Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) such as pharmaceutical hyoscyamine, anisodamine, and scopolamine, remains largely unknown. In this study, we characterized the physiological and metabolic functions of the ODC gene of Atropa belladonna (AbODC) and determined its role in TA production using metabolic engineering approaches. Feeding assays with enzyme inhibitors indicated that ODC, rather than arginine decarboxylase (ADC), plays a major role in TA biosynthesis. Tissue-specific AbODC expression analysis and β-glucuronidase (GUS) staining assays showed that AbODC was highly expressed in secondary roots, especially in the cylinder tissue. Enzymatic assays indicated that AbODC was able to convert ornithine to putrescine, with the highest activity at pH 8.0 and 30 °C. Additionally, AbODC showed higher catalytic efficiency than other plant ODCs, as evident from the Km, Vmax, and Kcat values of AbODC using ornithine as the substrate. In A. belladonna root cultures, suppression of AbODC greatly reduced the production of putrescine, N-methylputrescine, and TAs, whereas overexpression of AbODC significantly increased the biosynthesis of putrescine, N-methylputrescine, hyoscyamine, and anisodamine. Moreover, transgenic A. belladonna plants overexpressing AbODC showed a significantly higher production of hyoscyamine and anisodamine compared with control plants. These findings indicate that AbODC plays a key role in TA biosynthesis and therefore is a valuable candidate for increasing TA production in A. belladonna.

中文翻译：

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基于颠茄鸟氨酸中鸟氨酸脱羧酶代谢特征的工程对虾生物碱生产。

鸟氨酸脱羧酶（ODC）在各种生物过程中起着重要作用；然而，其在植物次生代谢中的作用，尤其是在药用羟乙胺，山iso碱和东碱等托烷生物碱（TAs）的生物合成中的作用仍然未知。在这项研究中，我们表征了颠茄ODC基因（AbODC）的生理和代谢功能，并使用代谢工程方法确定了它在TA生产中的作用。酶抑制剂的进料分析表明，ODC而非精氨酸脱羧酶（ADC）在TA生物合成中起主要作用。组织特异性AbODC表达分析和β-葡萄糖醛酸酶（GUS）染色分析表明，AbODC在次生根中，特别是在圆柱组织中高表达。酶促测定表明，AbODC能够将鸟氨酸转化为腐胺，在pH 8.0和30°C下具有最高的活性。此外，从鸟氨酸作为底物的AbODC的Km，Vmax和Kcat值可以明显看出，AbODC的催化效率高于其他植物ODC。在颠茄农杆菌的根培养物中，AbODC的抑制大大降低了腐胺，N-甲基putrescine和TA的产生，而AbODC的过表达显着提高了腐胺，N-甲基putrescine，hyoscyamine和山iso碱的生物合成。此外，与对照植物相比，过表达AbODC的转基因颠茄农杆菌植物显示出明显更高的羟胺和山iso碱产量。