iGEM 2024 — Team scu-china

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Parts | SCU-China 2024
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![Content Corner Image](https://static.igem.wiki/teams/5223/style/qingting.png)

A detailed notebook is provided.

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#### CONTENTS

![Sidebar Image](https://static.igem.wiki/teams/5223/style/plant.png)

![Content Corner Image](https://static.igem.wiki/teams/5223/style/qingting.png)

## The Synthesis of Resveratrol

### Cycle I

#### Design

In the design of resveratrol synthesis in our chassis, we discovered no resveratrol synthetic pathway in *Nicotiana benthamiana* after analyzing preliminary metabolic data and reviewing the relevant literature. However, the concentration of p-Coumaric acid in leaves of *Nicotiana benthamiana*, which is the substrate of many kinds of secondary metabolites, including resveratrol, is considerable. Consequently, we were willing to express the key enzyme needed to produce resveratrol -- Stilbene synthase (STS) in our chassis. Finally, we focused on the stilbene synthase gene in grape VvSTS, which has been tested and verified valid in wild organisms[1][2]. We wanted to see whether it could enable synthesis in *Nicotiana benthamiana*.

#### Build

We retrieved the VvSTS sequence on NCBI and contacted a professional company to synthesize and optimize the fragment. We constructed the pDONR207-VvSTS and pEAQ-DEST1-VvSTS using the Gateway method and then transferred the latter into *Agrobacterium* GV3101. After that, we conducted transient transfection to express the VvSTS.

#### Test

We analyzed the synthesis of resveratrol in the leaves of transiently transfected tobacco through LC-MS/MS. As shown in Fig.1-1, there was resveratrol produced in tobacco when VvSTS was successfully imported.

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| ![](https://static.igem.wiki/teams/5223/engineering/res1-1.jpg) |

Fig.1-1 Resveratrol synthesis and production in wild-type tobacco

CK: blank control; pEAQ:pEAQ-HT-DEST1 blank vector; VvSTS:pEAQ-HT-DEST1 with VvSTS

#### Learn

Compared to blank control and blank vector, we detected the resveratrol in the wild-type tobacco after importing VvSTS, inferring that VvSTS can express efficiently in our chassis. Notably, the yield of resveratrol was poor, which may be attributed to the factor that the central part of p-Coumaric acid is used to generate Chlorogenic acid and other secondary metabolites. Therefore, we tried to make the heterologous synthesis of resveratrol in our VessaTobacco. Meanwhile, our instructors said there may be negative feedback in the upstream and suggested improving the production by releasing or alleviating the feedback loop.

### Cycle II

#### Design

To verify the efficiency and strength of our chassis in the synthesis of secondary metabolites, we changed the wild-type tobacco into HQT-knockout tobacco and conducted transient transfection. We also found that the mutant of DAHP synthase YIARO3 in Saccharomyces can alleviate the repression of DAHP synthase from L-Phe in the upstream[3], as shown in Fig.1-2.

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| ![](https://static.igem.wiki/teams/5223/engineering/res1-2.png) |

Fig.1-2 YIARO3 alleviates the negative feedback

#### Build

We constructed the pDONR207-YIARO3 and pEAQ-DEST1-YIARO3 by Gateway and transferred the latter into *Agrobacterium* GV3101. By setting the group of single gene and double genes transfection, we hoped to observe the improvement in yield of resveratrol in VvSTS and YIARO3 simultaneously importing.

#### Test

We used LC-MS/MS to detect the resveratrol after injecting mutant tobacco. As we can see in Fig.1-3, ΔHQT tobacco produced more resveratrol than that of wild type. However, our anticipation that higher production of double gene groups than single gene groups did not appear.

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| ![](https://static.igem.wiki/teams/5223/engineering/res1-3.jpg) |

Fig.1-3 Production of resveratrol inΔHQT mutant

#### Learn

As shown above, HQT-knockout tobacco displayed a higher yield of resveratrol than wild type, indicating that knockout HQT dispersed the flow of p-Coumaric acid to resveratrol to some extent. Nevertheless, the effect was insignificant, and YIARO3 did not increase resveratrol production. More than that, the production of resveratrol decreased by nearly 50% compared with the data generated by the last batch of tobacco. We predicted these results may be due to the following factors:

1. Because different gene combinations injected different layers of leaves. Leaves underneath have grown for a longer time and have undergone the key phase of diversion of metabolism, which contributed to no improvement after injection of YIARO3 and even decreased.
2. The sample volumes were too small to keep the homogeneity of the samples. The status of plants significantly affected the results.
3. We delayed the sampling time for 24 hours, during which the resveratrol may be partially degraded.

In conclusion, we decided to enlarge the sample volume and fix the leaf layer to inject. The sampling time was controlled in 72 - 96 h after injection.

### Cycle III

#### Design

According to the experience in DBTL cycle Ⅱ, we placed importance on the following:

1. narrow the difference between samples with different genes injection
2. choose suitable tobacco to inject and adjust the sampling time
3. enlarge sample volume

#### Build

We chose suitable (four-week-age) tobaccos and injected 4 - 5 leaves for each plant. To narrow the difference between plants, we changed the method: one kind of gene group for the leaves from the same plant, and one gene group injected 2 - 3 plants. When sampling, we mixed samples with the same gene group and milled. Moreover, we extracted the metabolites using freeze-dried samples to reduce the influence of water content.

#### Test

Waiting for 72 - 96 h after injection, we harvested the samples freeze-dried for 24 h and then extracted metabolites. The same LC-MS/MS method was used to analyze resveratrol production. As shown in Fig.1-4, resveratrol production increased four times that of cycle Ⅱ. HQT mutant samples with only the VvSTS gene produced more resveratrol than the wild type. However, double genes make production lower than single VvSTS gene no matter mutants and wild types. HQT knockout plant decreased more apparent than wild type.

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| ![](https://static.igem.wiki/teams/5223/engineering/res1-4.jpg) |

Fig.1-4 The resveratrol production of ΔHQT mutant

#### Learn

Changing the injection method and sampling time and taking on freeze-dry helped increase resveratrol production and decrease the difference between groups. Thus, we will keep the parameters and conditions in our later investigation. Unfortunately, the expected improvement of resveratrol yield in VvSTS and YIARO3 groups did not come out. Moreover, the difference between HQT-knockout plants and wild-type plants is not apparent. We speculated that The competitive binding enzyme YIARO3 introduced into the feedback inhibition pathway may affect other growth-essential metabolic pathways, decreasing in synthetic yield. Moreover, we needed homozygous mutants to validate our design in the following experiments.

## The Synthesis of Phaselic Acid

The hydroxycinnamoyl transferase in tobacco has a low affinity for malic acid and a high affinity for quinic acid or shikimic acid. To verify the ability of the tobacco plant chassis to introduce accumulated caffeoyl-CoA into other metabolic reactions as a substrate for synthesis after the HQT gene is knocked out, we introduced the hydroxycinnamoyl transferase HCT-M from Trifolium pratense and codon-optimized it for expression in *Nicotiana benthamiana* to attempt the synthesis of phaselic acid, thereby verifying the chassis's ability to efficiently synthesize secondary metabolites downstream of the shikimate pathway.

### Cycle I

#### Design

We constructed a plasmid capable of expressing the exogenous HCT-M protein in plants using the Gateway cloning method. After transforming the plasmid into *Agrobacterium*, we used it to transfect *Nicotiana benthamiana*. The transfected tobacco was then subjected to LC-MS/MS analysis to verify the chassis's ability to synthesize phaselic acid.

#### Build

1. We amplified the HCT-M gene fragment with an aatB tag using PCR and connected it to the pDONR207 plasmid using BP reaction to construct the entry plasmid pDONR207-HCT-M.
2. The entry plasmid was subjected to an LR reaction with pEAQ-HT-DEST1 to construct the expression plasmid pEAQ-HT-DEST1-HCT-M.
3. The constructed plasmid was transformed into *Agrobacterium* GV3101, and single colonies were cultured in LB medium containing Rif and kanamycin for two days before being used to infect tobacco.
4. The infected tobacco region was excised, and metabolites were extracted using methanol for LC-MS/MS analysis.

#### Test

The data indicates a significant increase in the concentration of phaselic acid after the infiltration of *Agrobacterium*, suggesting that the efficiency of tobacco transformation has been enhanced following the activation of *Agrobacterium*, and consequently, the ability of *Nicotiana benthamiana* to synthesize phaselic acid has been substantially improved. We have standardized the collection of leaf samples, resulting in more uniform data among replicate experiments. Moreover, the level of phaselic acid synthesized by the mutant tobacco is higher than that of the wild-type tobacco, indicating that the *Nicotiana benthamiana* chassis, after the knockout of the HQT gene, possesses a greater capacity for the synthesis of phaselic acid.

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| ![](https://static.igem.wiki/teams/5223/engineering/pha-1.png) |

Fig.2-1 LC-MS/MS results in Cycle I

#### Learn

In the experiment, we cut the injected area of a single leaf as a group for LC-MS/MS detection. Due to the large differences in the status of the leaves, there was a large difference between the groups. Moreover, the specific experimental operation omitted the step of shaking activation, resulting in low *Agrobacterium* activity and thus an insignificant increase in phaselic acid. To improve the experimental operation details, we propose the following improvements:

1. Pick *Agrobacterium* from the plate, shake it for 48 hours, and then shake it overnight to inject tobacco in the best state;
2. After transforming the tobacco, only cut off the leaf area in good condition;
3. Mix the cut leaves with the same treatment before grouping to eliminate differences between groups;
4. Freeze-dry the leaves before LC-MS/MS extraction to eliminate the impact of leaf water content on the experimental results.

### Cycle II

#### Design

After transfecting the tobacco with activated *Agrobacterium*, we freeze-dried the transfected tobacco and performed LC-MS/MS analysis to verify the chassis's ability to synthesize phaselic acid.

#### Build

1. Pick single colonies of *Agrobacterium* with the target plasmid in LB medium containing Rif and kanamycin and culture for two days, then expand the culture overnight with a 1% inoculation ratio.
2. Excise the injected tobacco area, freeze-dry the leaves, extract metabolites using methanol, and perform LC-MS/MS analysis.

#### Test

The data showed a significant increase in the concentration of phaselic acid after injecting *Agrobacterium*, indicating that the exogenous HCT-M gene we injected can be expressed in tobacco and greatly improve the ability of *Nicotiana benthamiana* to synthesize phaselic acid. In addition, the content of phaselic acid synthesized by the mutant tobacco is higher than that of the wild-type tobacco, indicating that the *Nicotiana benthamiana* chassis after HQT knockout has a higher ability to synthesize phaselic acid.

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| ![](https://static.igem.wiki/teams/5223/engineering/pha-2.png) |

Fig.2-2 LC-MS/MS results in Cycle II

#### Learn

The ability of mutant tobacco to synthesize phaselic acid has improved to some extent compared to the wild type, but it may not have reached the best synthesis amount of phaselic acid for the chassis. This is because the mutant tobacco has missed the best time for transfection, and the chlorogenic acid in the mutant tobacco has begun to accumulate, reducing the ability to synthesize phaselic acid, and the difference with the wild type is no longer obvious.

## The Synthesis of Crocin

### Cycle I

#### Design

*ALDH* is one of the critical enzymes in the crocin synthesis pathway. Its primary function is to catalyze crocetindialdehyde to crocetin. In order to transform tobacco, we plan to use Gateway cloning to construct the plasmid vector pEAQ-DEST1-ALDH. The construction of this plasmid vector involves three transformations, which need to be screened in LB medium with gentamicin, LB medium with kanamycin, and LB medium with rifampicin and kanamycin, and then use agarose gel electrophoresis and sequencing and other bacterial testing methods to determine whether the target plasmid is successfully constructed.

