Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species
Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species
复制标题
DOI：
10.1111/j.1365-313x.2009.03980.x
复制
发表时间：
2009-11-01
期刊：
影响因子：
7.2
通讯作者：
Facchini, Peter J.
中科院分区：
文献类型：
--
作者：
Liscombe, David K.;Ziegler, Joerg;Facchini, Peter J.
为了发现和鉴定参与植物专化代谢的新基因，开发了一种综合方法，使用靶向代谢物图谱和适度的EST文库，每个文库包含大约3500个单基因。EST数据库已被建立用于产生物碱的加州棘球藻、苞叶罂粟和黄色唐松草的细胞培养物，是生物碱生物合成基因的丰富储存库。ESI-FTICR-MS和ESI-MS/MS分析有助于对每个系统中的生物碱进行明确的识别和相对定量。将每个EST文库中已知的和候选的生物合成基因与由经验代谢物图谱组装的苄基异喹啉生物合成网络进行手动整合，可以鉴定和鉴定四个N-甲基转移酶(NMT)。从黄曲霉中分离得到的一个基因编码的是一种对(+/-)-吡啶具有独特选择性的N-甲基转移酶(TfPavNMT)，它是第一个参与该生物碱分支途径的酶。特定生物碱的出现、编码已知的苄基异喹啉生物碱生物合成基因的EST的互补以及特定NMT的不同底物范围的相关性表明，双边预测酶功能和物种依赖的特定代谢物谱是可行的。
P>An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant-specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI-FTICR-MS and ESI-MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N-methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N-methyltransferase (TfPavNMT), which showed a unique preference for (+/-)-pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species-dependent specialized metabolite profiles.