Reconstructing Biosynthetic Pathway of the Plant-Derived Cancer Chemopreventive-Precursor Glucoraphanin in Escherichia coli,ACS Synthetic Biology

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Reconstructing Biosynthetic Pathway of the Plant-Derived Cancer Chemopreventive-Precursor Glucoraphanin in Escherichia coli
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2017-11-29 00:00:00 , DOI: 10.1021/acssynbio.7b00256
Han Yang _{1,

2} , Feixia Liu ₁ , Yin Li ₁ , Bo Yu ₁

Affiliation

Epidemiological data confirmed a strong correlation between regular consumption of cruciferous vegetables and lower cancer risk. This cancer preventive property is mainly attributed to the glucosinolate products, such as glucoraphanin found in broccoli that is derived from methionine. Here we report the first successful reconstruction of the complete biosynthetic pathway of glucoraphanin from methionine in Escherichia colivia gene selection, pathway design, and protein engineering. We used branched-chain amino transferase 3 to catalyze two transamination steps to ensure the purity of precursor molecules and used cysteine as a sulfur donor to simplify the synthesis pathway. Two chimeric cytochrome P450 enzymes were engineered and expressed in E. coli functionally. The original plant C–S lyase was replaced by the Neurospora crassa hercynylcysteine sulfoxide lyase. Other pathway enzymes were successfully mined from Arabidopsis thaliana, Brassica rapa, and Brassica oleracea. Biosynthesis of glucoraphanin upon coexpression of the optimized enzymes in vivo was confirmed by liquid chromatography–tandem mass spectrometry analysis. No other glucosinolate analogues (except for glucoiberin) were identified that could facilitate the downstream purification processes. Production of glucoraphanin in this study laid the foundation for microbial production of such health-beneficial glucosinolates in a large-scale.

中文翻译：

大肠杆菌中植物源性化学预防性前体葡萄糖素的重建生物合成途径

流行病学数据证实，经常食用十字花科蔬菜与降低癌症风险之间存在很强的相关性。该癌症的预防性质主要归因于芥子油苷产品，例如在西兰花中发现的蛋氨酸中的蛋黄素，其来源于蛋氨酸。在这里，我们从蛋氨酸报告莱菔子素的完整的生物合成途径的第一个成功的重建大肠杆菌通过基因选择，通路设计，和蛋白质工程。我们使用支链氨基转移酶3催化两个氨基转移步骤，以确保前体分子的纯度，并使用半胱氨酸作为硫供体来简化合成途径。工程改造了两种嵌合细胞色素P450酶并在大肠杆菌中表达功能上。最初的植物C–S裂解酶被Neurospora crassa的海西炔半胱氨酸亚砜裂解酶所取代。其他途径的酶成功地从拟南芥，芸苔（Brassica rapa）和甘蓝（Brassica oleracea）中提取。液相色谱-串联质谱分析证实了在体内共表达优化酶后的生物合成葡聚糖。没有发现其他可以促进下游纯化过程的硫代芥子油苷类似物（除葡糖苷外）。在这项研究中，生产葡糖甘露聚糖为大规模生产这种有益于健康的芥子油苷奠定了基础。

更新日期：2017-11-29

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