13C Tracer Reveals Phenolic Acids Biosynthesis in Hairy Root Cultures of Salvia miltiorrhiza
13C Tracer Reveals Phenolic Acids Biosynthesis in Hairy Root Cultures of Salvia miltiorrhiza
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DOI：
10.1021/cb3006962
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发表时间：
2013-07-01
影响因子：
4
通讯作者：
Chen, Wansheng
中科院分区：
文献类型：
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作者：
Di, Peng;Zhang, Lei;Chen, Wansheng
迷迭香酸（rosmarinic acid，RA）和紫草酸B（lithospermic acid，LAB）是两种典型的酚酸类化合物，具有显著的生物活性，可能与丹参的治疗作用有关。准确了解RA和LAB的生物合成途径是优化S.丹参。利用[环-C-13]-苯丙氨酸的体内同位素标记实验，结合UPLC/Q-TOF动态测量代谢产物水平，研究了S.丹参。这些数据表明体内酚类生物合成途径：来自一般苯丙素途径和酪氨酸衍生途径的两个中间体4-香豆酰-CoA和3，4-二羟基苯基乳酸（DHPL）通过酯形成酶迷迭香酸合酶（SmRAS）偶联形成4-香豆酰-3'，4'-二羟基苯基乳酸（4C-DHPL）。3-羟基通过细胞色素P450依赖性单加氧酶（SmCYP 98 A14）在途径后期引入，形成RA。随后，RA通过氧化转化为苯氧基自由基，并且两个苯氧基自由基自发结合形成LAB。这些结果揭示了S.丹参和扩大苯丙烷衍生的代谢途径的理解。这些关键酶的候选基因的揭示为今后通过代谢工程提高重要酚酸类化合物产量的后续研究奠定了坚实的基础。
Rosmarinic acid (RA) and lithospermic acid B (LAB) are two typical phenolic acids with significant bioactivities that may contribute to the therapeutic effects of Salvia miltiorrhiza. Precise knowledge of the biosynthetic pathway leading to RA and LAB is a necessary prerequisite to optimize the production of important phenolic compounds in S. miltiorrhiza. In vivo isotopic labeling experiments using [ring-C-13]-phenylalanine, combined with dynamic measurements of metabolite levels by UPLC/Q-TOF, were used to investigate the metabolic origin of phenolic acids in S. miltiorrhiza. These data indicate the in vivo phenolic biosynthetic pathway: two intermediates from the general phenylpropanoid pathway and the tyrosine-derived pathway, 4-coumaroyl-CoA and 3,4-dihydroxyphenyllactic acid (DHPL), are coupled by the ester-forming enzyme rosmarinic acid synthase (SmRAS) to form 4-coumaroyl-3',4'-dihydroxyphenyllactic acid (4C-DHPL). The 3-hydroxyl group is introduced late in the pathway by a cytochrome P450-dependent monooxygenase (SmCYP98A14) to form RA. Subsequently, RA is transformed to a phenoxyl radical by oxidation, and two phenoxyl radicals unite spontaneously to form LAB. The results indicate aspects of the complexity of phenolic acid biosynthesis in S. miltiorrhiza and expand an understanding of phenylpropanoid-derived metabolic pathways. The candidate genes for the key enzymes that were revealed provide a substantial foundation for follow-up research on improving the production of important phenolic acids through metabolic engineering in the future.