Instituto de Ecología, A.C. (INECOL), Red de Estudios Moleculares Avanzados (REMAV), Xalapa 91073, Veracruz, Mexico.
Instituto Mexicano del Seguro Social (IMSS), Centro de Investigación Biomédica del Sur (CIBIS), Xochitepec 62790, Morelos, Mexico.
Int J Mol Sci. 2020 Oct 14;21(20):7572. doi: 10.3390/ijms21207572.
This investigation cultured cells in suspension to evaluate the effect of nitrate deficiency on the growth and production of chlorogenic acid (CGA), a secondary metabolite with hypoglycemic and hypolipidemic activity that acts directly on type 2 diabetes mellitus. Using cell cultures in suspension, a kinetics time course was established with six time points and four total nitrate concentrations. The metabolites of interest were quantified by high-performance liquid chromatography (HPLC), and the metabolome was analyzed using directed and nondirected approaches. Finally, using RNA-seq methodology, the first transcript collection for was generated. HPLC analysis detected CGA at all sampling points, while metabolomic analysis confirmed the identity of CGA and of precursors involved in its biosynthesis. Transcriptome analysis identified differentially expressed genes and enzymes involved in the biosynthetic pathway of CGA. probably expresses a key enzyme with bifunctional activity, the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HQT/HCT), which recognizes shikimic acid or quinic acid as a substrate and incorporates either into one of the two routes responsible for CGA biosynthesis.
本研究通过悬浮培养细胞，评估硝酸盐缺乏对绿原酸（CGA）生长和产生的影响。CGA 是一种具有降血糖和降血脂活性的次生代谢物，可直接作用于 2 型糖尿病。通过悬浮细胞培养，建立了一个具有六个时间点和四个总硝酸盐浓度的动力学时间过程。采用高效液相色谱法（HPLC）定量测定感兴趣的代谢物，并采用有向和无向方法对代谢组进行分析。最后，采用 RNA-seq 方法，首次生成了 的转录组集合。HPLC 分析在所有采样点均检测到 CGA，而代谢组学分析则证实了 CGA 及其生物合成途径中涉及的前体的身份。转录组分析鉴定了差异表达的基因和参与 CGA 生物合成途径的酶。 可能表达一种具有双功能活性的关键酶，即 3-咖啡酰奎尼酸/3-咖啡酰莽草酸羟肉桂酰转移酶和 3-咖啡酰奎尼酸/莽草酸 5-羟肉桂酰转移酶（HQT/HCT），它可识别莽草酸或奎宁酸作为底物，并将其分别整合到负责 CGA 生物合成的两条途径之一中。
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