苏云金芽胞杆菌YBT-1532生物合成苏云金素的代谢调控
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摘要
苏云金素是苏云金芽胞杆菌产生的一种热稳定腺嘌呤衍生物，对鳞翅目等昆虫以及线虫和螨类具有良好的杀虫效果，其毒性比化学杀虫剂低的多，作为杀虫剂具有很大的市场潜力。本文对苏云金芽胞杆菌达姆斯塔特亚种YBT-1532菌株(YBT-1532)生物合成苏云金素的代谢调控进行了研究，结果如下：
1．通过提高高效液相色谱流动相中的磷酸二氢钾浓度和在流动相中加入1.0％乙酸的方法，优化了苏云金素的检测条件；运用离子交换法分离苏云金素，采用0.2mol／L的甲酸冲洗与0.1 mol／L的氯化钠溶液(pH2.0)洗脱相结合的方案，提高了苏云金素分离和纯化效率。
2．通过半合成培养基添加氨基酸和有机酸的刺激实验，发现腺嘌呤从头合成途径的底物，如天冬氨酸和谷氨酰氨等氨基酸，以及通过代谢对这些氨基酸合成有促进作用的有机酸，可提高苏云金素的发酵水平，证明苏云金素的生物合成与腺嘌呤从头合成途径有关。
3．在YBT-1532菌株分批培养中，添加2.0g／L的柠檬酸钠降低了丙酮酸激酶活性、丙酮酸含量和合成PHB的能力以及PHB对葡萄糖的得率，却提高了2-酮戊二酸、谷氨酸和腺嘌呤浓度，同时，也提高了6-磷酸葡萄糖脱氢酶活性、细胞合成苏云金素的能力和苏云金素对葡萄糖的得率。证明添加柠檬酸钠引起糖酵解途径减弱，磷酸戊糖途径代谢流量增强，从而使PHB合成底物丙酮酸的供量减少，苏云金素合成前体腺嘌呤的供量增加，实现了抑制PHB合成和促进苏云金素合成的调节。首次报道了苏云金素与聚-3-羟基丁酸(PHB)的代谢关系。
4．在YBT-1532菌株连续培养中，研究了细胞生长和代谢的动力学特征，并在稀释率为0.12 h~(-1)时，研究了柠檬酸钠对细胞代谢和苏云金素合成的影响，得到：
碳衡算方程：qs=1.51xμ+2.2xqp+0.049
菌体生长动力学方程：μ=0.63xS／(0.544+S)；
苏云金素生成动力学方程：dP／dt=0.0943xdX／dt+0.0146xX
当添加柠檬酸钠时，与对照相比丙酮酸激酶活性降低29.0％，而6-磷酸葡萄糖脱氢酶活性却提高42.1％，导致苏云金素的产量提高14.7％。进一步证实柠檬酸钠对细胞代谢和苏云金素合成的调节机制。
5．在YBT-1532菌株分批培养中，添加1.00g／L甲酸钠使腺嘌呤和苏云金素的产量提高。在静息培养中，当适量添加甲酸钠时，与对照相比NADH在胞内的含量增加19.6％，而甲酸脱氢酶、丙酮酸激酶和6-磷酸葡萄糖脱氢酶活性分别提高2.0倍、4.25倍和2.5倍，胞内天冬氨酸、丙酮酸、柠檬酸和腺嘌呤分别提高65.0％、75.0％、31.9％和71.4％，而苏云金素产量却提高了90％。结果表明甲酸钠对苏云金素合成的促进作用是通过提高与腺嘌呤合成相关的代谢途径的碳流量(糖酵解途径、三羧酸循环以及磷酸戊糖途径)，提高细胞对腺嘌呤与苏云金素的合成能力来实现的；甲酸钠不仅可作为腺嘌呤合成的前体，还可以作为碳源被用于细胞生长和代谢。
6．在YBT-1532菌株的分批培养中，添加DMSO(10mL／L)、SDS(0.10g／L)、青霉素(300IU／mL，9h加入)以及磷霉素(50U／mL，9h加入)均可显著提高苏云金素的产量。在青霉素作用下，胞内磷酸酯酶活性下降15.3％，胞内脱磷酸苏云金素提高25.0％，胞内苏云金素积累量减少12.0％，其向胞外分泌速率提高71.8％。表明细胞通透性增加后，苏云金素分泌增强，磷酸酯酶活性降低，导致胞内苏云金素积累减少，减少了胞内苏云金素合成的反馈调节，进一步促进了脱磷酸苏云金素合成。这些结果，国内尚未见报道。
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 KH_2PO_4 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.
