Yang Han, Liu Feixia, Li Yin, Yu Bo
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
University of Chinese Academy of Sciences , Beijing 100049, China.
ACS Synth Biol. 2018 Jan 19;7(1):121-131. doi: 10.1021/acssynbio.7b00256. Epub 2017 Nov 29.
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 coli via 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.
流行病学数据证实，经常食用十字花科蔬菜与较低的癌症风险之间存在密切关联。这种防癌特性主要归因于硫代葡萄糖苷产物，比如西兰花中发现的源自蛋氨酸的萝卜硫苷。在此，我们报告了首次通过基因筛选、途径设计和蛋白质工程，在大肠杆菌中成功重建了从蛋氨酸到萝卜硫苷的完整生物合成途径。我们使用支链氨基酸转氨酶3催化两个转氨步骤，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。设计并在大肠杆菌中功能性表达了两种嵌合细胞色素P450酶。用粗糙脉孢菌的组氨酸半胱氨酸亚砜裂解酶取代了原始的植物C-S裂解酶。从拟南芥、白菜和甘蓝中成功挖掘出了其他途径酶。通过液相色谱-串联质谱分析证实了体内共表达优化酶后萝卜硫苷的生物合成。未鉴定出其他可促进下游纯化过程的硫代葡萄糖苷类似物（除了葡糖芥苷）。本研究中萝卜硫苷的生产为大规模微生物生产这类有益健康的硫代葡萄糖苷奠定了基础。