#### Build

The *ALDH* gene fragment was connected to pDONR207 by BP reaction, and then the plasmid was transformed into *E. coli* TOP10. In the biosafety cabinet, 200μl of bacterial solution was applied to LB medium containing gentamicin and cultured overnight in a 37℃ incubator. The next day, a single colony was selected for expansion and culture, and then PCR and sequencing were performed. After successful Colony PCR, pEAQ-DEST1 was connected by LR reaction. After transformation, 200μl of bacterial solution was applied to LB medium containing kanamycin and cultured overnight in a 37℃ incubator. After Colony PCR, positive plasmids were taken for *Agrobacterium* transformation.

#### Test

The overnight culture of transformed *E. coli* showed poor growth, with few single and small colonies. After a single colony was expanded and sent for sequencing, the results showed that *ALDH* was not successfully connected to the plasmid vector.

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| ![](https://static.igem.wiki/teams/5223/engineering/image001.png) |

Fig.3-1 Bacteria growing on LB medium containing gentamicin

#### Learn

1. We analyzed the phenomenon of a few single colonies and small colonies in bacterial culture. This may be because we used a culture medium containing gentamicin to screen bacteria, which resulted in a relatively slow bacterial growth rate. Hence, we will extend the incubation time.
2. The poor sequencing results may be due to the unsuccessful BP reaction, which is related to the reaction time (overnight connection) and reaction temperature (16°C). Next time, we plan to adjust the reaction temperature to 25°C and the reaction time to 2 hours.

### Cycle II

#### Design

Considering that the first round of experiments may have failed due to operational errors or unsatisfactory reaction conditions, we plan to repeat the Gateway cloning experiment to construct the plasmid pEAQ-DEST1-ALDH. In this round, we will optimize the experimental steps and carefully check each step to ensure the successful construction of the plasmid.

#### Build

In the second round of experiments, we paid more attention to the standardization of experimental operations. We first optimized the conditions of the BP reaction. The reaction temperature was adjusted to 25°C, and the reaction time was adjusted to 2 hours to improve the connection efficiency. Subsequently, we used the optimized conditions to connect the *ALDH* gene fragment to the pDONR207 vector and transformed it into *E. coli* TOP10. In the biosafety cabinet, 200μl of the bacterial solution was spread on an LB medium containing gentamicin and cultured overnight in a 37°C incubator. The next day, we selected single colony for expansion culture and performed Colony PCR and sequencing.

#### Test

After optimizing the BP reaction conditions, we found that there was no bacterial we wanted growth on the plate, which indicated that other problems might affect the experiment's success.

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| ![](https://static.igem.wiki/teams/5223/engineering/image002.jpg) |

Fig.3-2 Bacteria growing on LB medium containing gentamicin

#### Learn

1. We analyzed that the failure of recombinant plasmid construction may be due to the incompatibility of the *ALDH* gene sequence with the Gateway vector. Gateway technology requires specific sites. If these sites are not contained in the target gene sequence or secondary structures in the sequence affect the recombination efficiency, the construction may fail.
2. We will re-evaluate the *ALDH* gene sequence to determine whether sequence features affect the Gateway reaction and consider using other vectors or methods for experiments.
3. We also plan to PCR amplify the *ALDH* gene and use other vectors for transformation to verify whether there are problems with the gene sequence itself. In the next round of experiments, we will adjust the experimental strategy based on the analysis results of this round of experiments and take corresponding measures to improve the success rate of the experiment.

### Cycle III

#### Design

After a detailed summary of the first two rounds of experiments, we decided to continue the construction of the *ALDH* gene recombinant plasmid using the Gibson assembly, as it is widely used for its high efficiency and flexibility and does not rely on specific restriction enzyme sites, and has a high efficiency and success rate when assembling large fragments of DNA.

#### Build

In this round of experiments, we designed specific primers for PCR amplification of the vector pEAQ-DEST1 and the *ALDH* gene. We added a His-tag sequence to the primer design to facilitate subsequent protein purification. After amplification, we used ClonExpress II One Step Cloning kit for recombination reaction at 50°C for 12 minutes. After transformation, 200 μl of the bacterial solution was spread on LB medium containing kanamycin and cultured overnight in a 37°C incubator.

#### Test

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| ![](https://static.igem.wiki/teams/5223/engineering/image003.png) |

Fig.3-3 pEAQ-DEST1 plasmid backbone amplification results

After PCR amplification of the *ALDH* gene and the pEAQ-DEST1 plasmid backbone, agarose gel electrophoresis and gel extraction were performed to verify the PCR products. The amplification results of the pEAQ-DEST1 plasmid backbone were poor, with evident mixed bands, and the plasmid concentration was low after gel extraction.

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| ![](https://static.igem.wiki/teams/5223/engineering/image010.png) |

Fig.3-4 Bacterial growth on LB medium containing kanamycin

After Gibson assembly and transformation into *E. coli* TOP10, we observed bacterial growth on an LB medium containing kanamycin. This indicates that the recombinant plasmid may have been successfully constructed and transformed. Further, Colony PCR also showed the expected target bands through nucleic acid electrophoresis. Unfortunately, the sequencing results showed that the plasmid was not wholly connected to the *ALDH* gene, and only part of the fragment was successfully recombined.

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| ![](https://static.igem.wiki/teams/5223/engineering/image006.jpg) |

Fig.3-5 Colony PCR results

#### Learn

1. The PCR vector shows strong mixed bands after running on the gel. Primer design problems, resulting in nonspecific binding amplification of primers and vectors. Or the vector itself may be contaminated, affecting the specificity of PCR amplification.
2. The vector fragment is too large, resulting in low recovery efficiency during gel extraction.
3. To solve these problems, we used the Phanta enzyme system to re-amplify to improve the amplification's specificity and efficiency. However, the gel extraction efficiency is still very low.
4. In the next round, we plan to redesign primers to avoid nonspecific binding, use a more efficient gel extraction kit.

### Cycle IV

#### Design

After a detailed summary of the previous round of Gibson assembly, we redesigned the primers and optimized the experimental conditions. To solve the problem of too large vector fragments and low gel extraction efficiency, we appropriately simplified the vector. We performed a multi-tube vector PCR system, and finally the products were extracted together. To solve the problem of vector contamination, we re-cultured the bacteria containing the vector and extracted its plasmid.

#### Build

In this round of experiments, we designed new specific primers for PCR amplification of the vector pEAQ-DEST1 and the *ALDH* gene and added a His-tag sequence to the primer design. After amplification, we used the ClonExpress II One Step Cloning Kit for the recombination reaction at 50°C for 2h. The cloned product was transformed into *E. coli* TOP10, the bacterial solution was then spread on an LB medium containing kanamycin and cultured overnight in a 37°C incubator. After bacterial inspection and sequencing, the plasmid was extracted for *Agrobacterium* transformation.

#### Test

Use new primers to obtain the target fragment through PCR and linearize the vector. After gel extraction, incubate the target fragment and linearized vector at 50°C for 2 hours, transform the cloned product into *E. coli* TOP10, and then spread the bacterial liquid on LB medium containing kanamycin and culture it in a 37°C incubator overnight.

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| ![](https://static.igem.wiki/teams/5223/engineering/image007.png) |

Fig.3-6 Bacterial growth on LB medium containing kanamycin

After overnight culture, there were uniform colonies on the plate. 28 single colonies were selected for amplification and Colony PCR.

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| ![](https://static.igem.wiki/teams/5223/engineering/image008.png) |

Fig.3-7 Colony PCR results

We observed bacterial growth on an LB medium containing kanamycin. This indicates that the recombinant plasmid may have been successfully constructed and transformed. Further electrophoresis also showed the expected target band. We sent the PCR product containing the target band for sequencing, and the sequencing results showed that the plasmid was successfully constructed. The plasmid was then extracted and transformed into *Agrobacterium*.

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| ![](https://static.igem.wiki/teams/5223/engineering/image009.png) |

Fig.3-8 Sequencing results

#### Learn

1. When designing primers, their specificity should be ensured to avoid the appearance of target bands during amplification.
2. Using multiple reaction systems to linearize the vector and then performing gel extraction together can increase the vector concentration.
3. Appropriately extending the Gibson assembly incubation time may increase the cloning efficiency.

## Directed Evolution

For the production of phaselic acid, the catalytic activity of HCT-M derived from Trifolium pratense is limited due to its weak affinity for caffeoyl CoA, instead favoring the binding of feruloyl CoA, leading to the production of a significant amount of feruloyl malic acid as a byproduct. To address this issue, we have conducted directed evolution of HCT-M to screen for mutants with high substrate affinity for caffeoyl CoA.

### Cycle I

#### Design

To construct plasmids containing various HCT-M mutants, we amplified the HCT-M gene, which includes an OmpA secretory tag and an ASA linker, using error-prone PCR to introduce mutations. Subsequently, we employed homologous recombination to ligate the mutated gene fragments into the pET-28a backbone.

#### Build

1. Design primers to amplify the HCT-M gene containing the OmpA secretory tag and ASA linker using error-prone PCR, and simultaneously amplify the pET-28a plasmid backbone using PCR. Recover and purify the amplified fragments from agarose gel electrophoresis.
2. Perform homologous recombination to ligate the randomly mutated HCT-M gene with the pET-28a backbone.
3. ransform the ligation products into DH5α.
4. Pick single colonies and inoculate them into LB broth containing kanamycin, culture overnight, perform colony PCR, and preserve and extract plasmids from PCR-positive cultures.

#### Test

Out of the 36 picked bacteria, only three were positive. Sequence some positive and negative colonies, confirming that the positive colonies have successfully ligated the targeted gene and the gene has random mutations.

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| ![](https://static.igem.wiki/teams/5223/engineering/evolution1.png) |

Fig.4-1 The results of colony PCR

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| ![](https://static.igem.wiki/teams/5223/engineering/evolution2.png) |

Fig.4-2 The sequencing for random mutations

#### Learn

This experiment revealed a low positive rate, and possible reasons include:

1. olThe low concentration of DNA after gel extraction, leading to low ligation efficiency.
2. 2. Insufficient electrophoresis time to separate linearized plasmids from the original plasmids. We will increase the concentration of fragments in the ligation reaction and extend the electrophoresis time appropriately to improve the positive rate.

### Cycle II

#### Design

We obtained HCT-M mutant gene expression plasmids with OmpA secretory tags and ASA linkers by increasing the concentration of fragments, and transformed them into BL21 for induced expression. After purifying the bacterial proteins, we used SDS-PAGE to verify protein expression.

#### Build

1. We amplified the HCT-M gene containing the OmpA secretory tags and ASA linker using error-prone PCR to introduce mutations, and then ligated the mutated gene fragments into the pET-28a backbone using homologous recombination. After transforming into DH5α, we picked single colonies, confirmed positive colonies by colony PCR. We extracted plasmids and transformed them into the BL21. We also constructed a non-mutated pET-28a-HCT-M without secretory tags as a control group.
2. We picked single colonies of BL21 and cultured them overnight, then scaled up the culture with a 2% inoculation ratio. When the OD reached 0.6-0.8, we induced expression for 16 hours with a final concentration of 0.2 mM IPTG.
3. After induction, we lysed the bacteria to extract total proteins and then purified the target protein.
4. We performed SDS-PAGE electrophoresis on the purified proteins.

