Study On Functions Of Gene Thu2 And ThuF In The Thuringiensin Biosynthetic Gene Cluster

The leading biopesticide, Bacillus thuringiensis, is a ubiquitous gram-positive, spore-forming bacterium that produces several toxins to a wide range of insect species. It is already a useful alternative or supplement to synthetic chemical pesticide, and also a key source of genes for transgenic expression to provide pest resistance in plants.Thuringiensin is a low molecular weight nucleotide insecticidal compound produced by some Bacillus thuringiensis strains. It is heat-stable and toxic to the larvae of a wide variety of invertebrate species and some species of nematode. Its molecular formula is C22H32O19N5P, molecular weight is 701 Da, composed of a phosphorylated allose diacid, a sugar moiety formed by D-glucose and an adenosine.In our study of Bacillus thuringiensis strain CT-43 that produces thuringiensin, we have analyzed the function of genes in the thuringiensin biosynthetic gene cluster, and then the thuringiensin biosynthesis pathway was deduced and clarified. The experiments of knockout and complementation of gene thuE, confirmed that the thuE is responsible to the production of thuringiensin. Clone and express the gene thuE to derive the purified protein ThuE. In vitro enzyme catalyzed experiment of ThuE proved that the biological function of ThuE is a phosphokinase, and responsible for the phosphorylation of thuringiensin at the last biosynthesis step.In this study, we foucsed on the functions of gene thu2 and thuF in the cluster. Based on the analysis of bioinformatics, we deduced that thu2 encode a kind of nonribosomal peptide synthetase, including an adenylation domain and a carrier protein domain, which can combine an allose diacid to start the biosynthesis of thuringiensin. We deduced that thuF encode a kind of glycosyltransferase, including the "ASP-X-ASP" motif, which can catalyze a glucose combine to allose diacid, forming the glucose allose diacid mitermediate of thuringiensin. In this study, we constructed plasmids, using temperature sensitive plasmid pHT304-TS, for thu2 and thuF gene knockout, respectively. We screened and obtained the thu2 knockout mutant CT-43-22, the thuF knockout mutant CT-43-F3. Detected by HPLC, both of the mutants could not produce thuringiensin.Then, we constructed two plasmids for complementation of thu2 and obtained complement recombinat strains CT-43-22b and CT-43-22c. Detected by HPLC, both of the recombinant strains restored the production of thuringiensin.We cloned and expressed the gene thu2 and thuF via vector pET28a, obtained the plasmid pEMB1440 for expressing protein Thu2, plasmid pEMB1441 for expressing truncated protein Thu2a and plasmid pEMB1442 for expressing ThuF. We also obtained purified phosphopantetheinyl transgerase Sfp.In vitro enzyme catalyzed experiment was carried out utilizing purified proteins. The first step was the phosphopantetheinylation of the Thu2 and Thu2a; the second step was the combination between allose diacid and the active Thu2, Thu2a obtained from the first step; the third step was adding ThuF and glucose donor after the accomplishment of the second step. All steps were detected by MS.In this study, the experiments of knockout and complementation of gene thu2 and thuF, respectively, demonstrated that the thu2 and thuF are both necessary to the production of thuringiensin. We have obtained the purified proteins and established the systems for enzyme catalyzed experiment in vitro, which was a solid foundation for next research work.