Jan Rahmatullah, Asaf Sajjad, Paudel Sanjita, Lee Sangkyu, Kim Kyung-Min
Division of Plant Biosciences, School of Applied Biosciences, College of Agriculture & Life Science, Kyungpook National University, 80 Dahak-ro, Buk-gu, Daegu 41566, Korea.
Natural and Medical Science Research Center, University of Nizwa 616, Nizwa 611, Oman.
Biology (Basel). 2021 Jan 6;10(1):32. doi: 10.3390/biology10010032.
Kaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in using naringenin as a substrate. was cloned into pRS42K yeast episomal plasmid (YEp) vector and the activity of the target gene was analyzed in engineered and empty strains. We confirmed a novel step of kaempferol and quercetin biosynthesis directly from naringenin, catalyzed by the rice flavanone 3-hydroxylase (). The results were confirmed through thin layer chromatography (TLC) followed by western blotting, nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry LCMS-MS. TLC showed positive results when comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed the lack of activity in empty strains and confirmed high expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that is responsible for the conversion of naringenin to both kaempferol and quercetin.
山奈酚和槲皮素是植物重要的次生代谢产物，在植物防御系统中具有多种生物学功能。本研究以柚皮素为底物，构建了山奈酚和槲皮素的生物合成途径。将其克隆到pRS42K酵母附加体质粒（YEp）载体中，并在工程菌株和空菌株中分析目标基因的活性。我们证实了水稻黄烷酮3-羟化酶催化从柚皮素直接合成山奈酚和槲皮素的新步骤。通过薄层色谱（TLC），随后进行蛋白质免疫印迹、核磁共振（NMR）和液相色谱-质谱联用（LCMS-MS）对结果进行了验证。将工程菌株中提取的两种化合物与标准对照品进行比较时，TLC显示出阳性结果。蛋白质免疫印迹证实空菌株中缺乏该活性，并证实在工程菌株中有高表达。核磁共振光谱仅证实了槲皮素，而LCMS-MS结果表明负责将柚皮素转化为山奈酚和槲皮素。
