Overview

In this section, we have presented all the significant experimental processes and outcomes. These include the successful validation of plasmid construction, the transient expression in Nicotiana benthamiana, and the achievement of rosmarinic acid production.

1.Clone of RAS10, CYP98A14 and CYP98A75

PCR amplification was performed using the cDNA library of S. miltiorrhiza as the template, and the primers were shown in Table 1. The product was detected by 1% agarose gel electrophoresis and showed 2 specific fragments of about 1500 bp, consistent with the expected results for the full length of the RAS10, CYP98A14, and CYP98A75 genes. The sequencing of the cloning result confirmed that it was identical to the full length of the sequence obtained from the already available studies.

Figure 1. PCR amplification of the RAS10 (3), CYP98A14 (1), and CYP98A75 (2) genes from S. mlitiorrhiza. M: marker.

2.Construction of the recombinant vectors

Xho I and Age I were chosen as the cleavage sites to design primers for the construction of RAS10/CYP98A14/CYP98A75-pEAQ-HT recombinant vectors. The CYP98A14/CYP98A75-pEAQ-HT vectors were successfully constructed by seamless cloning (DNA Assembly Mix Plus, REF: SB-EGR205S ShareBio, China), but failed with the RAS recombinant vector, so we found help from the authors of the article published in Plant Biotechnology Journal (2024, 22:1536-1548) and obtained the recombinant vector for RAS10.

Figure 2. Illustrative graph of gene- pEAQ-HT recombinant vector.

Figure 3. Recombinant vectors constructed by seamless cloning kit. M: marker, 1: linearised vectors digested by double restriction endonucleases (Xho I and Age I), 2-4: PCR-amplified DNA fragments of CYP98A14, CYP98A75 and RAS10.

3.Reconstitution of the rosmarinic pathway in N. benthamiana

After sequencing confirmation, the recombinant expression vectors were separately transformed into A. tumefaciens strain GV3101 competent cells. A. tumefaciens containing the constructed vectors were grown overnight at 28°C in YEB medium. The cells were collected via centrifugation, washed, and resuspended in infiltration buffer (MS buffer supplemented with 0.1 M MES and 0.1 M acetosyringone) to an OD600 of 0.5, followed by a 3-hour incubation at room temperature. The two different mixed solutions (as detailed in Table 3) were then infiltrated into the leaves of 4-5 week-old N. benthamiana plants. After a 24-hour dark incubation, the plants were subsequently cultivated under light conditions for another 24 hours. The substrate was diluted to 100 µM using MS liquid medium and injected into the tobacco leaves, followed by an additional 24-hour cultivation under light.

Figure 4. Tobacco leaves were infiltrated.

4. Determination of rosmarinic acid yield

Infiltrated leaves were harvested, freeze-dried, and ground. Then, 10 mg of each sample was placed into a 2 mL centrifuge tube, to which 1 mL of 75% ethanol solution was added. The mixture was ultrasonically extracted for 1 hour. After extraction, the samples were centrifuged at 14,000 rpm for 30 minutes at 4°C, and the supernatant was taken for detection.
Using rosmarinic acid standards as a reference, the content of rosmarinic acid in tobacco was determined by LC-MS/MS (TSQ Altis Plus, ThermoFisher, USA) to select the optimal exogenous recombination conditions.

Figure 5. MS fragment of rosmarinic acid.

Figure 6. Per thousand contents of rosmarinic acid.

5. Conclusion

In this project, tobacco was selected as the host system, leveraging the 4-coumaroyl-coenzyme A generated through its phenylalanine pathway as the acyl donor. We introduced exogenous danshensu along with RAS10 and CYP98A14, or 4-hydroxyphenyllactate together with RAS10, CYP98A14, and CYP98A75, to generate rosmarinic acid, with the aim of reconfiguring the salvianolic acid pathway. By comparing the yields of rosmarinic acid from these two approaches, we found no significant difference between them.

6. Future work

In the next phase of our laboratory work, we can further optimize the experiment by enhancing the catalytic performance of the enzymes and exploring new synthesis pathways, including the testing of novel enzymes.