Coding

Part:BBa_K5218002

Designed by: Amy Fu, Juliette, Lichuan Chen, Xiaojuan Wang   Group: iGEM24_BGI-MammothEdu-South   (2024-08-11)


AtC3H

p-coumaric acid 3-hdroxylase (C3H) gene from Arabidopsis thaliana.

Base Pairs：1524 bp

Function：p-coumaric acid 3-hdroxylase that catalyzes the formation of caffeic acid from p-coumaric acid.

Figure 1. C3H plays a role in the Caffeic acid cycle.
figure adopted from Tatiana et al., 2020[1]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 234
    Illegal NheI site found at 552
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 406
    Illegal XhoI site found at 749
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 132
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1193
    Illegal SapI site found at 387

Introduction

Cytochrome P450 family has been identified in all kingdoms of life, which can catalyze more than 20 types of reactions, including hydroxylation, epoxidation, cyclization, and so on [1]. One of its members, C3H, catalyzes the formation of caffeic acid from p-coumaric acid. This reaction is associated with lignin biosynthesis, which is vital for plant development [2]. Recently scientists have utilized Fungal Bioluminescence Pathway (FBP) to generate autoluminescent plant [3]. C3H, which produced the starting substance for caffeic acid cycle, is a crucial candidate gene for FBP genetical modification.

The engineering objective of this project is to generate brighter autoluminescent plants using synthetic biology approaches. Team BGI-MammothEdu-South 2024 selected a variety of candidate genes from different species and tested their functions in FBP via eGFP reporter system (pS1300-GFP plasmid). The AtC3H gene ID is AT2G40890, transcript ID is NM_180006.2 (NCBI). Team BGI-MammothEdu-South 2024 harvested the Arabidopsis thaliana flower, bud, and young stem samples to extract total RNA, reverse transcribe into cDNA and cloned the AtC3H CDS with specific primer pairs.


Team BGI-MammothEdu-South 2024 conducted the project based on Design-Build-Test-Learn principle (DBTL) principle to achieve a higher and more controllable bioluminescence in plants.

Figure 2. Engineering cycle of the project.

In total, three rounds of DBTL were carried out. In the first cycle, the concentration of substrates in the caffeic acid cycle was increased using the Tyrosine Ammonia-Lyase (TAL) and p-Coumarate 3-Hydroxylase (C3H) genes, thereby enhancing the plant bioluminescence intensitt, similar to adding more fuel to an engine to make it run faster. In the second cycle, we tested a regulatory module for key catalytic enzymes upstream of the caffeic acid pathway to further increase production, implementing a β-glucuronidase (GUS) staining module to enable the plant to respond to formaldehyde. In the third cycle, we combined the formaldehyde-responsive regulatory module with the enhanced bioluminescence genes, creating a plant that can respond to formaldehyde with increased light emission, much like adding an accelerator pedal to our engine.

Figure 3. Workflow of Registry part characterization in this project.


Characterisation

First DBTL Circle: transient expression RtTAL and AtC3H to enhance the self-luminescence brightness

The cloning strategy for AtC3H-GFP fusion gene is as follows: two rounds of primers pairs were designed. The first round reverse primer is located on the 3' UTR of AtC3H CDS for specificity. The second round PCR uses first round product as template, and the second reverse primer is located before the AtC3H CDS stop codon, and recombination overhang is added for cloning.

The non-stop-codon AtC3H CDS was amplified through 2 rounds of PCR, purified and cloned into pS1300-GFP vector via Gibson Cloning system. In the construct, AtC3H CDS, driven by a super promoter (CaMV 35S), is fused with eGFP reporter gene (3' end) followed by NOS terminator. The recombinant plasmid pS1300-AtC3H-GFP was transformed into E. coli TOP10 competent cells and verified through colony PCR and sequencing.

Figure 4. Cloning of AtC3H CDS into pS1300-GFP expression vector.
A. AtC3H CDS 2-round PCR product on agarose gel electrophoresis. Lane M, 1kb plus DNA ladder; first round PCR product (with stop codon, no recombination overhang) lane1, AtC3H, lane2, PpC3H; second round PCR product (without stop codon, recombination overhang) lane3, AtC3H, lane4, PpC3H;
B. Single colonies of pS1300-AtC3H-GFP transformants on LB kanamycin+ plate.
C. Colony PCR product on agarose gel electrophoresis. lane 1-12, 12 single colonies tested.