#### Test

In the control group, the purified target protein band was present, but it was not observed in the mutant bacteria. Additionally, we found that neither the control group nor the mutant bacteria had the target band in the supernatant, suggesting that the OmpA tag may not be functional.

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| ![](https://static.igem.wiki/teams/5223/engineering/evolution3.png) |

Fig.4-3 The results of protein purification in Cycle II

#### Learn

The inability to purify the mutant protein may be due to the addition of the secretion tag causing structural changes in the protein, preventing the exposure of the His-tag, or due to mutations at critical sites leading to the formation of a large number of inclusion bodies. In the next step, we will reconstruct the pET-28a-HCT-M plasmid containing the mutated HCT-M gene without the OmpA secretion tag and ASA linker.

### Cycle III

#### Design

We will remove the OmpA secretion tag from the plasmid through homologous recombination and transform it into BL21 bacteria for induced expression. After purifying the bacterial proteins, we will use SDS-PAGE to verify protein expression.

#### Build

1. We will amplify the HCT-M gene fragment from the mutated plasmid before mutation and the pET-28a plasmid backbone using PCR, and then connect them through homologous recombination to remove the OmpA secretion tag and ASA linker.
2. We will transform the pET-28a-HCT-M plasmid containing the mutated HCT-M gene into BL21, pick single colonies for cultivation, and then induce expression with IPTG.
3. We will extract and purify the proteins and perform SDS-PAGE detection.

#### Test

We performed SDS-PAGE detection on the purified proteins and successfully purified 9 out of 13 proteins.

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| ![](https://static.igem.wiki/teams/5223/engineering/evolution4.png) |

Fig.4-4 The results of protein purification in Cycle III

#### Learn

In the study, four mutant proteins still lack distinct bands, which may be due to the fact that after random mutation of the protein sequence, the proteins are more likely to form inclusion bodies or fail to expose the His-tag, leading to the failure of purification for these four mutant proteins.

## Semi-rational Design

Semi-rational design is based on a certain understanding of the physicochemical properties, three-dimensional structure, conformational relationship, catalytic mechanism and other information of proteins, and computer-assisted fixed-point mutation, saturation mutation, and combinatorial mutation of hotspot amino acids in the active centre or active pocket. This method is between irrational design and rational design, overcoming the shortcomings of both and reducing the technical requirements. To improve the yield of resveratrol and phaselic acid, we used a semi-rational design approach to optimize STS and HCT2 by screening favourable single-point mutation sites, and verified the effectiveness of the mutants in wet experiments. However, we encountered many problems during the semi-rational design and mutant validation process. Through multiple rounds of DBTL approach, we updated and iterated the design, mutation and purification methods, and finally completed the semi-rational design and validation of STS and HCT2, and obtained mutants possessing higher catalytic activity or better thermal stability.

### Cycle I

#### Design

Since the existing pre-trained protein language models and shared protein interaction design platforms are more maturely developed and there are studies to support their validity, we will use the shared computer platform to perform the initial screening of single-point mutation hotspots and confirm the stability of the mutant-ligand binding by molecular docking to exclude mutation sites that are not conducive to the stability of the protein-ligand binding, in order to obtain both stability and catalytic activity of the The mutants with improved stability and catalytic activity were obtained.

#### Build

1. The PDB files of STS and HCT2 were uploaded to Hotspot Wizard 3.0 (Lenka Sumbalova, et al., 2018) and SaprotHub (Jin Su, et al., 2024), respectively, to export the mutation hotspot scores and related data.
2. Alternative mutants were screened based on high or low scoring and the location of the mutation site (whether it was in the active pocket or not, etc.) (see protein modelling section for details).
3. PDB files of the mutants were obtained using ChimeraX 1.8.
4. STS was docked to p-coumaroyl coenzyme A and malonyl coenzyme A, and HCT2 to caffeoyl coenzyme A and malic acid molecules using [CB-Dock2](https://cadd.labshare.cn/cb-dock2/php/index.php)(Yang Liu, et al. 2022), and a vina score was obtained as a reference for docking results.
5. The obtained docking results were opened with PyMOL and polar bonds were visualised, eliminating mutants with less than 80% of the polar bonds of the wild type.

#### Test

Table 5-1: Number of polar bonds between molecularly docked Vina Score and corresponding protein-ligand complexes

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| ![](https://static.igem.wiki/teams/5223/engineering/table-1.png) |

Table 5-2: Mutants generated by this round of design

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| ![](https://static.igem.wiki/teams/5223/engineering/table-2.png) |

#### Learn

The round of design produced 18 STS mutants with 22 HCT2 mutants, considering the wet experiment time and workload, we need to continue the design scheme to screen fewer mutants into the validation session. In addition, since [CB-Dock2](https://cadd.labshare.cn/cb-dock2/php/index.php) does not support docking of a protein to two ligands at once, the reference value of Vina Score after the second docking is weak, so we need to apply a more accurate method to further screen the mutants.

### Cycle II

#### Design

Based on the mutants screened in the first round, we will use GROMACS to perform molecular dynamics simulations to study the movement mode of the protein-ligand-solvent system, and screen out mutant proteins with more stable system and faster convergence of the potential energy of the system to be constant through the simulations of the change of RMSD & RMSF, the change of the total potential energy of the system, the change of the radius of gyration of proteins, as well as the visualisation of the molecular movement trajectories.

#### Build

1. Open Babel converted the small molecule ligand format to .mol2, and the penalties were measured on CGenFF, which was too high for all four ligands and required ligand optimisation.
2. Small molecule ligands are generated on ORCA and Multifin with RESP atomic charges and optimised for chemical bonding, and finally the corresponding topology files are generated on sobtop.
3. Complexes were constructed using preprocessed protein and ligand topology files, periodic boundary conditions were set, energies were minimised, pre-equilibrated and the main simulation was completed.
4. After calibrating the system parameters, the corresponding RMSD, RMSF, change in total potential energy of the system, change curve of the radius of gyration of the protein, and molecular motion trajectory were output, and the corresponding indexes of the mutant were compared with those of the wild type to screen out the mutant proteins whose systems were more stable and whose potential energies tended to be constant more quickly.

#### Test

Table 5-3: RMSD, RMSF, Total Potential Energy Change and Regression Time

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| ![](https://static.igem.wiki/teams/5223/engineering/table-3.png) |

Table 5-4: Computer-assisted protein design screening results

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| ![](https://static.igem.wiki/teams/5223/engineering/table-4.png) |

Through molecular dynamics simulations, we screened a total of 12 STS proteins and 13 HCT2 proteins into the wet experiment validation session.

#### Learn

Molecular dynamics simulation is a very effective way to screen for mutants that make the system more stable and the potential to stabilise faster, and we obtained mutants with higher stability and enzyme activity at the computer prediction level through the above screening process.

### Cycle III

#### Design

Based on the mutants screened by computer-assisted design, we constructed the mutants by reverse PCR and performed primer design and point mutation according to the requirements of the toyobo point mutation kit SMK-101.

#### Build

1. Primer design: The mutation site is designed at or near the 5' end of the primer and the 3' end of the primer is complementary to the template by at least 20 bases. One primer is a mutant primer and the other primer is a conventional primer.
2. Point mutation PCR: Mix each set of dispensed PCR reaction solution according to the recommendations of the instruction manual and perform PCR amplification (5-10 cycles), adjusting the extension time and the number of cycles according to the size of the plasmid and the efficiency of the primers.
3. Digest the template plasmid DNA and ligate the PCR product.
4. The plasmid was transformed into the DH5α receptor state and extracted for sequencing after recovery.

#### Test

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/engineering/semi-1.png) |

Fig.5-1 Successfully constructed mutants

In total, three rounds of point mutations were performed (two rounds for STS and one round for HCT2), and only five STS mutants were successfully constructed, no HCT2 mutants were constructed, and most of the plasmids were sequenced as wild-type.

#### Learn

We hypothesised that the reason for the failure of the mutant construction was that one of the primers required for the Toyo Spinning kit did not carry the mutation site, which would have introduced a large number of wild-type plasmids. In addition, the insufficient number of PCR cycles also resulted in fewer mutant plasmids. Therefore, we need to improve the point mutation method to make the products of point mutation PCR purer and produce more mutant plasmids.

### Cycle IV

#### Design

In order to increase the efficiency and positivity of point mutation PCR, we improved the method of point mutation. This time, we used partially complementary primers with mutation sites in both primers for point mutation and increased the PCR cycle to 20 times.

#### Build

1. Primer design: Two primers of 25-45 base complementary lengths were designed with Tm values around 78°C. Both primers contained the expected mutation sites. In order to avoid the generation of primer dimers, the Tm values of the two primer pairs were kept below 50% as far as possible.
2. Point mutation PCR: The PCR enzyme was changed to the more efficient KOD One and 20 cycles were performed.
3. Digest template plasmid DNA.
4. The plasmid was transformed into the DH5α receptor state and extracted for sequencing after recovery.

#### Test

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/engineering/semi-2.png) |

Fig.5-2 Mutant sequencing results

After adjusting the point mutation PCR reaction, we constructed the corresponding mutants at once.

#### Learn

1. The success of point mutation is higher when both primers carry the mutation site, as there is a higher probability of picking positive bacteria.
2. Extending the number of PCR cycles and using more efficient PCR enzymes to increase the concentration of point mutant fragments are critical to the success of experiments to construct point mutant proteins.

### Cycle V

#### Design

Before the characterisation of the constructed mutant protein, the nature of the protein needs to be clarified, we learnt from the data that STS has low solubility and is easy to form inclusion bodies, so in the pre-experiment, we chose to express the wild-type STS by inducing it with 0.1 mM IPTG in LB medium at 24℃, 120 rpm for 16 h. The purpose is to avoid the formation of inclusion bodies to affect the subsequent purification and reaction. The solubility of HCT was better, so the wild-type HCT was induced in LB medium with 0.2 mM IPTG at 24℃, 150rmp for 16h to express wild-type HCT.

#### Build

1. Protein expression was induced under Design conditions.
2. HCT proteins were purified by non-denaturing nickel column purification, and STS proteins were purified by denaturing and non-denaturing nickel column purification, respectively, and the flow-through and each of the washes and eluents were collected.
3. The collected above liquid was run on SDS-PAGE.

#### Test

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/engineering/semi-3.png) |

Fig.5-3 SDS-PAGE results of STS protein after purification from non-denaturing nickel columns(M: marker; FT: Flow-through fluid; W: Wash fluid; E: Elution fluid)

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/engineering/semi-4.png) |

Fig.5-4 SDS-PAGE results of HCT protein after purification from non-denaturing nickel columns(M: marker; FT: Flow-through fluid; W: Wash fluid; E: Elution fluid)

The protein bands in the eluate after purification from the non-denaturing nickel column were extremely weak, but the bands corresponding to the size of STS protein in the denaturing purified eluate were stronger, indicating that most of the proteins formed inclusion bodies. The HCT protein bands were more obvious in the eluate after purification from the non-denaturing nickel column, indicating that the HCT protein was successfully purified.

#### Learn

The LB medium was able to carry too little bacteria, resulting in insufficient normal STS proteins being purified at the end, which needed to be replaced with a medium capable of carrying a larger amount of bacteria and further lowering the temperature to reduce inclusion body production and increase the amount of normal proteins.

