Uchida Kai, Fuji Yushiro, Tabeta Hiromitsu, Akashi Tomoyoshi, Hirai Masami Yokota
RIKEN Center for Sustainable Resource Science, 1-7-22 Yokohama, Kanagawa 230-0045, Japan.
Department of Applied Biological Sciences, Nihon University1866, Fujisawa, Kanagawa, 252-0880, Japan.
Plant Cell Physiol. 2025 Mar 31;66(3):304-317. doi: 10.1093/pcp/pcae151.
Soybean (Glycine max) is a leguminous crop cultivated worldwide that accumulates high levels of isoflavones. Although previous research has often focused on increasing the soybean isoflavone content because of the estrogen-like activity of dietary soy in humans, the rapidly increasing demand for soybean as a plant-based meat substitute has raised concerns about excessive isoflavone intake. Therefore, the production of isoflavone-free soybean has been anticipated. However, there have been no reports of an isoflavone-free soybean until now. Here, 2-hydroxyisoflavanone synthase (IFS), which is essential for isoflavone biosynthesis, was targeted for genome editing in soybean. A novel CRISPR/Cas9 system using Staphylococcus aureus Cas9 instead of the commonly used Streptococcus pyogenes Cas9 was established and customized. Through Agrobacterium rhizogenes-mediated transformation, IFS-edited hairy roots were generated in which all three IFS genes contained deletion mutations. Metabolome analyses of IFS-edited hairy roots revealed that isoflavone content significantly decreased, whereas levels of flavonoids, including a novel chalcone derivative, increased. A transcriptome analysis revealed changes in the expression levels of a large number of genes, including jasmonic acid-inducible genes. In addition, the functions of selected transcription factor genes (MYB14-L, GmbHLH112, and GmbHLH113), which were dramatically upregulated by IFS editing, were investigated by multiomics analyses of their over-expressing hairy root lines. They appear to be involved in flavonoid and triterpene saponin biosynthesis, salicylic acid metabolism, and central carbon metabolism. Overall, the results indicated that editing IFS genes caused the redirection of the metabolic flux from isoflavonoid biosynthesis to flavonoid accumulation, as well as dynamic changes in gene regulatory networks.
大豆（Glycine max）是一种在全球广泛种植的豆科作物，能积累高水平的异黄酮。尽管由于膳食大豆在人体内具有类似雌激素的活性，以往的研究常常聚焦于提高大豆异黄酮含量，但随着对大豆作为植物性肉类替代品的需求迅速增加，人们开始担忧异黄酮摄入过量的问题。因此，无异黄酮大豆的生产备受期待。然而，迄今为止尚未有关于无异黄酮大豆的报道。在此，对大豆中异黄酮生物合成所必需的2-羟基异黄酮合酶（IFS）进行了基因组编辑。建立并定制了一种新型的CRISPR/Cas9系统，该系统使用金黄色葡萄球菌Cas9而非常用的化脓性链球菌Cas9。通过发根农杆菌介导的转化，产生了编辑IFS的毛状根，其中所有三个IFS基因都含有缺失突变。对编辑IFS的毛状根进行代谢组分析发现，异黄酮含量显著降低，而包括一种新型查尔酮衍生物在内的黄酮类化合物水平升高。转录组分析揭示了大量基因表达水平的变化，包括茉莉酸诱导基因。此外，通过对过表达毛状根系进行多组学分析，研究了经IFS编辑后显著上调的选定转录因子基因（MYB14-L、GmbHLH112和GmbHLH113）的功能。它们似乎参与了黄酮类化合物和三萜皂苷的生物合成、水杨酸代谢以及中心碳代谢。总体而言，结果表明编辑IFS基因导致代谢通量从异黄酮生物合成转向黄酮类化合物积累，以及基因调控网络的动态变化。