Nicotiana benthamiana tobacco line FBP-22[4], in which the FBP (includes LUZ, H3H, CPH and HispS gene) was introduced, was proven to be autoluminescent. Yet the luminescence intensity was limited. Team BGI-MammothEdu-South 2024 foucsed on enhancing bioluminescence of plants and exploring further applications. Tobacco line FBP-22 was used as control and genetical engineering material for the functional varification of AtC3H in FBP.

The pS1300-AtC3H-GFP construct was transformed into Agrobacterium GV3101 strain, followed by transient transformation of tobacco leaf through injection. The transgenic tobacco leaves were examined under the fluorescence microscope. The results showed that AtC3H-GFP fusion protein signal was observed in the tobacco leaf epidermal cells, suggesting the engineering success of the construct.

Figure 5. Candidate gene-GFP fusion proteins expression in tobacco leaf epidermal cells.


The pS1300-AtC3H-GFP transgenic tobacco plants were also photographed in the dark room to record their bioluminescence intensity. Since the team could not get access to the plant in vivo imaging system, we used greyscale value of photos instead and quantified the data using ImageJ software. The results suggested that the pS1300-AtC3H-GFP lines were much more brighter compared to the control lines.

Figure 6. Enhanced bioluminescence of pS1300-AtC3H-GFP transgenic lines.

A. Picture of control and pS1300-AtC3H-GFP lines in the light. Control:FBP-22 only; vector control: FBP-22 transformed with pS1300-GFP.
B. Picture of control and pS1300-AtC3H-GFP lines in the dark.
C. Grayscale values of randomly circled areas in A and B. Value calculated with ImageJ software.

All the grey-scale value data were collected from the lines tested and bar charts plotted. The result showed that AtC3H CDS trangenic line had one of the highest bioluminescence intensity enhancement compared to the control lines.

Figure 7. Bioluminescence intensity of control and transgenic lines.
Greyscale value was used to represent bioluminescence intensity.

Figure 8. Bioluminescence intensity enhancement of different transgenic lines.
Increased greyscale value compared to vector control group.

Team BGI-MammothEdu-South 2024 focused on two genes, TALs and C3Hs from various species, tested their functions in the caffeic acid pathway. The results showed that the TAL and C3H genes we selected could all contribute to a range of bioluminescence intensity enhancement, presumably due to the FBP substance caffeic acid accumulation. These findings could provide valuable insights for future iGEM team or researchers in the field of bioluminescence plants.

Figure 9. Key components of caffeic acid pathway tested in our project.
Figure adopted from L. Li et al, 2021[5]

Besides wet lab cloning and validation, team BGI-MammothEdu-South 2024 also conducted in silico analysis of the C3Hs protein structure looking to explore the relationship between protein structure and function. Eight different C3Hs sequences from six plants were collected, their motif consensus, tertiary structure and predictive actives site were anylysed and compared. The result displayed that C3Hs held highly conserved structure both in motif and tertiary structure. For the light intensity discrepencies among each member, it is suspected that the active sites might have subtle structual changes that alters the catalytic activity. The results provide valuable insights for future research.

Figure 10. Protein structure analysis of 8 C3Hs from 6 plants
A. Motif consensus analysis using MEME model.
B. Tertiary structure prediction using SWISS model.
C. Predictive actives site from docking simulation of C3Hs with p-coumaric acid

We simulated the docking of p-coumaric acid, the substrate for C3H, using AutoDock Vina. Ligand-binding sites were identified using ConSurf, focusing on functional and structural residues surrounding the active site. The docking simulations revealed binding sites near conserved regions across the C3H homologs. Active sites for each protein homogy are calculated by Consurf. Key residues in the active sites, such as R436, P362, and G432 in At_CYP98A3, were identified as potential contributors to catalytic efficiency.

Figure 11. Illustration of 5 C3H homologs in three dimensions with ligand P-coumaric acid binding to its specific active site, visualized by Pymol.