### Cycle VI

#### Design

Compared to LB medium, TB medium is more nutrient-rich and able to carry more amount of bacteria, so we will choose TB as the medium for this round of experiments. In addition, we reduced the temperature at which we induced protein expression to 21°C, thereby reducing the amount of inclusion body formation. In order to increase the concentration of normal STS proteins, we will ultrafiltrate the samples after Akta non-deformed purification.

#### Build

1. TB medium, 0.1 mM IPTG, 21°C, 120 rpm, induction for 20 hours.
2. Akta non-denaturing purified protein, ultrafiltration.
3. Bradford assay for protein concentration.

#### Test

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/engineering/semi-5.png) |

Fig.5-5 Bradford protein concentration measurements

#### Learn

1. Compared with LB medium, TB medium was able to significantly increase the bacterial volume and was suitable for increasing protein production.
2. Lowering the temperature during IPTG-induced protein expression reduces the generation of inclusion bodies.
3. Ultrafiltration can significantly increase the concentration of proteins in solution.

### Cycle VII

#### Design

In order to characterise the enzyme activity of the purified wild-type and mutant HCT proteins, we used the L-malic acid assay kit (WST-8 method) of Biyun Tian to detect the remaining amount of malic acid in the reaction substrate. Firstly, we plotted the standard curve of malic acid concentration according to the method of the kit.

#### Build

1. Prepare a reaction buffer with 25 mM vitamin C and phosphate buffer to pH = 6.5.
2. Prepare the appropriate amount of colour development working solution according to the volume of 80 μl for each assay reaction. Mix 74 μl of Malate Assay Buffer, 2 μl of Enzyme Solution, 2 μl of Chromogen Solution and 2 μl of Substrate to make 80 μl of Working Solution. The working solution can be prepared into 80 μl of Working Solution.
3. Take 25 μl of Malic Acid Standard Solution (10 mM), add 475 μl of reaction buffer and mix well to formulate a concentration of 500 μM Malic Acid Standard Solution. Take 0, 1, 2, 4, 8 and 20 μl of 500 μM malic acid standard solution into the standard wells of 96-well plate respectively, and make up to 20 μl with the corresponding reaction buffer, at this time, the concentration of the standard curve is 0, 25, 50, 100, 200 and 500 μM.
4. Add 80 μl of colour development working solution to each well, mix well and react at 37ºC for 30 minutes.
5. Determine the absorbance at 450nm.

#### Test

Table 5-5: Results of malic acid concentration gradient determination (\* represents high OD value, exceeding the upper limit of detection of the zymography)

| Concentration(mM) | 0 | 25 | 50 | 100 | 200 | 500 |
| --- | --- | --- | --- | --- | --- | --- |
| OD450 | 3.900 | 3.954 | \* | 3.936 | \* | \* |

According to the kit instructions, the measured malic acid OD values should be between 0 and 1. In this case, the OD values of all six malic acid concentrations exceeded this range and did not form a gradient.

#### Learn

The detection principle of the kit is that L-malic acid is oxidised to oxalacetic acid (OAA) by malate dehydrogenase (MDH), and NAD+ is reduced to NADH in this reaction; the generated NADH is reduced to orange-coloured Formazan by the electron-coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium methyl sulfate). NADH is reduced to NADH by the electron coupling reagent 1-mPMS (1-Methoxyphenazinium methyl sulfate), and the generated NADH is reduced by WST-8 to form the orange-yellow colour of Formazan, with a maximum absorption peak at around 450 nm. And vitamin C also has reducing property, it is presumed that the vitamin C in the reaction buffer interfered with the colour development reaction.

### Cycle VIII

#### Design

In response to the previous round's speculation that vitamin C affects the colour development reaction of the malic acid assay, this speculation was verified in this round. We removed vitamin C from the reaction buffer and retained only the phosphate buffer at pH=6.5.

#### Build

1. Prepare a reaction buffer with phosphate buffer to pH = 6.5
2. Prepare a working solution for colour development in the same way as round 6.
3. Establish the malic acid concentration gradient using the new reaction buffer without vitamin C.
4. 80 μl of colour developing working solution was added to each well, mixed well and reacted at 37ºC for 30 minutes. Absorbance was measured at 450nm.

#### Test

Table 5-6: Results of malic acid concentration gradient determination

| Concentration(mM) | 0 | 25 | 50 | 100 | 200 | 500 |
| --- | --- | --- | --- | --- | --- | --- |
| OD450 | 0.143 | 0.187 | 0.211 | 0.287 | 0.387 | 0.610 |

The OD values for this round of tests all fell between 0 and 1 with a clear gradient.

#### Learn

Vitamin C does interfere with the colour development reaction, suggesting that vitamin C has a much greater ability to reduce NAD+ than malic acid, and interferes with the colour development in the assay principle. By removing vitamin C from the reaction buffer, the malic acid concentration can be measured normally.

---

References

[1] He, X., et al., Overexpressing fusion proteins of 4-coumaroyl-CoA ligase (4CL) and stilbene synthase (STS) in tobacco plants leading to resveratrol accumulation and improved stress tolerance. Plant Biotechnology Reports, 2018. 12(5): p. 295-302.

[2] Jeandet, P., C. Clément and E. Courot, Resveratrol production at large scale using plant cell suspensions. Engineering in Life Sciences, 2014. 14(6): p. 622-632.
Liu, M., et al., Remodelling metabolism for high-level resveratrol production in Yarrowia lipolytica. Bioresource Technology, 2022. 365: p. 128178.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% set show\_hero = true %}
{% set image\_url = 'https://static.igem.wiki/teams/5223/background-pics/experiment-background.png' %}
{% set title = 'Experiments' %}
{% set subtitle = 'Project' %}
{% block page\_content %}

#### CONTENTS

![Sidebar Image](https://static.igem.wiki/teams/5223/style/plant.png)

![Content Corner Image](https://static.igem.wiki/teams/5223/style/qingting.png)

## Molecular Cloning

Molecular cloning was applied to the construction of all plasmids in our project.

This section includes:

1. Plasmid Extraction
2. Agarose Gel Electrophoresis
3. Gel Extraction
4. Heat-Shock Transformation
5. Error-Prone PCR(Beyotime QuickMutation)
6. Gibson Assembly
7. Gateway Assembly

## Protein Expression

In the enzyme functional validation experiment, we need to use IPTG to induce protein expression and lyse bacteria to extract the target protein. In terms of protein purification methods, we combined two affinity chromatography purification methods, AKTA Purifier and Ni NTA Sefinose Resin (Pre Packaged Gravity Column), to purify the target protein with His Tag.

This section includes:

1. TB Medium Preparation
2. IPTG Concentrate Preparation
3. Bacteria Culture Method
4. IPTG Induction Protocol
5. Protein Lysis Protocol(For Ni-Resin affinity chromatography gravity column method)
6. Protein Purification Protocol(For Ni-Resin affinity chromatography gravity column method)
7. Protein Staining Protocol(Coomassie brilliant blue R-250)
8. Modified Bradford Protein Assay Kit Protocol
9. Protein Purification Protocol(Using AKTA-Purifier)

## Tobacco Cultivation

We transiently transformed our target plasmids into specific tobacco leaves to express our target proteins and synthesize the corresponding metabolites, and we tested the synthesis yields of metabolites by LC-MS/MS.

This section includes:

1. Tobacco cultivation
2. Tobacco transient transfection
3. LC-MS/MS

---

{% endblock %}

{% extends "layout.html" %}

Results | SCU-China 2024
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% set show\_hero = true %}
{% set image\_url = 'https://static.igem.wiki/teams/5223/background-pics/experiment-background.png' %}
{% set title = 'Results' %}
{% set subtitle = 'Project' %}
{% block page\_content %}

#### CONTENTS

![Sidebar Image](https://static.igem.wiki/teams/5223/style/plant.png)

![Content Corner Image](https://static.igem.wiki/teams/5223/style/qingting.png)

## Tasks

1. Constructing HQTs knockout *Nicotiana benthamiana*.
2. Validating the chassis function through the heterologous synthesis of phaselic acid, resveratrol, and crocin.
3. Optimizing key enzymes involved in the synthesis of target products through semi-rational design and directed evolution methods.

## 1.Construction of HQT knockout plants

This part of the experiment was designed to reduce chlorogenic acid levels in tobacco, thereby enabling
greater metabolic flow to other metabolites, thus creating a high-quality tobacco synthetic biology
chassis. We identified target genes by analyzing genes related to chlorogenic acid synthesis and the
metabolic flow of the pathway, and subsequently knocked them out. Metabolic flow analysis of the target
products was then conducted in the knockout plants to confirm that the constructed knockout plants
served as efficient expression chassis for the target products.

### Identification of Genes for Knockout

During the metabolic flux analysis, we conducted knockout simulations on the three synthetic pathways
of chlorogenic acid, comparing a total of six knockout methods involving the knockout of one gene and
the knockout of two genes. The simulation results indicated that knocking out the HQT pathway is the
most effective way to significantly reduce the production of the byproduct chlorogenic acid and increase
the flux of the target product synthesis.

[See
details](metabolic-flow-analysis#Chlorogenic acid production simulation)

### Design of sgRNA for Knockout

We accessed the genome database of *Nicotiana benthamiana* (https://www.nbenth.com/) and
downloaded the sequences of four HQT genes contained in *Nicotiana benthamiana*: NbL07g01330.1,
NbL10g00550.1, NbL13g01170.1, and NbL16g00540.1. Subsequently, we utilized the online software CRISPR-P
2.0 (http://crispr.hzau.edu.cn/CRISPR2/) to predict the gene editing targets. For sgRNA design, we
selected 20 bp exon sequences adjacent to the PAM site (-NGG) located near the 5'-end of the genes as
the basis.

Table 1-1: Designed sgRNA Sequences

| Gene | sgRNA-1 | sgRNA-2 |
| --- | --- | --- |
| *HQT2+4* | GTCACTTGTGATCTTAAACA | CAACATGTAACTTTGTCAAT |
| *HQT2* | ATAAGTACTACCCTCGTGCT | ACTACCCTCGTGCTCGGACT |
| *HQT3* | AATGTGGTGGAGTCTCCATA | ACATGGTCAGAAGTTGCCCG |
| *HQT4* | AATGTGGTGGAGTCTCCATA | ACATGGTCAGAAGTTGCCCG |

### Construction of Knockout plasmids

Using homologous recombination, vectors were constructed by designing target site sequences and BsmBI
recognition sites into primers. The target site sequences were incorporated between the U6 promoter and
gRNA sequences via PCR. Subsequently, BsmBI endonuclease was used to insert the target site sequences
into the BGFastCas9 SS Plant vector, resulting in the construction of CRISPR/Cas9 single knockout
vectors for NbHQT1, NbHQT2, NbHQT3, and NbHQT4. To enhance knockout efficiency, in addition to
constructing single mutants for each of the four genes, vectors were also constructed for the
simultaneous knockout of NbHQT1 and NbHQT3, as well as NbHQT2 and NbHQT4, generating double knockout
CRISPR/Cas9 vectors for NbHQT1+3 and NbHQT2+4.

