Summary

The establishment of leonurine biosynthesis was carried out through a stepwise engineering process. We first reconstructed the biosynthetic pathway in tobacco hairy roots and later switched to E. coli for in-vitro enzymatic catalysis. Developing the hairy root system normally requires a long period of time; however, thanks to the sterile tobacco seedlings provided by our advisor’s research group, we were able to shorten part of the experimental cycle. Even so, this extended process left a strong impression on our team. The overall workflow included plasmid construction, Agrobacterium-mediated hairy root induction, transcriptional and metabolic validation, as well as heterologous protein expression and purification in E. coli. Our detailed experimental timeline is provided below. For specific procedural steps and experimental outcomes, please refer to the Protocols and Engineering Success sections.

1 April

● Visit to the Science and Research Center of Shanghai Chen Shan Botanical Garden

● Brainstorming

2 May

● Plant expression vectors were constructed through a hierarchical assembly from Level 0 to Level 1 to Level 2.

● The successfully verified plant expression vectors were introduced into Agrobacterium tumefaciens strain Ar.1193 via electroporation.

● The confirmed positive Agrobacterium strains were used to infect sterile tobacco seedlings.

3 June

● Upon the emergence of hairy roots from the leaves of sterile seedlings, GFP fluorescence signals were observed under a laser lamp. Hairy roots exhibiting GFP signals were individually excised and cultured for two weeks.

● Following genotypic PCR confirmation, the positive tobacco hairy roots were transferred onto solid MS medium and cultured for an additional week.

4 July

● The positive hairy roots were then transferred to liquid MS medium and subjected to dark culture for three weeks.

● Additional liquid MS medium was supplemented, and the culture was continued for one more week.

5 August

● The MS liquid medium was replaced, and after five days of further culture, the samples were harvested to analyze the expression of the target gene and the leonurine content in the hairy roots.

● The prokaryotic expression vectors were constructed.

● The target proteins were expressed in a prokaryotic system and purified.

● In vitro enzyme activity assays were performed to evaluate the efficiency of leonurine production in the prokaryotic system.