iGEM 2024 — Team bioplus-shanghai

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# Background

Danshen, derived from the root of *Salvia miltiorrhiza*, is an important traditional Chinese herbal medicine. The phenolic acid compounds contained in it, such as caffeic acid, danshensu, rosmarinic acid, and salvianolic acid B, have multiple
pharmacological activities such as anti-platelet aggregation, anti-thrombosis, improving microcirculation, promoting tissue recovery, scavenging free radicals, and anti-lipid peroxidation, and are used to treat cardiovascular and cerebrovascular
diseases (Zhao et al., 2008). The biosynthetic pathway of phenolic acid components mainly uses 4-coumaroyl-CoA generated by the phenylalanine pathway as an acyl donor, and 4-hydroxyphenyllactic acid or danshensu generated by the tyrosine pathway
as an acyl acceptor. Rosmarinic acid is generated under the catalysis of rosmarinic acid synthase and cytochrome P450 family 98A subfamily monooxygenase (CYP98A) in sequence, and further generates various complex phenolic acid substances(Trócsányi
et al., 2020). Here, we use tobacco as a chassis cell to produce phenolic acid components of *Salvia miltiorrhiza* to alleviate the dependence on the cultivation of *Salvia miltiorrhiza* resources and improve the production efficiency
of *Salvia miltiorrhiza* active substances.

# Design

![Figure 1](https://static.igem.wiki/teams/5214/eng/change-eng-1.png)

Fig. 1 Project working mode diagram

Our project employs synthetic biology techniques to produce rosmarinic acid, an important component of *Salvia miltiorrhiza*. The design of our experiment is illustrated in Fig. 1. We selected *Nicotiana benthamiana* as the host cell and
utilized its own phenylalanine pathway to produce 4-coumaroyl-CoA as the acyl donor. By introducing substrates and key enzymes in the synthesis pathway, we reconstructed the salvianolic acid pathway, enabling the synthesis of rosmarinic acid.
Our experiment involved two synthetic pathways: one involved the introduction of danshensu, SmRAS10, and SmCYP98A14, while the other involved the introduction of 4-hydroxyphenyllactic acid, SmRAS10, SmCYP98A14, and SmCYP98A75. Experimental cloning
of three key enzymes was required, namely SmRAS10 (BBa\_K5214000), SmCYP98A14 (BBa\_K5214004), and SmCYP98A75 (BBa\_K5214005) (Fig. 2A).

Using *Salvia miltiorrhiza* cDNA as a template, we cloned the target gene fragments and constructed them into the plant expression vector pEAQ-HT using homologous recombination principles, resulting in the recombinant vectors SmRAS10-pEAQ-HT,
SmCYP98A14-pEAQ-HT, and SmCYP98A75-pEAQ-HT (Fig. 2B). The pEAQ-HT vector utilizes the “CPMV-HT” super translation system derived from the cowpea mosaic virus, which achieves high-yield production of recombinant proteins through its efficient translation
efficiency (Peyret & Lomonossoff, 2013). Subsequently, we transferred the recombinant vectors into *Agrobacterium tumefaciens* GV3101 and used Agrobacterium-mediated transient gene expression technology to express the key enzymes in *Nicotiana benthamiana*,
reconstructing the salvianolic acid pathway and synthesizing rosmarinic acid. By comparing the yields of the two pathways, we laid the foundation for the sustainable development and utilization of Danshen.

![Figure 2](https://static.igem.wiki/teams/5214/eng/eng2.png)

Fig. 2 Plasmid construction. (A) Diagram of our three parts. (B) Schematic diagram of vector construction strategy.

# Build

First, we used the cDNA of *Salvia miltiorrhiza* as a template and cloned the gene fragments of SmCYP98A14 and SmCYP98A75 by PCR. In order to connect the gene to the vector by homologous recombination, we designed primers and performed another
PCR to obtain a gene fragment with homologous arm sequences at both ends (Fig. 3A). Then it was connected with the linearized pEAQ-HT vector, transformed into Escherichia coli Trans1-T1 competent cells, and positive clones were screened by colony
PCR (Fig. 3B). The plasmids in the positive bacteria were extracted and sent for sequencing. Finally, the sequence alignment results showed that we successfully constructed the recombinant vectors SmCYP98A14-pEAQ-HT and SmCYP98A75-pEAQ-HT (Fig.
3C). In addition, the SmRAS10-pEAQ-HT recombinant vector was obtained from Dr. Zhou (Plant Biotechnology Journal, 2024, 22:1536-1548).

