Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus's geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.

中文翻译：

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长春花属玫瑰悬液培养物在质体或胞质溶胶中过表达香叶醇合酶的代谢变化。

先前的研究表明，香叶醇可能是长春花细胞和毛状根培养物中单萜类途径产生萜类吲哚生物碱（TIA）的上游限制因素。前体可用性的不足可能是由于（1）香叶醇香叶醇合酶的有限表达导致该酶催化香叶基二磷酸转化为香叶醇的活性低；（2）香叶醇从质体向胞质溶胶运输的限制。因此，在这项研究中，玫瑰色念珠菌的香叶醇合成酶（CrGES）基因在非TIA产生的玫瑰色念珠菌细胞系的质体或胞质溶胶中过表达。证实了CrGES在质体或细胞质中的表达，并成功建立了组成型转化系。使用HPLC进行的目标代谢物分析表明，转化的细胞系除非产生茉莉酸作为其亲本细胞系，否则不会产生TIA或虹彩化合物前体。这表明需要表达额外的诱导途径基因以在该细胞系中产生TIA。有趣的是，使用基于NMR的代谢组学进行的进一步分析表明，如果在质体或胞质溶胶中表达，CrGES的过表达对初级代谢的影响会不同。在过表达质体的细胞系中，缬氨酸，亮氨酸和一些源自the草酸途径的代谢产物（即苯丙氨酸和酪氨酸）的水平明显较高，而在胞浆-CrGES中则较低。该结果表明，质体或细胞质中CrGES的过表达引起与植物细胞生长和发育有关的初级代谢的改变。必须进行全面的组学分析才能揭示代谢工程的全部效果。