Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex.
Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex.
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DOI：
10.1186/1475-2859-11-42
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发表时间：
2012-04-04
影响因子：
6.4
通讯作者：
Yan Y
中科院分区：
文献类型：
--
作者：
Lin Y;Yan Y
咖啡酸（3，4-dihydroxycinnamic acid）是一种天然酚类化合物，来源于植物类苯丙烷途径。咖啡酸及其苯乙酯（CAPE）因其丰富的药理活性和保健作用而备受关注。如今，通过微生物方法大规模生产药物或药物前体为化学合成和从植物来源提取提供了有前途的替代方案。我们首先鉴定了大肠杆菌天然羟化酶复合物，其先前表征为4-羟基苯乙酸3-羟化酶（4 HPA 3 H），能够有效地将对香豆酸转化为咖啡酸。在植物中由膜结合的细胞色素P450酶，对香豆酸3-羟化酶（C3 H）催化的这一关键酶促步骤难以在原核系统中功能性表达。此外，还比较了两种红细菌酪氨酸氨裂解酶（TAL）在大肠杆菌中过表达后的性能。杆菌结果表明，鲎试剂对红曲霉毒素的敏感性较好。capsulatus（Rc）对酪氨酸和L-多巴都具有较高的活性。基于这些发现，我们进一步设计了由酶4 HPA 3 H和RcTAL组成的从酪氨酸到咖啡酸的双途径。这种异源途径延长了E.大肠杆菌天然酪氨酸生物合成机制，并能在低盐培养基中产生咖啡酸（12.1 mg/L）。进一步提高产量是通过促进E.大肠杆菌，其中涉及缓解酪氨酸诱导的反馈抑制和碳通量重定向。摇瓶培养48 h，咖啡酸的效价达到50.2mg/L。我们已经成功地建立了一个新的途径，并构建了一个E。大肠杆菌菌株生产咖啡酸。这项工作为进一步提高产量奠定了基础，并开辟了微生物合成咖啡酸衍生的更复杂的植物次级代谢产物的可能性。此外，我们已经确定TAL是该途径中的限速酶。因此，通过生物勘探和蛋白质工程方法探索更有活性的TAL对于进一步提高咖啡酸产量是必要的。
Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE) have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H), is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs) from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc) possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L) in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from caffeic acid. In addition, we have identified that TAL is the rate-limiting enzyme in this pathway. Thus, exploration for more active TALs via bio-prospecting and protein engineering approaches is necessary for further improvement of caffeic acid production.
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