![Figure 3](https://static.igem.wiki/teams/5214/eng/eng3.png)

Fig. 3 Plasmid construction. (A) Agarose gel identification of linearized vector pEAQ-HT and target gene fragments amplified by PCR. M: marker; 1: Linearized vector pEAQ-HT; 2: PCR-amplified DNA fragments of CYP98A14; 3: PCR-amplified DNA fragments
of CYP98A75 (B) Colony PCR screening of positive colonies. M: marker, 1-4: CYP98A14-pEAQ-HT, 5-8: CYP98A75-pEAQ-HT. (C) Sequence alignment between SmCYP98A14 and positive clone sequencing. (D) Sequence alignment between SmCYP98A75 and positive
clone sequencing.

# Test

We plan to express key enzymes in tobacco through Agrobacterium-mediated transient expression technology, and then supply substrates exogenously to achieve the production of rosmarinic acid in tobacco. We consider using two different synthetic pathways
to produce rosmarinic acid and compare the yields of the two methods. In detail, one method uses danshensu as a substrate and expresses two enzymes, SmRAS and SmCYP98A14, in tobacco to achieve the synthesis of rosmarinic acid, hereinafter referred
to as the DSS method. The other method uses 4-hydroxyphenyllactic acid as a substrate and uses three enzymes, SmRAS, SmCYP98A14 and SmCYP98A75, to achieve the purpose of synthesizing rosmarinic acid, hereinafter referred to as the HPA method.

1.Transient Expression in *Nicotiana 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 to an OD600 of 0.5, followed by a 3-hour incubation at room temperature. The two different mixed solutions (as detailed in results) 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](https://static.igem.wiki/teams/5214/eng/eng4.png)

Fig. 4 Tobacco leaves were infiltrated.

2.Mass spectrometry verification of rosmarinic acid production in tobacco

In order to ensure that the mass spectrometry analysis is optimal in terms of sensitivity, specificity and quantitative accuracy, we used rosmarinic acid standards to obtain its peak diagram (Fig. 5) and optimized and verified the Multiple Reaction
Monitoring (MRM) parameters (Table 1). This can avoid errors caused by matrix interference when performing mass spectrometry analysis on complex plant samples, ensuring the accuracy and reliability of the experimental results

![Figure 5](https://static.igem.wiki/teams/5214/eng/eng5.png)

Fig. 5 MS fragment of rosmarinic acid

![Table 1](https://static.igem.wiki/teams/5214/eng/change-eng-2.png)

Table 1. MRM parameter of rosmarinic acid.

Subsequently, the tobacco leaves were freeze-dried and crushed, ethanol was added for ultrasonic extraction, and the supernatant was centrifuged. We used the above MRM parameters to perform DSS method and DSS method to obtain mass spectrometry analysis
of the sample solution. Each sample was repeated three times to obtain the rosmarinic acid content per thousand (Table 2), and a column chart was drawn. The results showed that there was no significant difference in the rosmarinic acid content
between the two methods, but the HPA method had a slightly higher content (Fig. 6).

![Table 2](https://static.igem.wiki/teams/5214/eng/change-eng-3.png)
![Figure 6](https://static.igem.wiki/teams/5214/eng/eng8.png)

Fig. 6 Per thousand contents of rosmarinic acid.

# Learn

We utilized *Nicotiana benthamiana* as the host cell and established two different pathways for synthesizing rosmarinic acid using Agrobacterium-mediated transient expression technology. Through mass spectrometry, we successfully detected the
presence of rosmarinic acid. Statistical results indicated that the HPA method had a slightly higher yield. 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.

# References

[1] Peyret, H., & Lomonossoff, G. P. (2013). The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant molecular biology, 83, 51-58.
[2] Trócsányi, E., György, Z., & Zámboriné-Németh, É. (2020). New insights
into rosmarinic acid biosynthesis based on molecular studies. Current plant biology, 23, 100162.
[3] Zhao, G.-R., Zhang, H.-M., Ye, T.-X., et al. (2008). Characterization of the radical scavenging and antioxidant activities of danshensu and
salvianolic acid B. Food and Chemical Toxicology, 46(1), 73-81.

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# 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.

![table1](https://static.igem.wiki/teams/5214/result/res1.png)![img1](https://static.igem.wiki/teams/5214/result/res2.png)

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.

![img2](https://static.igem.wiki/teams/5214/result/res3.png)

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

![img](https://static.igem.wiki/teams/5214/result/change-res-1.png)![img4](https://static.igem.wiki/teams/5214/result/res5.png)

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.

![img](https://static.igem.wiki/teams/5214/result/change-res-2.png)![img4](https://static.igem.wiki/teams/5214/result/res7.png)

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.

![img](https://static.igem.wiki/teams/5214/result/change-res-3.png)![img5](https://static.igem.wiki/teams/5214/result/res9.png)

Figure 5. MS fragment of rosmarinic acid.

![img](https://static.igem.wiki/teams/5214/result/change-res-4.png)![img6](https://static.igem.wiki/teams/5214/result/res11.png)

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.

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