大肠杆菌酪氨酸转运系统基因敲除对酪氨酸生产的影响

Effect of gene knockout of L-tyrosine transport system on L-tyrosine production in Escherichia coli

摘要:

酪氨酸是三大芳香族氨基酸之一，广泛用于食品、医药和化工等领域。转运系统工程为代谢工程改造大肠杆菌选育酪氨酸生产菌株提供了一种重要的研究策略。大肠杆菌中酪氨酸胞内转运主要通过aroP和tyrP基因编码的通透酶进行调控。以酪氨酸生产菌株HGXP为出发菌株，利用CRISPR-Cas9技术成功构建了aroP和tyrP基因敲除菌，并通过发酵试验考察了调节转运系统对酪氨酸生产的影响。发酵结果表明，aroP和tyrP基因敲除菌酪氨酸产量分别达到3.74 g/L和3.45 g/L，较出发菌株酪氨酸产量分别提高了19%和10%。对诱导温度进行了优化，结果表明38 ℃为最佳诱导温度。在3 L发酵罐上进行了补料分批发酵，aroP和tyrP基因敲除菌酪氨酸产量进一步提高至44.5 g/L和35.1 g/L，较出发菌株酪氨酸产量分别提高了57%和24%。研究结果对代谢工程强化大肠杆菌生产酪氨酸具有重要的参考价值。

Abstract:

L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.