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

作　　者：罗诗琪 魏婉清 吴静 宋伟 胡贵鹏 刘立明 LUO Shiqi;WEI Wanqing;WU Jing;SONG Wei;HU Guipeng;LIU Liming(School of Food Engineering,Anhui Science and Technology University,Fengyang 233100,Anhui,China;School of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122,Jiangsu,China;Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]安徽科技学院食品工程学院,安徽凤阳233100 [2]江南大学生命科学与健康工程学院,江苏无锡214122 [3]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2024年第8期2444-2456,共13页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2021YFC2102700);轻工业技术与工程国家一级学科计划(QJGC20230101)。

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,Ec Tna A)和噬甲基菌(Methylophaga aminisulfidivorans)来源的黄素依赖性单加氧酶(flavin-dependent monooxygenase,Ma FMO)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Ma FMO进行蛋白质工程改造,获得了有益突变体Ma FMO^(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(1288.59±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Ma FMO活性提高的突变体,为靛蓝的工业化生产奠定了基础。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(1288.59±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.

关 键 词：大肠杆菌 靛蓝 蛋白质工程改造 双酶级联 L-色氨酸 

分 类 号：TQ613[化学工程—精细化工] TQ203.2