Table 1-2: Design of Single Knockout CRISPR
Primers

| Gene | Forward Primer | Reverse Primer |
| --- | --- | --- |
| *NbHQT1* | CAGCTAGAGTCGAAGTAGTG  ATTGACGGCGGGACGGCGAC  GGAGGTTTCAGAGCTATGC | CTGTTTCCAGCATAGCTCTG  AAACGACATACTTTGCTAGC  CCTACAATCACTACTTCGA |
| *NbHQT2* | CAGCTAGAGTCGAAGTAGTG  ATTGATAAGTACTACCCTCG  TGCTGTTTCAGAGCTATGC | CTGTTTCCAGCATAGCTCTG  AAACAGTCCGAGCACGAGGG  TAGTCAATCACTACTTCGA |
| *NbHQT3* | CAGCTAGAGTCGAAGTAGTG  ATTGAATGTGGTGGAGTCTC  CATAGTTTCAGAGCTATGC | CTGTTTCCAGCATAGCTCTG  AAACCGGGCAACTTCTGACC  ATGTCAATCACTACTTCGA |
| *NbHQT4* | CAGCTAGAGTCGAAGTAGTG  ATTGAATGTGGTGGAGTCTC  CATAGTTTCAGAGCTATGC | CTGTTTCCAGCATAGCTCTG  AAACCGGGCAACTTCTGACC  ATGTCAATCACTACTTCGA |
| *NbHQT1+3* | CAGCTAGAGTCGAAGTAGTG  ATTGAAGTAACCTGGTGGCA  AAGGGTTTCAGAGCTATGC | CTGTTTCCAGCATAGCTCTG  AAACTTGGTGTAGCTTGAAC  ACACAATCACTACTTCGA |
| *NbHQT2+4* | CAGCTAGAGTCGAAGTAGTG  ATTGGTCACTTGTGATCTTA  AACAGTTTCAGAGCTATGC | CTGTTTCCAGCATAGCTCTG  AAACATTGACAAAGTTACAT  GTTGCAATCACTACTTCGA |

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/result-fig-1.png) |

Fig.1-1 Electrophoresis results showing successful cloning of
the intermediate fragment (approximately 400bp)

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/result-fig-2.png) |

Fig.1-2 Knockout plasmids containing sgRNA (taking HQT2+4 as an
example)

The plasmid was transformed into *E. coli DH5α* competent cells, spread onto plates, and incubated
overnight. Single clones were picked for further culture. A pair of primers was designed based on the
upstream and downstream sequences corresponding to the sgRNA. Using the DNA exposed after bacterial
lysis as a template, PCR was performed with the aforementioned primers. The amplified bands represented
the region near the two target sequences on the plasmid, with a length of approximately 300bp, which was
consistent with the theoretical length. The sequencing results of the amplified products were correct,
confirming the successful transformation of the knockout plasmid into *E. coli DH5α.*

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/result-fig-3.png) |

Fig.1-3 Colony PCR Results of CRISPR Carrier

Lane: 1, 11, and 14 are DL2000 markers; 2-4 are NbHQT1-CRISPR
plasmids;
5-7 are NbHQT2-CRISPR plasmids; 8-10 are NbHQT3-CRISPR plasmids;
12-13 are
NbHQT4-CRISPR plasmids; 15-16 are NbHQT1+3-CRISPR plasmids;
and 17-18 are NbHQT2+4-CRISPR plasmids.

### Construction of Knockout plant

#### *Agrobacterium tumefaciens* Construction

We successfully transformed the target plasmid into Agrobacterium tumefaciens, obtaining the desired strains: single-gene knockout strains *NbHQT1-EHA105*, *NbHQT2-EHA105*, *NbHQT3-EHA105*, and *NbHQT4-EHA105* (Fig.1-4, Fig.1-5); and double-gene knockout strains *NbHQT1+3-EHA105* and *NbHQT2+4-EHA105* (Fig. 1-5).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/res-1-4-1.png) |

Fig.1-4 Transformed *Agrobacterium tumefaciens*

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/res-1-4-2.png) |

Fig.1-5 *Agrobacterium* strains produced and stored in batches

#### Stable Transfection, Plant Tissue Culture, Budding, Rooting, Acclimatization, Transplanting, and Cultivation to Obtain the T0 Generation

The single-gene knockout stable transformation callus of *NbHQT2*, *NbHQT3*, *NbHQT4* and the double-gene knockout stable transformation callus of *NbHQT2+4* could be induced and grow normally (Fig.1-6). In contrast, the callus of NbHQT1 and *NbHQT1+3* gradually became enlarged and vitrified and could not differentiate into resistant buds usually (Fig.1-7).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/res-1-4-3.png) |

Fig.1-6 Callus growth after stable transformation (partial)

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/res-1-4-4.png) |

Fig.1-7 Resistant buds that have just been transplanted (partial)

The first batch of resistant shoots from callus tissue was transplanted onto rooting medium. Compared to the wild-type, the edited *Nicotiana benthamiana* plants exhibited slower root development, with only a small fraction of plants forming roots after approximately four weeks of culture. Additionally, the time required for rooting varied depending on the specific genes that were edited. The double-gene knockout plants demonstrated slower growth overall. In general, the single-gene knockout plants (*NbHQT2*, *NbHQT3*, and *NbHQT4*) rooted more quickly than the double-gene knockout plants (*NbHQT2+4*). (Fig.1-8)

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/res-1-4-5.png) |

Fig.1-8 Growing *Nicotiana benthamiana* CRISPR plants

The DNA from the CRISPR-edited tissue culture seedlings was extracted, and the Cas9 editing region was amplified by PCR (Fig.1-9) and subsequently sent for sequencing.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/res-1-4-6.png) |

Fig.1-9 PCR results of CRISPR strain tissue culture seedlings

After the sequencing results are returned, they are compared with the correct gene sequence. Comparison of the sequencing results with the gene sequences revealed that the corresponding target *NbHQT* gene had been knocked out in some plants of each CRISPR strain, and single deletion mutants of *NbL10g00550.1* (*NbHQT2*), *NbL13g01170.1* (*NbHQT3*), and *NbL16g00540.1* (*NbHQT4*) were successfully obtained (Fig.1-10, Fig.1-11, and Fig.1-12).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/sup-const7.png) |

Fig.1-10 Sequencing results of partial plants from the *NbL10g00550.1*-CRISPR line

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/sup-const8.png) |

Fig.1-11 Sequencing results of partial plants from the *NbL10g00550.1*-CRISPR line

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/sup-const9.png) |

Fig.1-12 Sequencing results of partial plants from the *NbL13g01170.1*-CRISPR line

The sequencing results were compared with the gene sequences of *NbHQT2* and *NbHQT4*, respectively. It was found that *NbHQT2* and *NbHQT4* had been knocked out in plant No. 5 of the *NbHQT2+4*-CRISPR strain, successfully resulting in the creation of the *NbHQT2+4* double-gene knockout mutant (Fig.1-13).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/sup-const10.png) |

Fig.1-13 Sequencing results of partial plants from the *Nbl13g01170.1+NbL16g00540.1-*CRISPR line

The CRISPR-positive *Nicotiana benthamiana* plants with established root systems underwent hardening and transplanting (Fig.1-14).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/sup-const11.png) |

Fig.1-14 CRISPR-positive plants in seedling hardening

#### Sowing T1 generation and then sequencing plants

Wild-type, mutant 2-4, and mutant 2-8 tobacco seedlings were transplanted into new pots, with two plants per pot, and placed in an incubator after transplantation (Fig.1-15). Based on the band size, the primers successfully amplified the gene. However, it was unclear whether the gene had been successfully knocked out, so the remaining PCR products were sent for sequencing (Fig.1-16).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/sup-const12.jpeg) |

Fig.1-15 Tobacco after transplanting

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/construction/sup-const13a.png) ![](https://static.igem.wiki/teams/5223/result/construction/sup-const13b.png) |

Fig.1-16 Sequencing results

### Conclusion

*NbHQT* is a key gene involved in chlorogenic acid synthesis in tobacco. Based on our modeling results, we successfully knocked out the HQT gene in the *Nicotiana benthamiana* genome in various combinations, constructing a chassis with reduced chlorogenic acid levels. This breakthrough addresses the issue of high chlorogenic acid concentrations diverting significant metabolic flow away from the production of secondary metabolites, which previously limited the synthetic capacity of the chassis. By lowering chlorogenic acid levels, we have created a high-quality chassis for plant synthetic biology, enhancing the production potential of *Nicotiana benthamiana* and paving the way for further exploration of its ability to synthesize a wide range of high-value-added compounds.

## 2.Synthesis of Phaselic Acid

### Background and Introduction

Phaselic acid is a common acylated acid found in plants, formed by the combination of Caffeoyl-CoA and
malic acid. In order to produce this compound in our gene-edited *Nicotiana benthamiana*,
hydroxycinnamoyl transferase (HCT) is required. An enzyme called HCT-M, derived from red clover, has a
high affinity for malic acid. Introducing this gene into *Nicotiana benthamiana* provides the
necessary activity for the synthesis of phaselic acid. Additionally, codon optimization of the
expression system in *Nicotiana benthamiana* was carried out to verify the plant's ability to
synthesize the target compound downstream of the shikimate pathway.

### Plasmid Construction and Codon Optimization

We first performed PCR amplification of the codon-optimized *HCT-M* gene for *E. coli*, obtaining
the target gene, followed by gel extraction for further use.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-1.png) |

Fig.2-1 Electrophoresis Results of HCT-M Gene Amplification

We selected the pDONR207 and pEAQ-HT-DEST1 to facilitate the efficient introduction of the gene
into *Agrobacterium*.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-2-n1.png) | ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-3-n.png) |

Fig.2-2 Plasmid Maps of pDONR207-HCT-M and pEAQ-HT-HCT-M

Using pDONR207 as the vector, the *HCT-M* gene was inserted through a BP reaction. After the BP reaction, the product was transformed into Top10 competent cells and plated on LB plates with gentamycin resistance to screen for positive clones. After inoculating the selected clones into LB broth, the plasmids were extracted to obtain the entry clone plasmid. Agarose gel electrophoresis was performed on the extracted plasmids, and the plasmids were sent for sequencing. The results confirmed that the *HCT-M* gene was successfully inserted into the vector.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-4.png) | ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-5.png) |

Fig.2-3 Colony Formation and Colony PCR Detection Results After
BP Reaction

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-6-n1.png) |

Fig.2-4 Plasmid Sequencing Results After BP Reaction

After the BP reaction, we extracted plasmids and sequenced them. Once the BP reaction was successful, we performed the LR reaction using the entry clone plasmid and destination vector to obtain the expression plasmid. The product was plated on kanamycin-resistant LB plates to screen for positive clones. After inoculating the selected clones into LB broth, the plasmids were extracted to obtain expression plasmids. Agarose gel electrophoresis was performed on the extracted plasmids, and the plasmids were sent for sequencing. The results confirmed that we successfully constructed the expression plasmid.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-7.png) | ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-8.png) |

Fig.2-5 Colony Formation and Colony PCR Detection Results After
LR Reaction

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| --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-9-n.png) |

Fig.2-6 Plasmid Sequencing Results After LR Reaction

We transformed the expression plasmid into *Agrobacterium*. The transformed *Agrobacterium*
was cultured in LB medium containing antibiotics, where single colonies successfully grew. We performed
colony PCR and agarose gel electrophoresis. The results confirmed that the target gene was successfully
introduced into *Agrobacterium*.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-10.png) |

Fig.2-7 Culture Results of *Agrobacterium*

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| --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-11.png) |

Fig.2-8 Electrophoresis Results

### Transient Transformation

The *Agrobacterium* culture was prepared into a suspension, with the OD600 adjusted to
approximately 0.9, for transient transformation in tobacco plants. Using a syringe, the suspension was
injected into the underside of tobacco leaves. The injected plants were covered with black plastic bags
and placed in an incubator for cultivation. The injection sites were marked.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-12.png) | ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-13.png) |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-14.png) | ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-15.png) |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-16.png) | ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-17.png) |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-18.png) | ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-19.png) |

Fig.2-9 Tobacco Injection Results

### LC-MS/MS Results

The injection areas of the tobacco leaves were cut, freeze-dried for 24 hours, and metabolites were
extracted using 80% methanol for LC-MS/MS analysis. The results showed a significant increase in phaselic
acid concentration after *Agrobacterium* injection. This indicates that the introduced *HCT-M* gene
was successfully expressed in tobacco and catalyzed the synthesis of phaselic acid from endogenous malic
acid and caffeoyl-CoA. Additionally, the mutant tobacco plants produced more phaselic acid than the
wild-type plants, suggesting that *Nicotiana benthamiana* with HQT knockout has a higher capacity
for phaselic acid synthesis.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/phaselic-acid-20.png) |

Fig.2-10 Phaselic Acid Content Measurement

### Conclusion

By introducing the exogenous *HCT-M* gene, *Nicotiana benthamiana* was able to synthesize phaselic
acid. Furthermore, the mutant tobacco demonstrated an enhanced ability to synthesize phaselic acid
compared to the wild-type tobacco. This indicates that after knocking out the *HQT* gene, *Nicotiana
benthamiana* gained an increased capacity to synthesize target products downstream of the shikimate
pathway. However, since the mutant tobacco missed the optimal time for transfection, chlorogenic acid
had already begun to accumulate, reducing the plant's ability to synthesize phaselic acid. In future
experiments, earlier transfection times should be considered to achieve better synthesis performance.

