et al cloned nine integrity open reading frames (ORFs, the sequence is orf3,orf1,zmaR,orf2,orf4,orf5...orf8), including Zwittennicn A self-resistance gene (zmaR)and partial orf9. In this lab, we cloned Zwittermicn A biosynthetic cluster (which was 64kb in length) from B. thuringiensis G03 with high toxicity to lepidopteran insect pests.Sequence analysis revealed integrity orf9 (called tzwM) and five new ORFs, namely tzwA,tzwB, tzwC, tzwN and tzwO.Sequence analysis showed that: the length of tzwM was 1,074 bp and encoded a 40.5 kDprotein with similarity to monooxygenase enzyme of the alkanesulfonate, the length oftzwO was 1,077 bp and encoded a 42.0 kD protein with high similarity to FkbH (FkbH isinvolved in the formation of MM-ACP, an extender unit needed for the biosynthesis ofFK520). The tzwM and tzwO genes were cloned into fusing-expression vector pET-21band overexpressed in E. coli and then the recombined proteins were purified respectively.To test the function oftzwMand tzwO, the alleles containing an in-frame deletion wereconstructed and were cloned into the temperature-sensitive shuttle vector pRN5101,respectively. Allelic exchange was performed in G03, and three mutants of tzwM (namelyG03ΔtzwM-101, G03ΔtzwM-140 andΔG03tzwM-262) and three mutants of tzwO(namely G03ΔtzwO-89, G03ΔtzwO-144 and G03ΔtzwO-184) were obtained separately.The delitions of the mutants G03ΔtzwM-101 and G03ΔtzwO-184 were confirmed bysouthern blotting. The cultural supernatant of all mutants did not inhibit the growth ofErwinia herbicola. Furthermore, Zwittennicin A products from mutants could not bedetected by the mass spectrogram analysis. Then, tzwM and tzwO were introduced into theG03ΔtzwM-101 and G03ΔtzwO-184 separately, both transformants restored to produceZwittermicin A respectively. These results indicated that tzwM and tzwO were necessaryfor Zwittennicin A biosynthesis. The identification of the two genes further provides cluesof Zwittrmicin A biosynthesis pasway.