Second DBTL Circle: formaldehyde-responsive promoter - GUS staining test

Formaldehyde (HCHO) is a common environmental and occupational pollutant, widely used in both industrial and consumer products. Exposure to formaldehyde can result in serious health issues, including upper respiratory illnesses and cancer. Therefore, developing effective monitoring and purification technologies for HCHO is essential.

Plant 14-3-3 proteins are key regulators that play significant roles in plant growth, development, and stress response processes [6]. GRF3 proteins are reported to regulate the response of plants to HCHO stress by interacting with AtMDH1 and AtGS1 [7].

Figure 12. HCHO stress response pathway in Arabidopsis.
Figure adopted from X. Zhao et al, 2023 [7]

The engineering objective of this project is to generate brighter autoluminescent plants using synthetic biology approaches, and explore its potential applications. Team BGI-MammothEdu-South 2024 selected a set of promoter elements candidate and tested their regulatory functions in Fungal Bioluminescence Pathway (FBP) via eGFP and GUS reporter system (pS1300-GFP, pS1300-GUS plasmid). The AtGRF3 gene ID is AT5G38480, transcript ID is NM_123209.4 (NCBI). Team BGI-MammothEdu-South extracted the Arabidopsis thaliana leaf genomic DNA and cloned the AtGRF3 promoter with specific primer pairs.


The expression pattern of AtGRF3 was firstly investigated on the AtGenExpress eFP database. The result showed that AtGRF3 was highly expressed in imbibed seed and shoot apex; moderately expressed in leaf and flowers.

Figure 13. AtGRF3 expression pattern.

The cloning strategy for p1300-GRF3-GUS construct is as follows: The pS1300-GUS vector was first digested with BamH I to remove the super promoter and get linear p1300-GUS, which was ligated with HCHO-responsive GRF3 promoter fragment via Gibson Cloning system. In the construct, GUS reporter gene was driven by GRF3 promoter, followed by NOS terminator. The recombinant plasmid p1300-GRF3-GUS was transformed into E. coli TOP10 competent cells and verified through colony PCR and sequencing.

Figure 14. Generation of p1300-GRF3-GUS construct.

A. PCR and digestion product on agarose gel electrophoresis. Lane M, 5000bp DNA marker; lane 1, cicular pS1300-GUS plasmid; lane 2, BamH I digestion of pS1300-GUS; lane 3, GRF3 promoter PCR prodoct.
B. Single colonies of p1300-GRF3-GUS transformants on LB kanamycin+ plate.
C. Colony PCR product on agarose gel electrophoresis. Lane 1-16, 16 single colonies tested.
D. GRF3 promoter sequence validated through sequencing.
E. Single colonies of p1300-GRF3-GUS transformants on LB kanamycin+ rifampicin+ plate.
F&G. Colony PCR of GV3101 transformants product on agarose gel electrophoresis. Lane 1-8, 8 single colonies tested.

The p1300-GRF3-GUS construct was transformed into Agrobacterium GV3101 strain, followed by transient transformation of tobacco leaf through injection. The transgenic tobacco plants were stressed with 2mM HCHO (treatment) or H2O (control) for 36 hours, and leaf samples were collected 12 hours after treatment for GUS staining procedure. After destaining, the leaf tissues were photographed.

The result displayed that for the negative control 0.5x PBS, no GUS signal was found in either HCHO or H2O group. When transgene was introduced, GUS signal was detected in H2O group, in which MDH1-GUS signal being the strongest, follwed by vector control pS1300-GUS, GRF3-GUS and GS1-GUS. However, after HCHO stress, 3 promoter-GUS showed different levels of signal reduction, compared to the enhanced signal in vector control pS1300-GUS. The result indicated that the three promoter candidates in this project were negatively responsive to HCHO stress, and GRF3 had the strongest phenotype.

Figure 15. Promoter candidates negatively responded to HCHO stress in dissected leaf GUS staining.


Third DBTL Circle: Creating a Controlled Bioluminescence Intensity Regulation Module

Since AtC3H has been shown to increase bioluminescence intensity in FBP plants, we further explored the potential of these enhanced bioluminescent plants for applications in biological monitoring, such as formaldehyde (HCHO) detection. For this reason, team BGI-MammothEdu-South 2024 generated a new construct, p1300-GRF3-AtC3H-GFP, in which we used pS1300-GFP backbone, replaced super promoter with a formaldehyde-responsive promoter, pGRF3[7] to regulate the expression of AtC3H. We then transformed the construct into Agrobacterium GV3101 strain followed by infiltration to generate transient expression lines.

