Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Metab Eng. 2022 Jan;69:15-25. doi: 10.1016/j.ymben.2021.10.010. Epub 2021 Oct 29.
Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6'-O-β-xylopyranosyl)-O-β-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction.
植物化学物质是药物和营养保健品的丰富资源。获取这些宝贵化合物的一个关键挑战可能会对开发造成重大瓶颈。肉桂醇二糖也称为罗萨文，是著名药用植物红景天的主要生物活性成分。肉桂基-(6'-O-β-吡喃木糖基)-O-β-吡喃葡萄糖苷（罗萨文 E）是一种天然罗萨文类似物，其阿拉伯吡喃糖单元被其差向异构体木糖取代，仅从红景天中微量分离得到。在此，我们描述了大肠杆菌中罗萨文 E 的从头合成。1,6-葡糖基转移酶 CaUGT3 被工程改造为木糖基转移酶，通过将 T145 残基替换为缬氨酸，将肉桂醇单葡糖苷（松香）转化为罗萨文 E。通过添加突变 N375Q，酶活性进一步提高了 2.9 倍。通过将变体 CaUGT3、来自苜蓿中华根瘤菌 1021 的 UDP-木糖合酶（SmUXS）和松香生物合成酶与苯丙氨酸过量产生的大肠杆菌菌株结合，从葡萄糖合成罗萨文 E，达到了 92.9mg/L 的浓度。通过共过表达拟南芥 UDP-木糖合酶（AtUXS3）和 SmUXS，进一步提高了罗萨文 E 的产量，在 5L 生物反应器中进行分批补料发酵，产量达到 782.0mg/L。这项工作代表了通过糖基转移酶工程和人工途径构建生产具有二糖链的天然产物的一个极好范例。
