Function Identification Of ThuE Gene In The Thuringiensin Biosynthesis Cluster Of Bacillus Thuringiensis Strain CT-43

Thuringiensin (short for Thu) is a nucleotide insecticidal toxin produced by some Bacillus thuringiensis strains. The biosyntheis cluster of Thu was cloned from CT-43 by our group, the cluster was sequenced and analysed. The function of thuE gene showed high homologous with serine kinase by BLAST analysis. So, the function of thuE gene was proposed responsible for the phosphorylation of Thu. The thuE gene was knocked out by homologous double-crossover in strain CT-43. The resulted mutant strain CT-43-A3 could not produce Thu. In this study, the complementation experiment and the extracellular function identification of thuE gene were performed.In this study, the thuE gene was subcloned into pHT304 and transformed into mutation strain CT-43-A3 for complementation experiment. The supernatant of recombinant strain was identified with HPLC. The research result indicated recombinant transformant can synthesis Thu. The thuE gene has relationship with the production of Thu in strain CT-43.The thuE gene was got by PCR and subcloned into vector pGEX-6p-1, the resulted vector was transformed into E.coli BL21 (DE3). ThuE was purified use GSTrap FF column. The predicted precursor C was obtained by digesting purified Thuringiensin with Alkaline Phosohatase (CIAP), and then identified by HPLC-MS. The HPLC revealed a new peak with retention time of about 5.3 min. The LCMS-IT-TOF was performed to determine the presence of precursor C whose molecular weight is 620.1593 under the [M-H]-pattern. The purified ThuE was incubated with precursor C in reaction buffer and analyzed by HPLC. The HPLC result showed the conversion of precursor C to thuringiensin after 3 hours incubation.Allomucic acid is a key component of Thu. In this study, allomucic acid was treated by ThuE protein to detecte its substrate specificity. The reaction mixture was analysed by HPLC. A substance whose retention time was different with allomucic acid was got and subsequently detected by MS. The ThuE protein was proved to catalyze the allomucic acid to a different chemical compound. However, the chemical structure of this product is pending.In this study, the gene thuE is proved responsible for the phosphorylation of Thu at the last step of biosynthesis. This result confirmed the last step of deduced synthesis pathway of Thu.