## 3.Synthesis of Resveratrol

This part aims to validate whether the increased p-Coumaryl-Coenzyme A, an essential chemical in the MEP pathway, could be used to synthesize medicinal secondary metabolite resveratrol in bulk. Considering that *Nicotiana benthamiana* lacks the key enzyme -- Stilbene synthase in catalyzing p-Coumaryl-Coenzyme A to produce resveratrol, we focus on the widely verified stilbene synthase gene VvSTS originating from grapes. We transferred it into tobacco and analyzed the production of resveratrol. Moreover, in the upstream of p-Coumaryl-Coenzyme A, L-Phe represses the DHAP synthase through a negative feedback loop, resulting in a reduction in p-Coumaryl-Coenzyme A. DHAP synthase gene YIARO3 from *Saccharomyces* is the mutant of ARO3 and can inhibit the negative feedback. Thus, we hope to improve the resveratrol production by over-expressing YIARO3 in our chassis. Finally, we conducted LC-MS/MS to analyze resveratrol production.

### Construction of Expression Plasmid

VvSTS and YIARO3 fragments were synthesized and optimized by a professional company. We added attB sequences at both ends of the genes. We used Gateway clone and exerted BP reaction first to attach VvSTS and YIARO3 to vector pDONR207 to form entry clones.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/resveratrol/1.png) |

Fig.3-1 pDONR207-YIARO3, pDONR207-VvSTS,pEAQ-DEST1-YIARO3 and pEAQ-DEST1-YIARO3 plasmids

The sequencing results showed that the critical regions were utterly consistent with the standard sequences, and no mutations occurred, indicating success in constructing entry clones.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/resveratrol/2.png) |

Fig.3-2 Sequencing results of pDONR207-YIARO3 and pDONR207-VvSTS

Next, we conducted an LR reaction to attach VvSTS and YIARO3 to expression vector pEAQ-HT-DEST1 and to regulate expression by CaMV35S strong promoter. The sequencing results showed no mutation in critical regions, indicating that pEAQ-DEST1-YIARO3 and pEAQ-DEST1-YIARO3 expression plasmids were constructed successfully.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/resveratrol/3.png) |

Fig.3-3 Sequencing results of pDONR207-YIARO3 and pDONR207-VvSTS

To enable expression in *Nicotiana benthamiana*, we transferred the pEAQ-DEST1-YIARO3 and pEAQ-DEST1-YIARO3 plasmids into *Agrobacterium* GV3101. The sequencing results showed no mutation in critical regions, indicating successful transfers.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/resveratrol/4.png) |

Fig.3-4 *Agrobacterium* positive clone screening results

a) YIARO3; b) VvSTS; c) pEAQ-HT-DEST1

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/resveratrol/5.png) |

Fig.3-5 Sequencing results of *Agrobacterium* bacterial solution

### LC-MS/MS Analysis to Resveratrol Production

We transiently transfected the tobacco with *Agrobacterium*, carrying targeted genes, and ran an LC-MS/MS non-target metabolism analysis. We drew the standard curve with the data and calculated the hqt - knockout plants and wild-type resveratrol production.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/resveratrol/6.png) |

Fig.3-6 LC-MS/MS Resveratrol standard curve versus hqt2 knockout tobacco resveratrol yield

a) LC-MS/MS analysis of resveratrol standard curve; b) T0 generation hqt2 knockout tobacco resveratrol yield.
WT: wild-type; CR-nbhqt2: T0 generation hqt2 knockout

Above all, we drew the standard curve according to the correspondence between the area and concentration of resveratrol standards. Compared to wild-type tobacco, T0-generation hqt2 - knockout plants increased resveratrol production somewhat, regardless of VvSTS single gene transfer and VvSTS and YIARO3 two gene transfer. These results matched our anticipation.

However, we found that resveratrol production of mutant and wild-type tobacco transferred by VvSTS and YIARO3 was slightly lower than that of single VvSTS. We speculated that the sample volume was too small and that the plant difference significantly influenced the results. Consequently, we enlarged the sample volume when transferring T1 - generation hqt2 - knockout tobacco. We mixed the samples from the same gene combination group to narrow the difference between different plants and leaf layers. We observed that hqt2 - knockout plants produced more resveratrol than the wild-type when injected with a single VvSTS (p < 0.05), which was what we expected.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/resveratrol/7.png) |

Fig.3-7 T1 generation HQT2 knockout tobacco resveratrol yield

WT: wild-type; CR-nbhqt2: T1 generation hqt2 knockout

Nevertheless, hqt2 - knockout plants transferred with VvSTS and YIARO3 were lower than that of wild-type. Similarly to T0 - generation, resveratrol production transferred with two genes was significantly decreased compared to VvSTS transfer, and it was more evident in hqt2 - knockout plants. We suspected that DHAP synthase encoded by YIARO3 was competitively combined with DHAP, resulting in possible influence in other metabolic pathways and finally reducing resveratrol production. The speculation needed to be validated.

In conclusion, we successfully used Gateway to construct VvSTS and YIARO3 expression plasmids and transferred them into hqt2- knockout plants through *Agrobacterium* to verify resveratrol production increase in hqt2 - knockout plants. This part proved that our chassis has advantages in synthesizing secondary metabolites. However, it remains to test whether YIARO3 can improve resveratrol production.

## 4.Synthesis of Crocin

We chose crocin as the synthetic product for analysis to evaluate the metabolic changes in other metabolic pathways such as mevalonate pathway (MEP pathway) besides the shikimate pathway. By reading the relevant literature, we selected the multiple gene superposition synthesis method, which expresses four genes related to crocin synthesis (*GjCCD4a*, *GjALDH2C3*, *GjUGT74F8*, and *GjUGT94E13*) in *Nicotiana Benthamiana*.

### Plasmid construction

Integrating multiple genes into the plasmid will make the plasmid too large resulting in the reduction of plasmid transformation efficiency and stability and increasing experimental operation complexity and difficulty. We planned to construct four plasmids for each gene. Thus, the efficiency of plasmid construction and the expression of exogenous genes in the host would be improved. We cloned *GjCCD4a*, *GjALDH2C3*, *GjUGT74F8* and *GjUGT94E13* into the pEAQ-DEST1 plasmid vector and used the 35S promoter to constitutively express the exogenous gene.

### Construct *GjCCD4a*、**GjUGT74F8** and *GjUGT94E13* expression vector by gateway clone.

Gateway consists of 2 main reactions: BP reaction and LR reaction. We commissioned
Sangon Biotech (Shanghai) Co. Ltd to synthesize CDS sequences for four genes, which sequenced was codon-optimized. And these sequences contain attB sites at both ends. By BP enzyme, we recombined the target fragment into the gateway cloning vector pDONR207 (Fig.4-1). Then the constructed plasmid was transformed into *E. coli* TOP10 (Fig.4-2). Finally, the colony PCR products were sent for sequencing (Fig.4-3). The sequencing results showed that three entry plasmids successfully constructed.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc1.png) |

Fig.4-1 pDONR207-CCD4a plasmid, pDONR207-UDP1 plasmid and pDONR207-UDP2 plasmid

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc2.png) |

Fig.4-2 BP reaction-transformation

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| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc3.png) |

Fig.4-3 BP reaction sequencing result

After confirmation of the successful construction of the entry plasmids, in the LR reaction, the entry plasmid pDONR207-gene was recombined with the expression vector pEAQ-DEST1 under the action of LR enzyme to form the final expression clone. The constructed expression plasmid was transfected into *E. coli* TOP10 (Fig.4-4). Then the constructed plasmids were transformed into *E. coli* TOP10 (Fig.4-5). Subsequently, the PCR products were sent for sequencing (Fig.4-6). The sequencing results showed that three expression plasmids successfully constructed.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc4.png) |

Fig.4-4 pEAQ-DEST1-CCD4a plasmid, pEAQ-DEST1-UDP1 plasmid, pEAQ-DEST1-UDP2 plasmid

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc5.png) |

Fig.4-5 LR reaction-transformation

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc6.png) |

Fig.4-6 LR reaction sequencing result

### Add a His-tag to the vector by Gibson assembly

Since no His-tag was added at the end of the gene sequence when we designed. We added His-tag to each gene end by Gibson assembly (Fig.4-7). Then the target fragment is obtained by PCR, and the vector was linearized.

The target bands were cut, and Gibson assembly was performed using a ClonExpress II One Step Cloning Kit, incubated at 50°C for 5 min, and the constructed plasmids were inserted into *E. coli* TOP10 (Fig.4-8). Finally, the PCR products were sent for sequencing (Fig.4-9).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc7.png) |

Fig.4-7 pEAQ-DEST1-CCD4a + His-tag plasmid, pEAQ-DEST1-UDP1 + His-tag plasmid, pEAQ-DEST1-UDP2 + His-tag plasmid

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc8.png) |

Fig.4-8 LR reaction-transformation and colony PCR

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| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc9.png) |

Fig.4-9 Sequencing result

So far, the expression vector of *GjCCD4a*, **GjUGT74F8**  and *GjUGT94E1* had been built successfully.

### Construct *GjALDH2C3* expression vector by Gibson assembly.

After several failed BP reactions to *GjALDH2C3*, we realized that not all genes could be used to construct expression vectors by Gateway cloning, so we used Gibson assembly for expression vector construction.

