黄色短杆菌TK0303的L-亮氨酸生物合成途径分析

Pathway analysis for production of L-leucine by Brevibacterium flavum TK0303

摘要:

应用途径分析方法分析了在拟稳态时黄色短杆菌(Brevibacterium flavum)TK0303由葡萄糖发酵生产L-亮氨酸的代谢途径,确定了L-亮氨酸合成的最佳途径和最大理论产率。通过比较途径分析所获得的反应模型,确定了丙酮酸和乙酰辅酶A是L-亮氨酸合成途径的关键节点。在此基础上改变外界环境因子,强化L-亮氨酸生物合成途径中丙酮酸和乙酰辅酶A两个关键节点的代谢流,以期进一步提高L-亮氨酸产率。结果表明,经过谷氨酸以及醋酸铵的调节,代谢途径流量发生显著变化,L-亮氨酸产量有明显提高。

Abstract:

Brevibacterium flavum is used for the production of a number of amino acids in the biotechnology industry. The yield of producing a metabolite is ultimately limited by the ability of the central metabolism and the desired biosynthesis pathway. Pathway analysis is a very useful tool for metabolic engineering, which can be applied to increase the yield of a metabolite or channeling a metabolite into desired pathways. It does not require any kinetic parameters and only uses the Stoichiometric equations. Pathway analysis for production of L-leucine by Brevibacterium flavum TK0303 at steady state was conducted in this paper. Theoretical yield and flux distribution for optimal pathway were determined.It is also concluded that pyruvate and acetyl-coenzyme A are the key nodes of the L-leucine biosynthesis pathway by analyzing the flux distributions of different modes. According to the pathway analysis, the production of L-leucine is expected to be raised by strengthening the flux of the key nodes (pyruvate and acetyl-coenzyme A) through changing the environmental factors. Because the flux of TCA cycle in Brevibacterium flavum TK0303 is weak, the production of L-leucine must be provided enough amido by adding glutamic acid to the fermentation medium. NH₄Ac is both a carbon source and a nitrogen source, which could be helpful to the production of L-leucine. The effects of glutamic acid and NH₄Ac on the production of L-leucine were further studied. The production of L-leucine increased 56% by adding glutamic acid. By improving the concentration of NH4Ac, the biosynthesis of L-leucine was greatly strengthened too. The results indicate that the flux of L-leucine can be largely increased by changing the chemical regulatory factors such as NH₄Ac and glutamic acid and the modes established by pathway analysis prove to be efficient to describe the metabolic network of L-leucine production by Brevibacterium flavum TK0303.