Self-Sufficient In Vitro Multi-Enzyme Cascade for Efficient Synthesis of Danshensu from l -DOPA

ACS Synthetic Biology, vol. 12, pp. 277–286

Abstract

Danshensu (DSS), a traditional Chinese medicine, is widely used for the treatment of cardiovascular and cancer diseases. Here, a one-pot multi-enzyme cascade pathway was designed for DSS synthesis from l -DOPA using tyrosine aminotransferase from Escherichia coli ( Ec TyrB) and d -isomer-specific 2-hydroxyacid dehydrogenase from Lactobacillus frumenti ( Lf D2-HDH). Glutamate dehydrogenase from Clostridium difficile ( Cd gluD) was also introduced for a self-sufficient system of α-ketoglutaric acid and NADH. Under optimal conditions (35 °C, pH 7.0, Ec TyrB: Lf D2-HDH: Cd gluD = 3:2:1, glutamate:NAD + = 1:1), 98.3% yield (at 20 mM l -DOPA) and space-time yield of 6.61 g L –1 h –1 (at 40 mM l -DOPA) were achieved. Decreased yields of DSS at elevated l -DOPA concentrations (100 mM) could be attributed to an inhibited Cd gluD activity caused by NH 4 + accumulation. This developed multi-enzyme cascade pathway (including Ec TyrB, Lf D2-HDH, and Cd gluD) provides an efficient and sustainable approach for the production of DSS from l -DOPA.

Authors 9

1. Ruizhi Han Aachen Institute of Biotechnology

   Jiangnan University · RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen52074, Germany

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China

2. Ke Gao

   Jiangnan University

   Affiliation as printed

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China

3. Yulin Jiang

   Jiangnan University

   Affiliation as printed

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China

4. Jieyu Zhou

   Jiangnan University

   Affiliation as printed

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China

5. Guochao Xu

   Jiangnan University

   Affiliation as printed

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China

6. Jinjun Dong

   Jiangnan University

   Affiliation as printed

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China

7. Ulrich Schwaneberg Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen52074, Germany

8. Yu Ji corresponding Aachen Institute of Biotechnology

   Jiangnan University · RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, Aachen52074, Germany

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China

9. Ye Ni corresponding

   Jiangnan University

   Affiliation as printed

   Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi214122, China