Due to the large size of the vector, to increase the efficiency of linearized PCR of the vector, we removed unnecessary sequences when designing the primers (Fig.4-10).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc10.png) |

Fig.4-10 pEAQ-DEST1-ALDH + His-tag plasmid

We obtained the target linearized gene by PCR and performed the Gibson assembly after gel extraction. Then we transformed the product into *E. coli* TOP10. Then 28 single colonies for colony PCR were selected randomly (Fig.4-11).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc11.png) |

Fig.4-11 Colony PCR

Electrophoresis results showed that the target bands appeared in 12 lanes, and we confirmed that the pEAQ-DEST1-ALDH was successfully constructed after sequencing.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc12.png) |

Fig.4-12 pEAQ-DEST1-ALDH expression vector sequencing result

After the expression plasmids of all genes were constructed, we inserted them into Agrobacterium GV3101 by chemical transformation, and then selected single colonies for colony PCR (Fig.4-13) and sent the PCR products for sequencing. Sequencing results showed that the plasmid was successfully transferred into Agrobacterium(Fig.4-14).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc13.png) |

Fig.4-13 Colony PCR

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| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc14.png) |

Fig.4-14 Colony PCR Sequencing result

### Transient transfection and metabolome extraction

We mixed different strains of Agrobacterium tumefaciens (*GjCCD4a*-GV3101, *GjALDH2C3*-GV3101, *GjUGT74F8* -GV3101, and *GjUGT94E13*-GV3101) and added them to the suspension solution for injection into the leaves (Fig.4-15). After injection, the leaves were subjected to a 24-hour dark treatment.

After 48 hours of light cultivation, the leaves were cut for metabolome extraction (Fig.4-16). A round of concentrated extraction was performed. A total of five blank control tubes and nine experimental tubes of samples were prepared.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc15.png) |

Fig.4-15 A: tobacco transfection(Front of leaf), B: tobacco transfection(Leaf reverse side)

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc16.png) |

Fig.4-16 A:75% methanol extract, B: Crocin I sample, C: Samples from all groups

### Crocin LC-MS/MS Result

We generated a standard curve for crocin I according to the LC-MS/MS results of the crocin I standard (Fig.4-17). Our samples were divided into four groups: Wild-type control, CR-nbhqt2 control, wild-type experimental, and CR-nbhqt2 experimental. Since the amount of each sample bottle in the WT experimental group could not reach the detection standard, we performed a sample mixing, which means there is only one result for the WT experimental group. Moreover, the value obtained after analysis can be used as the theoretical average of a one-sample t-test.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc17.png) |

Fig.4-17 Crocin I standard curve

After analyzing the sample results (Fig.4-18), we found that crocin I was not detectable in the WT and CR-nbhqt2 control groups. However, crocin I was detected in both the WT and CR-nbhqt2 experimental groups. Compared to the WT experimental group, the CR-nbhqt2 experimental group showed a significant increase in crocin content. These results indicate that we successfully transfected the crocin metabolic pathway into *Nicotiana Benthamiana* and achieved its expression.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/crocin1/crc18.png) |

Fig.4-18 Crocin I LC-MS/MS result

### Summary

In conclusion, by transiently expressing four essential genes for crocin synthesis in *Nicotiana Benthamiana*, we found that the improved *Nicotiana Benthamiana* could synthesize more crocin I than wild-type *Nicotiana Benthamiana*, which means when the chlorogenic acid synthesis is turned down, the MEP synthesis will increase. In our experiment, we successfully synthesized crocin I. It indicates that the modified *Nicotiana Benthamiana* has a high application value. The crocin and other rare compounds with terpenoids as precursors are expected to be produced in large quantities in our *Nicotiana Benthamiana*.

## 5.Directed Evolution

For the production of resveratrol, wild VvSTS enzyme derived from grapes still has significant room for improvement in its catalytic activity. Therefore, we have further optimized the enzymatic activity and thermal stability of VvSTS through semi-rational design. For the production of phaselic acid, the catalytic activity of HCT-M derived from red clover is limited due to its weak affinity for caffeoyl-CoA, which tends to bind with feruloyl-CoA instead, resulting in a large amount of feruloyl malic acid by-products. To address these issues, on one hand, we have optimized the catalytic activity and thermal stability of the enzyme through semi-rational design. On the other hand, we have conducted directed evolution of the enzyme, hoping to screen for mutants with high substrate affinity for caffeoyl-CoA.

### Design Principle and Process

In order to screen for mutants with high substrate affinity for caffeoyl-CoA, we conducted directed evolution of HCT-M and performed enzyme activity assays on various variants.

To construct plasmids carrying different HCT-M mutants, we performed error-prone PCR to introduce random mutations into the HCT-M gene and then used homologous recombination to ligate the mutated gene fragments into the pET-28a plasmid backbone. The constructed plasmids were transformed into *E.coli BL21*, and protein expression was induced with IPTG. The mutated HCT-M proteins were purified and the enzyme activities were tested to screen for mutants with higher catalytic activity.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/4-1-n.png) |

Fig.5-1 pET-28a-HCT-M plasmid

### Plasmid Construction

#### Control Plasmid Construction

PCR amplification was performed on the *HCT-M* fragment and the pET-28a plasmid to obtain the *HCT-M* gene fragment and the pET-28a backbone.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/4-2-n.png) |

Fig.5-2 *HCT-M* fragment and pET-28a backbone

After agarose gel electrophoresis of the PCR fragments and gel extraction, homologous recombination was used to ligate the pET-28a-HCT-M plasmid, which was then transformed into *E.coli DH5α*. Colony PCR and sequencing results indicated successful ligation of the *HCT-M* gene to the vector. The plasmid from *E.coli DH5α* was extracted and transformed into *E.coli BL21.*

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/sup-5-3.png) |

Fig.5-3 colony PCR for control bacteria

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/4-4-n.png) |

Fig.5-4 sequencing for control plasmid

#### Random Mutant Plasmid Construction

Error-prone PCR was performed on the *HCT-M* to obtain randomly mutated *HCT-M* gene fragments; PCR was performed on the pET-28a plasmid backbone to obtain the pET-28a backbone.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/4-5-n.png) |

Fig.5-5 random mutant *HCT-M* fragment and pET-28a backbone

After agarose gel electrophoresis of the PCR fragments and gel extraction, homologous recombination was used to ligate the pET-28a-HCT-M plasmid containing random mutations, which was then transformed into *E.coli DH5α*. Colony PCR indicated successful ligation of the *HCT-M* gene to the vector. The plasmid from *E.coli DH5α* was extracted and transformed into *E.coli BL21*.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/4-6-n.png) |

Fig.5-6 colony PCR for mutant bacteria

### Protein Expression and Activity Assay

#### Protein Purification

Single colonies were picked and cultured in LB medium containing kanamycin at 37 ℃, 220 rpm overnight. The bacterial culture was transferred into 50mL LB medium with kanamycin resistance at a 1% inoculation rate, cultured at 37 ℃, 220 rpm for 3 hours to achieve absorbance between 0.6-0.8. IPTG was added to a final concentration of 0.2mM, and the culture was induced at 24 ℃, 150 rpm for 16 hours to express the HCT-M protein.

After centrifugation and lysis of the bacteria, total bacterial protein was extracted. SDS-PAGE results showed that out of 13 proteins, 9 were successfully purified. Four mutant proteins still did not show clear bands, which may be due to the fact that after random mutations in the protein sequence, proteins may have been formed inclusion bodies or are unable to expose the His-tag, leading to the failure of purification of these four mutant proteins.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/4-7-n.png) |

Fig.5-7 SDS-PAGE for control protein and mutant protein

#### Enzyme Activity Assay

Protein concentration was determined using the Bradford protein concentration detection kit, with absorbance measured at 595 nm to calculate the protein concentration. In the reaction system, the concentration of tartaric acid was 300 μM, the concentration of caffeoyl-CoA was 300 μM, and the enzyme concentration was 100 ng/mL, with the reaction carried out at 30 ℃ for 30 minutes. The results showed that compared to the control group, the enzyme activity of mutant3 was increased, while the enzyme activities of mutant5, mutant6, and mutant7 were decreased, and the rest of the mutants did not show significant enzyme activity. This indicates that directed evolution can change the catalytic activity of the enzyme by altering key residues of the enzyme.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/evolution/4-8.png) |

Fig.5-8 The detection of enzyme activity

## 6.Semi-rational Design

Although directed evolution has achieved some success, it requires lots of experimental screening and has a low probability of obtaining favorable mutations. Therefore, we adopted a semi-rational design approach to screen for mutation sites through molecular docking, dynamic simulation, and other methods. Then, we established a mutation library and verified its effectiveness through wet experiments. The results showed that among the 7 HCT-M mutants, 2 had higher enzyme activity and thermal stability than the wild type. And among the 12 VvSTS mutants, 5 mutants had stronger enzyme activity at the optimal temperature than the wild type, and 4 mutants had stronger thermal stability than the wild type. Our work will help increasing the production of resveratrol and caffeoyl-CoA in tobacco chassis, and demonstrate that semi-rational design is a efficient protein optimization strategy with broad application prospects in plant synthetic biology.

### Design Principles and Processes

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-0.png) |

Fig.6-1 Process of semi-ration deisgn

### Semi-rational design and codon optimization

#### Semi-rational design

Through three rounds of semi-reational design, we successfully screened 13 HCT-M mutants and 12 VvSTS mutants for validation. Firstly, we simulated single point saturation mutations of HCT-M and VvSTS on a computer, and used a pre trained protein language model to score all mutants in terms of stability and ligand affinity. We selected the mutants with higher scores. On this basis, protein and ligand molecular docking was performed to compare the binding stability of mutant and wild-type proteins and ligands, and to exclude mutants that are not conducive to stable binding. Finally, molecular dynamics simulations were conducted on the mutants selected in the first two rounds to evaluate the energy and stability of the entire reaction system from a physical perspective, and obtain mutants for validation by wet experiments

#### Codon Optimization

Considering that we will heterologous express HCT-M from Trifolium repens and VvSTS from grapes in Escherichia coli, it is highly likely that using the original sequence directly will result in a decrease in protein expression due to differences in amino acid coding preferences among different species. The common strategy for codon optimization is to replace codons with those preferred by the host organism and adjust their GC content to increase mRNA stability and adapt to the host organism's level. The genes of HCT-M and VvSTS were both codon optimized and synthesized by Sangon Biotech, and we conducted experiments at [IDT](https://sg.idtdna.com/pages/tools/codon-optimization-tool).The effectiveness of codon optimization has been verified.

### Plasmid Construction and Primer Design

The synthesized HCT-M and VvSTS gene both carry Histag and are loaded onto the pET-28a (+) plasmid, which was synthesized by Sangon Biotech. The reason for choosing pET-28a (+) is that it is a plasmid vector with a lac operon and T7 promoter, which can induce overexpression of exogenous genes through IPTG, effectively controlling the expression of exogenous genes and reducing their harmful effects on host cells. At the same time, high concentrations of expressed proteins help us perform enzyme function testing.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-1.png) |

Fig.6-2 pET-28a (+) - HCT-M wild-type plasmid map

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| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-2.png) |

Fig.6-3 pET-28a(+)-VvSTS wild-type plasmid map

In order to perform site directed mutagenesis on wild-type genes, we designed primers based on the selected mutation sites. The primers carry corresponding mutation sites, which are marked in green in the table. The naming convention for mutants is: original amino acid abbreviation - mutation site - mutated amino acid abbreviation. For example, H294L is mutated from histidine at position 294 to leucine. The naming convention for primers is: mutation site of amino acid - abbreviation of mutated amino acid - F (forward primer)/R (reverse primer).

