Thu1 And Thut Are Key Genes For Synthesis And Secretion Of Thuringiensin In Bacillus Thuringiensis CT43

Bacillus thuringiensis is a Gram-positive bacteria that are widely distributed around the world and can form spores to resist harsh environments, it can produce a variety of active substances intracellular and extracellular and thuringiensin is one of thoses which is a thermostable small molecular that is toxic to demodicid mite, some species of insect and nematode. Thuringiensin consists of adenosine, glucose, allose acid and phosphate groups in a ratio of 1:1:1:1, and the formula is C22H32O19N5P.Our group discovered the first international thuringiensin biosynthetic gene cluster of 12Kb, and deduced Thu biosynthesis’ pathway. The cluster comprised 11 ORFs in which the most critical genes are thu2(Acyl Carrier Protein), thuF(Glycosyltransferase), thul(S-adenosylmethionine synthetase),thuE(homoserine kinase), and thuT(transport protein). Knockout and complementary experiments in vivo and enzymatic experiments in vitro have confirmed that gene thuE’s function is to phosphorylate precursor C to form a dynamic thuringiensin in the process of thuringiensin synthesis. Meanwhile, knockout experiments in vivo of thu2 and thuF also shows that thuF and thu2 are the key genes in the synthesis of thuringiensin biosynthesis. Therefore, this study focus in the function of thuT and thul.thuT is one of the main secretion genes for thuringiensin. thuT is the sole gene associated with the transporter protein in the thuringiensin biosynthetic gene cluster, and is one of the members of major facilitater superfamily protein which is responsible for transporting the metabolites primarily. Therefore, we deduced thuT function as secreting the thuringiensin. In this study, we complete in-frame deletion of thuT using in-frame deletion system constructed by our laboratory, we detect the ability for thuringiensin production of the mutation by HPLC. HPLC results show that the mutation lose the ability of thuringiensin production, Q-TOF result shows that extracellular accumulation of precursors C increases about 76 times, which indicate that thuT is essential for thuringiensin synthesis. In order to confirm that phosphorylation and translocation of thuringiensin is collaborative by thuE and thuT, we express and purify ThuE protein to verify the interaction between ThuE and ThuT. To confirm that whether virB4-2 is responsible for thuringiensin’s secretion that located downstream of thuringiensin biosynthetic gene cluster, so we konck out it in CT43, which shows that gene deletion does not influence the secretion of thuringiensis. Thus we think thuT is one of the main secretion genes for thuringiensin. Besides, bioinformatics analysis showed that ThuE located in the cell and experimental results show that there is not mature thuringiensin intracellular in strain CT43, according which we propose a model for thuringiensin secretion.thul is the essential gene for thuringiensin synthesis. Thul is an S-adenosylmeth-ionine synthetase, and its traditional function is to synthesize S-adenosyl methionine acid using L-methionine (L-Met) and adenosine triphosphate as substrate, and in this study, we believe that it is related with thuringiensin’s assembly for adenosine, interesting, the func-tion is different with traditional function of S-adenosylmethionine synthase. Therefore, we complete in-frame deletion of thul using in-frame deletion system, HPLC result shows that the mutation lose the ability for thuringiensin production which indicate that thul is the essential gene for thuringiensin synthesis. To verify the novel function of Thul, we expresse and purify Thul of high concentrationin in E.coli for further research.Therefore, this study mainly focuses on two aspects:the first part is the in-frame deletion of thuT and the detection for corresponding accumulated substance which proves that thuT is one of the main secretion genes for thuringiensin and we speculate function of thuT is related with thuE. Therefore, we propose a model for thuringiensin secretion and verify it preliminary. The second part is in-frame deletion of thul which proves that thul is the essential gene for thuringiensin synthesis. To verify the function of Thul, we expr-esse and purify it of high concentrationin in E.coli for further research.