School of Bioengineering, Henan University of Technology, Lianhua Street 100, Zhengzhou High-technology Zone, Henan Province 450001, China.
School of Bioengineering, Henan University of Technology, Lianhua Street 100, Zhengzhou High-technology Zone, Henan Province 450001, China.
Gene. 2024 Nov 30;928:148815. doi: 10.1016/j.gene.2024.148815. Epub 2024 Aug 2.
Rehmannia glutinosa produces many phenylethanoid glycoside (PhG) compounds, including salidroside, which not only possesses various biological activities but also is a core precursor of some medicinal PhGs, so it is very important to elucidate the species' salidroside biosynthesis pathway to enhance the production of salidroside and its derivations. Although some plant copper-containing amine oxidases (CuAOs), phenylacetaldehyde reductases (PARs) and UDP-glucose glucosyltransferases (UGTs) are thought to be vital catalytic enzymes involved in the downstream salidroside biosynthesis pathways, to date, none of these proteins or the associated genes in R. glutinosa have been characterized. To verify a postulated R. glutinosa salidroside biosynthetic pathway starting from tyrosine, this study identified and characterized a set of R. glutinosa genes encoding RgCuAO, RgPAR and RgUGT enzymes for salidroside biosynthesis. The functional activities of these proteins were tested in vitro by heterologous expression of these genes in Escherichia coli, confirming these catalytic abilities in these corresponding reaction steps of the biosynthetic pathway. Importantly, four enzyme-encoding genes (including the previously reported RgTyDC2 encoding tyrosine decarboxylase and the RgCuAO1, RgPAR1 and RgUGT2 genes) were cointegrated into Saccharomyces cerevisiae to reconstitute the R. glutinosa salidroside biosynthetic pathway, achieving an engineered strain that produced salidroside and validating these enzymes' catalytic functions. This study elucidates the complete R. glutinosa salidroside biosynthesis pathway from tyrosine metabolism in S. cerevisiae, establishing a basic platform for the efficient production of salidroside and its derivatives.
地黄产生许多苯乙醇苷（PhG）化合物，包括红景天苷，它不仅具有多种生物活性，而且还是一些药用 PhG 的核心前体，因此阐明该物种红景天苷的生物合成途径对于提高红景天苷及其衍生物的产量非常重要。尽管一些植物含铜胺氧化酶（CuAOs）、苯乙醛还原酶（PARs）和 UDP-葡萄糖葡萄糖基转移酶（UGTs）被认为是参与下游红景天苷生物合成途径的重要催化酶，但迄今为止，这些蛋白质或地黄中的相关基因尚未得到表征。为了验证从酪氨酸开始的假定地黄红景天苷生物合成途径，本研究鉴定并表征了一组编码 RgCuAO、RgPAR 和 RgUGT 酶的地黄基因，用于红景天苷的生物合成。通过在大肠杆菌中异源表达这些基因来测试这些蛋白质的功能活性，在生物合成途径的相应反应步骤中证实了这些催化能力。重要的是，将四个酶编码基因（包括先前报道的编码酪氨酸脱羧酶的 RgTyDC2 和 RgCuAO1、RgPAR1 和 RgUGT2 基因）共整合到酿酒酵母中，以重建地黄红景天苷生物合成途径，实现了产生红景天苷的工程菌株，并验证了这些酶的催化功能。本研究阐明了从酿酒酵母中天冬氨酸代谢途径到红景天苷的完整地黄红景天苷生物合成途径，为高效生产红景天苷及其衍生物建立了一个基本平台。
