双酶级联催化L-色氨酸合成靛蓝

A dual-enzyme cascade for production of indigo from L-tryptophan

摘要:

靛蓝(indigo)作为一种水溶性非偶氮类着色剂，广泛应用于纺织、食品、制药等工业领域。目前靛蓝主要采用化学法合成，存在环境污染、安全隐患等问题，亟须寻找更安全、更绿色的合成方法。本研究利用大肠杆菌(Escherichia coli)来源的色氨酸酶(tryptophanase, EcTnaA)和噬甲基菌(Methylophaga aminisulfidivorans)来源的黄素依赖性单加氧酶(flavin-dependent monooxygenase, MaFMO)构建双酶级联路径，以L-色氨酸为底物合成靛蓝，导入E. coli中获得重组菌株EM-IND01。通过对限速酶MaFMO进行蛋白质工程改造，获得了有益突变体MaFMO^D197E，比酶活和k_cat/K_m比野生型分别提高了2.36倍和1.34倍；将其引入菌株EM-IND01中获得重组菌株EM-IND02，并进行发酵条件优化，在5 L发酵罐中靛蓝产量为(1 288.59±7.50) mg/L，转化率为0.86 mg/mg L-色氨酸，生产强度为26.85 mg/(L·h)。本研究通过蛋白质工程改造，获得MaFMO活性提高的突变体，为靛蓝的工业化生产奠定了基础。

关键词:大肠杆菌;靛蓝;蛋白质工程改造;双酶级联;L-色氨酸

Abstract:

Indigo, as a water-soluble non-azo colorant, is widely used in textile, food, pharmaceutical and other industrial fields. Currently, indigo is primarily synthesized by chemical methods, which causes environmental pollution, potential safety hazards, and other issues. Therefore, there is an urgent need to find a safer and greener synthetic method. In this study, a dual-enzyme cascade pathway was constructed with the tryptophan synthase (tryptophanase, EcTnaA) from Escherichia coli and flavin-dependent monooxygenase (flavin-dependent monooxygenase, MaFMO) from Methylophaga aminisulfidivorans to synthesize indigo with L-tryptophan as substrate. A recombinant strain EM-IND01 was obtained. The beneficial mutant MaFMO^D197E was obtained by protein engineering of the rate-limiting enzyme MaFMO. MaFMO^D197E showed the specific activity and k_cat/K_m value 2.36 times and 1.34 times higher than that of the wild type, respectively. Furthermore, MaFMO^D197E was introduced into the strain EM-IND01 to construct the strain EM-IND02. After the fermentation conditions were optimized, the strain achieved the indigo titer of (1 288.59±7.50) mg/L, the yield of 0.86 mg/mg L-tryptophan, and the productivity of 26.85 mg/(L·h) in a 5 L fermenter. Protein engineering was used to obtain mutants with increased MaFMO activity in this study, which laid a foundation for industrial production of indigo.

Key words:Escherichia coli;indigo;protein engineering;dual-enzyme cascade;L-tryptophan