Table6-1: HCT-M Primer Sequence Carrying Mutation Sites

| Mutation | Primer | Sequence (5' to 3') |
| --- | --- | --- |
| H294L | 294L-F | TCGTTTCCTGGCTGATATCCGCCGTCGTATCA |
| 294L-R | TATCAGCCAGGAAACGAACAACGCTTTCCTGGTC |
| H294Y | 294Y-F | CGTTTCTATGCTGATATCCGCCGTCGTATCAAC |
| 294Y-R | TATCAGCATAGAAACGAACAACGCTTTCCTGGTCATC |
| A419T | 419T-F | CACGATACCGCGTACATTACCCTGAGCCCG |
| 419T-R | ATGTACGCGGTATCGTGCGGAGACACACCT |
| A389N | 389N-F | CCAGGTTAACTCCTGGACCGGTATGCC |
| 389N-R | TCCAGGAGTTAACCTGGAAGTTCGGGTTACC |
| T162Y | 162Y-F | CGATGGTTATGGTGCTGTTAAATTCATTAACTCCTGGG |
| 162Y-R | CAGCACCATAACCATCGCTCAGGCTATG |
| K26A | 26A-F | TGTCTGATGCGGATCAGGTGGCGACCCAGC |
| 26A-R | CCTGATCCGCATCAGACAGCCACAGACGACC |
| K26G | 26G-F | TGTCTGATGGCGATCAGGTGGCGACCCAGC |
| 26G-R | CCTGATCGCCATCAGACAGCCACAGACGACC |
| K26I | 26I-F | TGTCTGATATTGATCAGGTGGCGACCCAGC |
| 26I-R | CCTGATCAATATCAGACAGCCACAGACGACC |
| K26L | 26L-F | TCTGATCTGGATCAGGTGGCGACCCAGCAC |
| 26L-R | CCTGATCCAGATCAGACAGCCACAGACGACC |
| K26F | 26F-F | TCTGATTTTGATCAGGTGGCGACCCAGCAC |
| 26F-R | CCTGATCAAAATCAGACAGCCACAGACGACC |
| K26S | 26S-F | TCTGATAGCGATCAGGTGGCGACCCAGCAC |
| 26S-R | CCTGATCGCTATCAGACAGCCACAGACGACC |
| K26V | 26V-F | TCTGATGTGGATCAGGTGGCGACCCAGCAC |
| 26V-R | CCTGATCCACATCAGACAGCCACAGACGACC |
| K26Y | 26Y-F | TCTGATTATGATCAGGTGGCGACCCAGCAC |
| 26Y-R | CCTGATCATAATCAGACAGCCACAGACGACC |

Table6-2: VvSTS Primer Sequence Carrying Mutation Sites

| Mutation | Primer | Sequence (5' to 3') |
| --- | --- | --- |
| P269N | 269N\_F | CTGTGGAACAACGTTCCGACCCTGATCAGCG |
| 269N\_R | CGGAACGTTGTTCCACAGGTGGAAGGTCAGACC |
| P269S | 269S\_F | ACCTGTGGAGCAACGTTCCGACCCTGATCAGC |
| 269S\_R | AACGTTGCTCCACAGGTGGAAGGTCAGACCAACTTC |
| P269K | 269K\_F | CCTGTGGAAAAACGTTCCGACCCTGATCAGCG |
| 269K\_R | TGGAAGGTCAGACCAACTTCGCGCAGGTTACCCG |
| P269R | 269R\_F | CCTGTGGCGCAACGTTCCGACCCTGATCAGC |
| 269R\_R | TGGAAGGTCAGACCAACTTCGCGCAGGTTACCCG |
| W268L | W268L\_F | TCCACCTGCTGCCGAACGTTCCGACCC |
| W268L\_R | CGTTCGGCAGCAGGTGGAAGGTCAGACCA |
| W268S | 268S\_F | CTTCCACCTGAGCCCGAACGTTCCGACCC |
| 268S\_R | CGTTCGGGCTCAGGTGGAAGGTCAGACCAACTTCG |
| S220A | 220A\_F | GGCTCTGCGGCGGTGATCGTGGGCTCTGATCCG |
| 220A\_R | GTCACCGAACAGAGCCTGGCCAACCAGGCTATCC |
| S219A | 219A\_F | TGACGGCGCGAGCGCGGTGATCGTGG |
| 219A\_R | CGCTCGCGCCGTCACCGAACAGAGCCTG |
| S219C | 219C\_F | ACGGCTGCAGCGCGGTGATCGTGGGC |
| 219C\_R | CGCGCTGCAGCCGTCACCGAACAGAGCC |
| T173I | 173I\_F | CTGCGTATTGCAAAAGATCTGGCGGAAAACAACGCAGGCGCT |
| 173I\_R | AACGGTACCGCCCGCGTAGCAACCCTGGTGG |
| L114I | 114I\_F | GCGATTAAAGAATGGGGCCAGCCGAAAAGCAAAATCACCCACC |
| 114I\_R | TTTCAGCGCCGCATCACGGCCCAGACGC |
| N57Q | 57Q\_F | AAGAAATTCCAGCGTATCTGCGATAAAAGCATGATCAA |
| 57Q\_R | GCAGATACGCTGGAATTTCTTTTTCAGTTCGGTCATG |

### Construction of Point Mutation Plasmids

We used mutation primers to perform PCR reaction on wild-type plasmids and obtained linearized plasmids with mutation sites. After digesting the wild-type plasmids with dpn1 enzyme, the mutation products were transferred into competent *E. coli* DH5α, and plasmids were extracted and sequenced from *E. coli* DH5α after expanded cultivation.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-3.png) |

Fig.6-4 Sequencing results of 13 mutants of HCT-M

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-4.png) |

Fig.6-5 Sequencing results of 12 mutants of VvSTS

As shown in the sequencing results, we have successfully constructed all the required mutants.

### Enzyme Expression and Activity Detection

We induced protein expression and purification of the successfully constructed mutant HCT-M gene and VvSTS gene. The activity validation of HCT-M enzyme is characterized by detecting the remaining amount of malic acid, while the activity validation of VvSTS enzyme is characterized by directly detecting the amount of resveratrol.

#### HCT-M

For HCT-M, due to time and budget constraints, seven mutants including H294L, H294Y, A419T, A389N, K26F, K26S, and K26V were selected for protein characterization. After inducing IPTG expression, in order to unify the concentrations of various enzymes in subsequent enzymatic reactions, we measured the purified protein concentration using the Bradford method.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-5.png) |

Fig.6-6 HCT-M protein concentration determination (a1-b9:standard curve, b10-12: H294L, c1-c3:H294Y, c4-c6:A419T, c7-c9:A389N, c10-c12: K26F, d1-d3:K26S, d4-d6 :K26V)

By analyzing the absorbance data, the concentration of mutant protein was determined as shown in the table below.

Table6-3: Protein concentration

| Sample | ng/ul |
| --- | --- |
| MU-1 | 101.10 |
| MU-2 | 60.85 |
| MU-3 | 408.48 |
| MU-4 | 1067.57 |
| MU-10 | 171.67 |
| MU-11 | 340.82 |
| MU-12 | 77.18 |

The mutant protein and wild-type protein were diluted to the same concentration and subjected to the following enzymatic reaction: 300μM caffeoyl-CoA, 300μM malic acid, 50ng/μL HCT-M enzyme, and phosphate buffer to adjust the reaction system to pH 6.5. The reaction time is 30 minutes, and three temperature gradients are set to verify thermal stability. The temperature gradients are 30 ℃ (the optimal temperature for HCT-M enzyme), 35 ℃, and 40 ℃, respectively. Using specific enzyme activity as a measure of activity.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-6.png) |

Fig.6-7 Comparison of HCT-M enzyme activity

For the seven mutants, only three have activity, namely A419T, A389N, and K26F. At 30 ℃, the activity of A419T is lower than that of the wild type, while the activity of A389N and K26F is higher than that of the wild type. When the temperature is 35 ℃, the wild-type HCT-M loses its activity, but A419T, A389N, and K26F still have activity. At 40 ℃, A389N and K26F remain active. This indicates that semi-reational design can effectively construct enzymes with higher enzyme activity and higher thermal stability.

#### VvSTS

Due to the low solubility of VvSTS, inclusion bodies are easily formed. In order to increase the amount of normal VvSTS collected, we used TB medium as the source of bacterial nutrition and induced expression conditions of 0.1 mM IPTG, 21 ℃, 120 rpm, and 20 h. Finally, we successfully obtained sufficient protein concentration for the reaction through gravity column purification and ultrafiltration.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-7.png) |

Fig.6-8 VvSTS protein concentration determination (A: Bradford method for detecting protein concentration; B: standard curve; C: VvSTS protein concentration)

Afterwards, an enzyme activity assay experiment was conducted with a system of 50 μ L, consisting of the following components: VvSTS 0.4 μ g, coumarin-CoA 50 μ mol/L, malonyl-CoA 100 μ mol/L, pH 7.0, reaction time 60 min, and three temperature gradients (50 ℃, 55 ℃, 60 ℃).

After the reaction, the concentration of the product resveratrol was detected using the Resveratrol Immune Competition Kit (ELISA), and the results are as follows:

|  |
| --- |
| ![](https://static.igem.wiki/teams/5223/result/semi-8.png) |

Fig.6-9 Resveratrol concentration of all enzyme reaction

The reaction activity of wild-type VvSTS did not change significantly under three temperature gradients, indicating that VvSTS itself has good heat resistance. In terms of thermal stability, mutant W268S showed better reaction activity than WT at 55 and 60 ° C, while S219A and P269K showed better reaction activity than WT at 55 ° C, indicating that the semi-reational design constructed enzymes with better thermal stability. In terms of catalytic activity, the mutants with stronger catalytic activity than WT at the optimal temperature include S219A, W268L, S220A, and L114I, indicating that semi-reational design has constructed mutants with higher catalytic activity.

## Outlook

The high chlorogenic acid accumulation in *Nicotiana benthamiana* limits its potential for synthesizing other products. To develop tobacco as a versatile chassis for the short-term, efficient synthesis of desired plant natural products, we first constructed a genome-scale metabolic model (GSMM) and used COBRA algorithms to formulate the best metabolic flow adjustment strategies. Subsequently, the critical gene NbHQTs for chlorogenic acid synthesis in tobacco was knocked out using CRISPR/Cas9, and a low chlorogenic acid level tobacco chassis was obtained. We then introduced heterologous synthetic pathways for phaselic acid, resveratrol, and crocin into the chassis and improved the synthesis capability by engineered critical enzymes. The chassis could quickly synthesize the desired functional products to fulfill different requirements and is expected to be developed into an efficient biological reactor for the biopharmaceutical industry, thereby promoting the green transformation and upgrading of the traditional tobacco cultivation industry.

During the iGEM competition cycle, the number of repetitions and technical iterations of our experiments is limited. Currently, the validation results for the potential of chassis synthesis are very limited and the credibility is questionable. It is necessary to select more types of compounds from other metabolic pathways, such as upstream and downstream in the MVA pathway, for exogenous synthesis to functionally validate our tobacco chassis. In addition, we only selected two key enzymes, VvSTS and HCT-M, for thermal stability and catalytic activity modification in order to increase the production of phaselic acid and resveratrol. However, other key enzymes used for synthesizing other products such as crocin were not optimized during the experimental period. For other compounds such as crocin, more efforts need to be made in enzyme modification and metabolic pathway design before applying our chassis for efficient production.

In the future, we will further optimize the chassis of *Nicotiana benthamiana* by knocking out more genes such as nicotine synthesis genes that occupy a large amount of metabolic flow, in order to release more metabolic flow potential for the synthesis of other downstream secondary compound products.

In terms of factory production, we will use soilless cultivation techniques to cultivate our chassis plants on a large scale in artificially controlled greenhouses, achieving the large-scale and systematic construction of plant synthetic biology "bioreactors" based on *Nicotiana benthamiana* as the chassis organism. We will also actively cooperate with biopharmaceutical companies to strive for the industrial implementation of the project.

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