Institute of Biosciences and Bioresources, National Research Council of Italy, 06128 Perugia, Italy.
Department of Biomolecular Sciences, University Carlo Bo, 61029 Urbino, Italy.
Int J Mol Sci. 2021 May 12;22(10):5102. doi: 10.3390/ijms22105102.
Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of (tobacco) transformed by with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.
植物的基因工程已被证明是一种有吸引力的方法来生产各种次生代谢物。在这里,我们试图在转化的 (烟草) 植物中产生犬尿氨酸,这是一种促进健康的代谢物,该植物转化的基因编码人类吲哚胺 2,3-双加氧酶 1 (IDO1),该酶负责犬尿氨酸的产生,因为色氨酸降解。通过 PCR 证实了转基因植物中存在 IDO1 基因,但未检测到蛋白质。为了赋予异源人 IDO1 蛋白更高的稳定性,并提供更灵敏的方法来检测感兴趣的蛋白,我们克隆了编码 IDO1-GFP 的基因构建体。对瞬时转染的烟草原生质体的分析表明,IDO1-GFP 基因导致可检测蛋白的表达,并在原生质体培养基中产生犬尿氨酸。有趣的是,人 IDO1 在植物细胞中的细胞内定位与在哺乳动物细胞中发现的相似,主要在细胞质中,但也在内体早期中。据我们所知,这是首次报道在植物细胞中表达能够分泌犬尿氨酸的人 IDO1 酶。