Poly-β-hydroxybutyrate On Metabolism Of Bacillus Thuringensis Bmb171Cry1Ac

Bacillus thuringiensis is a widely spread Gram-positive bacterium in nature and can largely synthesize insecticidal crystal proteins, which have insecticidal activity and form crystals during sporulation phase. The poly-p-hydroxybutyric acid (PHB) will be accumulated in Bacillus thuringiensis when it grows in a nutrient-rich medium, which while will be used quickly during sporulation phase in lack of nutrient.Many studies indicated that the BMB171PHB-cry1Ac whose gene of phb was knocked out can not form spore and ICPs during mid-post-stationary phases. In this study, nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) were used to detect the metabolites in intra-cellular and extra-cellular of BMB171crylAc and BMB171PHB-cry1Ac, which will provide a good theoretical support to the analysis of metabolism in future.First of all, according to the analysis of metabolites changed between strains, the comprehensive metabolic network diagram about the lack of PHB influence on the intra-cellular metabolism was constructed. Second, we found the nucleoside metabolism and pentose-phophate pathway changed dramatically in mutations from the nuclear magnetic resonance test results. So we assume that the lack of PHB is likely to affect the energy metabolism cycle, and then, affecting the formation of the crystal and spore,and give rise to a series of physiological reaction. Therefore, the method of high performance liquid chromatography (HPLC) was used to detect the content of main energy material (ATP、ADP、AMP、NAD+and NADP) inside the cell. The test condition was optimized before testing which monitor this five physical at the same time from the mobile phase composition, velocity, pH etc, and the method of samples pretreatment were also optimized.The results of the study show that deletion of PHB gene not only impede normal formation of spores and crystal, but also cause a lot of metabolic changes inside the cell, for example, a large number of organic acids are producted, pH changes significantly, aerobic metabolic pathways are suppressed, the fatty acids are consumpted excessivly, amino acid metabolism changes greatly, nucleoside metabolism abnormalities, the consumption rate of glucose accelerates sharply and bacteria growth speeds in the early logarithmic phase etc. we think the reason may be that PHB particles in the mutations failing to be synthetized influence intracellular nucleoside metabolism, and then the energy cycle in strains is restrained, it also leads to many metabolic changes mentioned above.