The Metabolic Regulation Of Thuringiensin Biosynthesis By Bacillus Thuringiensis YBT-1532

Thuringiensin is a kind of adenine derivative produced by some strains of Bacillusthuringiensis. It has showed the broad-spectrum insecticidal activity against nematodes,mites and insects such as squama, and it has been much lower toxicity than that of manyother chemical pesticides. Therefore, thuringiensin has displayed a more attractive andpromising market prospect. The biosynthesis and metabolic regulation of thuringiensinproduction were explored in the thesis, and the main results are as follows:1. The quantitative analysis method of HPLC for thuringiensin was optimized byimproving KH₂PO₄ concentration in mobile phase and by adding 1.0% of acetic acid intomobile phase. The ion exchange method used to isolate thuringiensin was optimized as wellby eluting orderly with 0.2 mol/L of formic acid and 0.1 mol/L of NaCl solution (pH2.0).The above technique improvements significantly enhanced the efficiency of thuringiensindetection, separation and purification.2. A stimulation test had been carried out by the addition of some amino acids(including asparagines and glutamine, which serve as the precursors of de novo pathwayof adenine) or organic acids (their metabolism could also facilitate the above amino acidsynthesis) into the semisynthetic culture media. The results showed that thuringiensinfermentative level could be increased by the addition of the above compounds, and theseproved thuringiensin synthesis was relative with de novo pathway of adenine synthesis.3. In a batch cultivation system of Bacillus thuringiensis YBT-1532, 2.0 g/L citrateaddition significantly decreased pyruvate kinase activity, pyruvate content, the ability tosynthesize PHB and transformation from glucose to PHB as well, whereas obviouslyenhanced the production of 2-ketoglutarate, adenine and glutamate as well asglucose-6-phosphate dehydrigenase activity, cell ability to synthesize thuringiensin andtransformation from glucose to thuringiensin. The results above demonstrated that the citrateaddition attenuated glycolytic flux, and increased the carbon metabolic flux in the pentosephosphate pathway, respectively. The changes were obviously in favor of more substratessupplying for thuringiensin synthesis and less for pyruvate production, which consequentlyincreased the thuringiensin synthesis level and decreased the PHB production, respectively.The metabolic relationships between thuringiensin and PHB were reported for the first timein this work.4. The dynamic characteristies of cell growth and metabolite were investigated in acontinuous culture system of B. thuringiensis YBT-1532. The influence of citrateaddition on cell growth and thuringiensin synthesis was also studied at a dilution rate of0.12 h^-1. The results could be described as following: q_S=1.51×μ+2.2×q_P+0.049;μ=0.63×S/(0.544+S); dP/dt=0.0943×dX/dt+0.0146×XThe activities of pyruvate kinase and glucose-6-phosphate dehydrogenase with citrateaddition at the dilution rate of 0.12 h^-1 were 29.0% lower and 42.1% higher than thoseof the control, respectively. Thuringiensin synthesis yield increased by 14.7% whencompared with control. The results further verified the regulatory mechanism of citrateaddition on thuringiensin synthesis in continuous culture.5. The yields of adenine and thuringiensin were increased significantly with additionof 1.0 g/L formate in batch culture of B. thuringiensis YBT-1532. Resting cell of thestrain was also cultivated with appropriate formate addition. NADH concentrationincreased by 19.6%, intracellular enzymes activities of formate dehydrogenase, pyruvatekinase and glucose-6-phosphate dehydrogenase enhanced by 2-fold, 4.25-fold and2.5-fold when compared with control, respectively. Intracellular production of aspartate,pyruvate, citrate and adenine were 65.0%, 75.0%, 31.9% and 71.4% higher than thoseof the control, respectively, and thuringiensin yield increased by 90% as wall. The resultsdemonstrated that the formate addition could facilitate carbon metabolic flux inglycolysis, tricarboxylic acid cycle and the pentose phosphate pathway, and finallyfavored the syntheses of intracellular adenine and thuringiensin production. The formatenot only could serve as the precursor of adenine biosynthesis, but also might play roles ofcarbon source for cell growth and metabolism.6. Appropriate addition of DMSO (10 mL/L), SDS (0.10 g/L), penicillin (300 U/mL,9 h) and fosformycin (50 U/mL, 9 h) facilitated thuringiensin synthesis that was 30.7%,34.1%, 71.4% and 85.2% higher than that of the control, respectively, in the batchculture of B. thuringiensis YBT-1532. At the case of penicillin addition, intracellularthuringiensin and activity of intracellular phosphatase decreased by 12.0% and 15.3%,respectively. Intracellular dephosphorylated thuringiensin and thuringiensin secretionenhanced by 25.0% and 71.8% compared to control. The adding penicillin enhanced cellpermeability, facilitated thuringiensin excrete, and decreased intracellular thuringiensinconcentration and phosphatase avtivity, which decreased the feedback inhibition onthuringiensin synthesis and favored dephosphorylated thuringiensin synthesis.