1．通过提高高效液相色谱流动相中的磷酸二氢钾浓度和在流动相中加入1.0％乙酸的方法，优化了苏云金素的检测条件；运用离子交换法分离苏云金素，采用0.2mol／L的甲酸冲洗与0.1 mol／L的氯化钠溶液(pH2.0)洗脱相结合的方案，提高了苏云金素分离和纯化效率。
2．通过半合成培养基添加氨基酸和有机酸的刺激实验，发现腺嘌呤从头合成途径的底物，如天冬氨酸和谷氨酰氨等氨基酸，以及通过代谢对这些氨基酸合成有促进作用的有机酸，可提高苏云金素的发酵水平，证明苏云金素的生物合成与腺嘌呤从头合成途径有关。
3．在YBT-1532菌株分批培养中，添加2.0g／L的柠檬酸钠降低了丙酮酸激酶活性、丙酮酸含量和合成PHB的能力以及PHB对葡萄糖的得率，却提高了2-酮戊二酸、谷氨酸和腺嘌呤浓度，同时，也提高了6-磷酸葡萄糖脱氢酶活性、细胞合成苏云金素的能力和苏云金素对葡萄糖的得率。证明添加柠檬酸钠引起糖酵解途径减弱，磷酸戊糖途径代谢流量增强，从而使PHB合成底物丙酮酸的供量减少，苏云金素合成前体腺嘌呤的供量增加，实现了抑制PHB合成和促进苏云金素合成的调节。首次报道了苏云金素与聚-3-羟基丁酸(PHB)的代谢关系。
4．在YBT-1532菌株连续培养中，研究了细胞生长和代谢的动力学特征，并在稀释率为0.12 h~(-1)时，研究了柠檬酸钠对细胞代谢和苏云金素合成的影响，得到：
碳衡算方程：qs=1.51xμ+2.2xqp+0.049
菌体生长动力学方程：μ=0.63xS／(0.544+S)；
苏云金素生成动力学方程：dP／dt=0.0943xdX／dt+0.0146xX
当添加柠檬酸钠时，与对照相比丙酮酸激酶活性降低29.0％，而6-磷酸葡萄糖脱氢酶活性却提高42.1％，导致苏云金素的产量提高14.7％。进一步证实柠檬酸钠对细胞代谢和苏云金素合成的调节机制。
5．在YBT-1532菌株分批培养中，添加1.00g／L甲酸钠使腺嘌呤和苏云金素的产量提高。在静息培养中，当适量添加甲酸钠时，与对照相比NADH在胞内的含量增加19.6％，而甲酸脱氢酶、丙酮酸激酶和6-磷酸葡萄糖脱氢酶活性分别提高2.0倍、4.25倍和2.5倍，胞内天冬氨酸、丙酮酸、柠檬酸和腺嘌呤分别提高65.0％、75.0％、31.9％和71.4％，而苏云金素产量却提高了90％。结果表明甲酸钠对苏云金素合成的促进作用是通过提高与腺嘌呤合成相关的代谢途径的碳流量(糖酵解途径、三羧酸循环以及磷酸戊糖途径)，提高细胞对腺嘌呤与苏云金素的合成能力来实现的；甲酸钠不仅可作为腺嘌呤合成的前体，还可以作为碳源被用于细胞生长和代谢。
6．在YBT-1532菌株的分批培养中，添加DMSO(10mL／L)、SDS(0.10g／L)、青霉素(300IU／mL，9h加入)以及磷霉素(50U／mL，9h加入)均可显著提高苏云金素的产量。在青霉素作用下，胞内磷酸酯酶活性下降15.3％，胞内脱磷酸苏云金素提高25.0％，胞内苏云金素积累量减少12.0％，其向胞外分泌速率提高71.8％。表明细胞通透性增加后，苏云金素分泌增强，磷酸酯酶活性降低，导致胞内苏云金素积累减少，减少了胞内苏云金素合成的反馈调节，进一步促进了脱磷酸苏云金素合成。这些结果，国内尚未见报道。
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 KH_2PO_4 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.
引文
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