基于地衣芽孢杆菌表达系统搭建左旋多巴新合成体系  

Preparation of L-DOPA from tyrosine by biotransformation using Bacillus licheniformis strains expressed tyrosine 3-monooxygenase andsepiapterin reductase

作　　者：李乐云 李由然 许银彪[1,2] 张梁 顾正华 丁重阳[1,2] 石贵阳 LI Leyun;LI Youran;XU Yinbiao;ZHANG Liang;GU Zhenghua;DING Zhongyang;SHI Guiyang(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]粮食发酵工艺与技术国家工程实验室(江南大学),江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2020年第11期23-31,共9页Food and Fermentation Industries

基　　金：轻工业技术与工程国家一流学科计划(LITE2018-22)。

摘　　要：左旋多巴(3,4-dihydroxylphenylalanine,L-DOPA)是一种L-酪氨酸(L-tyrosine,L-Tyr)的氨基酸类似物,在临床医学、美容保健等领域具有广阔的应用前景。通过生物信息学方法挖掘到Streptosporangium roseum DSM 43021来源的酪氨酸羟化酶(tyrosine hydroxylase,TH)基因(srth)在地衣芽孢杆菌(Bacillus licheniformis)中重组表达,结合墨蝶呤还原酶重组菌以L-Tyr为底物进行全细胞联合转化合成L-DOPA,并初步探究其催化过程。结果显示,srth具有保守的Fe 2+及底物喋呤的氨基酸结合位点,与人来源的TH亲缘关系较远。SrTH蛋白胞内粗酶活力为30.7 U/mL,胞外酶活力为22.79 U/mL。利用srth重组菌Bl/pMH与墨蝶呤还原酶重组菌Bl/pMF全细胞联合转化L-Tyr,成功合成L-DOPA。TH催化合成L-DOPA过程中,四氢生物蝶呤(tetrahydrobiopterin,BH4)是限制酶反应进行的重要因素,L-Tyr的消耗量与BH4的需求量之比约为1∶2。研究首次实现了TH在地衣芽孢菌中的表达,成功基于B.licheniformis表达系统搭建L-DOPA新合成体系,对拓展酶催化法高效合成L-DOPA具有重要的指导意义和应用潜力。3,4-Dihydroxylphenylalanine(L-DOPA)is an analog of L-tyrosine(L-Tyr),which has broad application prospects in clinical medicine,cosmetic,health care and other fields.A gene(srth)encoding a tyrosine 3-monooxygenase from Streptosporangium roseum DSM 43021 was cloned and expressed in Bacillus licheniformis,and the recombinant srth was obtained.The intracellular and extracellular activities of the recombinant tyrosine 3-monooxygenase were 30.7 U/mL and 22.79 U/mL,respectively.The recombinant srth and a strain expressed sepiapterin reductase were efficiently used for production of L-DOPA from L-Tyr by whole-cell biotransformation.Tetrahydrobiopterin(BH4)was found a restrictive factor in the process and the ratio of the consumption of L-Tyr to the demand of BH4 was about 1∶2.The results provide a novel biotransformation routine for L-DOPA preparation.

关 键 词：左旋多巴 地衣芽孢杆菌 酪氨酸羟化酶 全细胞转化 L-酪氨酸 四氢生物蝶呤 

分 类 号：Q939.9[生物学—微生物学]