Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, Anhui, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; National Center of Technology Innovation for Synthetic Biology, Tianjin, China; Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; National Center of Technology Innovation for Synthetic Biology, Tianjin, China; Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China; University of Chinese Academy of Sciences, Beijing, China.
Metab Eng. 2024 Mar;82:274-285. doi: 10.1016/j.ymben.2024.02.017. Epub 2024 Feb 28.
Rosavin is the characteristic component of Rhodiola rosea L., an important medicinal plant used widely in the world that has been reported to possess multiple biological activities. However, the endangered status of wild Rhodiola has limited the supply of rosavin. In this work, we successfully engineered an Escherichia coli strain to efficiently produce rosavin as an alternative production method. Firstly, cinnamate: CoA ligase from Hypericum calycinum, cinnamoyl-CoA reductase from Lolium perenne, and uridine diphosphate (UDP)-glycosyltransferase (UGT) from Bacillus subtilis (Bs-YjiC) were selected to improve the titer of rosin in E. coli. Subsequently, four UGTs from the UGT91R subfamily were identified to catalyze the formation of rosavin from rosin, with SlUGT91R1 from Solanum lycopersicum showing the highest activity level. Secondly, production of rosavin was achieved for the first time in E. coli by incorporating the SlUGT91R1 and UDP-arabinose pathway, including UDP-glucose dehydrogenase, UDP-xylose synthase, and UDP-xylose 4-epimerase, into the rosin-producing stain, and the titer reached 430.5 ± 91.4 mg/L. Thirdly, a two-step pathway derived from L-arabinose, composed of L-arabinokinase and UDP-sugar pyrophosphorylase, was developed in E. coli to further optimize the supply of the precursor UDP-arabinose. Furthermore, 1203.7 ± 32.1 mg/L of rosavin was produced from D-glucose and L-arabinose using shake-flask fermentation. Finally, the production of rosavin reached 7539.1 ± 228.7 mg/L by fed-batch fermentation in a 5-L bioreactor. Thus, the microbe-based production of rosavin shows great potential for commercialization. This work provides an effective strategy for the biosynthesis of other valuable natural products with arabinose-containing units from D-glucose and L-arabinose.
罗萨文是红景天的特征成分，红景天是一种在世界范围内广泛使用的重要药用植物，已被报道具有多种生物活性。然而，野生红景天的濒危状况限制了罗萨文的供应。在这项工作中，我们成功地构建了一种大肠杆菌菌株，能够高效生产罗萨文，作为一种替代生产方法。首先，我们从贯叶连翘中选择肉桂酰辅酶 A 连接酶、从黑麦草中选择肉桂酰辅酶 A 还原酶和来自枯草芽孢杆菌的尿苷二磷酸（UDP）-糖基转移酶（UGT）（Bs-YjiC），以提高大肠杆菌中松香的含量。随后，鉴定了来自 UGT91R 亚家族的四个 UGT 来催化松香转化为罗萨文，其中番茄 UGT91R1 表现出最高的活性水平。其次，通过将 SlUGT91R1 和 UDP-阿拉伯糖途径（包括 UDP-葡萄糖脱氢酶、UDP-木糖合酶和 UDP-木糖 4-差向异构酶）整合到产生松香的菌株中，首次在大肠杆菌中实现了罗萨文的生产，产量达到 430.5±91.4mg/L。第三，在大肠杆菌中开发了一个源自 L-阿拉伯糖的两步途径，由 L-阿拉伯糖激酶和 UDP-糖磷酸化酶组成，以进一步优化前体 UDP-阿拉伯糖的供应。此外，使用摇瓶发酵从 D-葡萄糖和 L-阿拉伯糖生产了 1203.7±32.1mg/L 的罗萨文。最后，通过在 5L 生物反应器中进行分批补料发酵，罗萨文的产量达到 7539.1±228.7mg/L。因此，基于微生物的罗萨文生产具有很大的商业化潜力。这项工作为从 D-葡萄糖和 L-阿拉伯糖生物合成含有阿拉伯糖单元的其他有价值的天然产物提供了一种有效的策略。
