Sevilla A, Vera J, Díaz Z, Cánovas M, Torres N V, Iborra J L
Departamento de Bioquímica y Biología Molecular B, Facultad de Química, Universidad de Murcia, 30100 Murcia, España.
Biotechnol Prog. 2005 Mar-Apr;21(2):329-37. doi: 10.1021/bp0497583.
In this work metabolic engineering strategies for maximizing L-(-)-carnitine production by Escherichia coli based on the Biochemical System Theory and the Indirect Optimization Method are presented. The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of L-(-)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in L-(-)-carnitine production (74% increase in production rate), in close agreement with the model's predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the L-(-)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value.
本文提出了基于生化系统理论和间接优化方法，通过大肠杆菌最大化生产L-(-)-肉碱的代谢工程策略。该模型使用幂律形式主义整合了代谢水平和生物反应器水平。基于S-系统模型应用间接优化方法，得到了与细胞生理学和生物反应器系统操作条件均兼容的参数值曲线。这确保了它们的生存能力和适应性，并提高了L-(-)-肉碱的生产速率。使用高细胞密度反应器的实验结果与间接优化方法的优化预测进行了比较。当在实际实验系统中将两个参数（稀释率和初始巴豆甜菜碱浓度）直接改变为规定的最佳值时，系统在L-(-)-肉碱生产方面表现出更好的性能（生产率提高74%），与模型预测非常吻合。该模型显示了在宏观（反应器操作）和微观（分子）水平上的控制点，在这些点上可以提高转化率和生产率。根据优化解决方案，提高L-(-)-肉碱生产率的下一个合理步骤将涉及通过过表达肉碱转移酶CaiB的活性以及负责底物和产物进出细胞运输的蛋白质载体CaiT，对大肠杆菌菌株进行代谢工程改造。通过这种方式，预计产量可能提高到原来的三倍。