Figure 16. p1300-pGRF3-AtC3H-GFP plasmid map.


Figure 17. Generation of p1300-pGRF3-RtTAL-GFP and p1300-pGRF3-AtC3H-GFP construct.

A. PCR product on agarose gel electrophoresis. Lane M, 1kb plus DNA marker; lane 1&2, MDH1 promoter; lane 3&4, GS1 promoter; lane 5&6, GRF3 promoter. Lane 1/3/5 products contain RtTAL recombination overhang; lane 2/4/6 products contain AtC3H recombination overhang;
B. Single colonies of p1300-pGRF3-RtTAL-GFP transformants on LB kanamycin+ plate.
C. Single colonies of p1300-pGRF3-AtC3H-GFP transformants on LB kanamycin+ plate.
D. Single colonies of p1300-pGRF3-RtTAL-GFP transformants on LB kanamycin+ rifampicin+ plate.
E. Single colonies of p1300-pGRF3-AtC3H-GFP transformants on LB kanamycin+ rifampicin+ plate.
F. Colony PCR of p1300-pGRF3-RtTAL-GFP in E.coli. Lane M, 5000bp DNA marker. Lane 1-24, 24 single colonies tested.
G. MDH1 promoter sequence validated through sequencing.
H. Colony PCR of p1300-pGRF3-AtC3H-GFP in E.coli. Lane M, 5000bp DNA marker. Lane 1-24, 24 single colonies tested.
I. MDH1 promoter sequence validated through sequencing.
J. Colony PCR of p1300-pGRF3-RtTAL-GFP in GV3101. Lane M, 5000bp DNA marker. Lane 1-8, 8 single colonies tested.
K. Colony PCR of p1300-pGRF3-AtC3H-GFP in GV3101. Lane M, 5000bp DNA marker. Lane 1-8, 8 single colonies tested.


Upon 2mM HCHO treatment, the bioluminescence intensity was clearly repressed in pS1300-GFP control lines, whereas the pS1300-AtC3H-GFP and p1300-pGRF3-AtC3H-GFP lines could compensate the repression. The results showed that AtC3H could stably enhance the bioluminescence of plants, and GRF3 is indeed responsive to HCHO. Yet the enhancement of light intensity by promoter GRF3 is limited.

Figure 18. pGRF3-AtC3H in response to HCHO.



Reference

1.Jung S T, Lauchli R, Arnold F H. (2011) Cytochrome P450: taming a wild type enzyme[J]. Current opinion in biotechnology, 22(6): 809-817.

2.Barros J, Escamilla-Trevino L, Song L, Rao X, Serrani-Yarce JC, Palacios MD, Engle N, Choudhury FK, Tschaplinski TJ, Venables BJ, Mittler R, Dixon RA. 4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. (1994) Nature Communication, 2019 Apr 30;10(1).

3.Mitiouchkina, T., Mishin, A.S., Somermeyer, L.G. et al. Plants with genetically encoded autoluminescence. (2020) Nature Biotechnology, 38, 944–946.

4.Zheng, P., Ge, J., Ji, J., Zhong, J., Chen, H., Luo, D., Li, W., Bi, B., Ma, Y., Tong, W., Han, L., Ma, S., Zhang, Y., Wu, J., Zhao, Y., Pan, R., Fan, P., Lu, M. and Du, H. (2023), Metabolic engineering and mechanical investigation of enhanced plant autoluminescence. Plant Biotechnol J, 21: 1671-1681.

5.Li LL, Liu X, Qiu ZT, et al. (2021) Microbial synthesis of plant polyphenols. Chinese Journal of Biotechnology, 37(6): 2050-2076.

6.Zhao, X., Li, F., Li, K. (2021) The 14-3-3 proteins: regulators of plant metabolism and stress responses. Plant Biol 23, 531–539.

7.Xing Zhao, Xueting Yang, Yunfang Li, Hongjuan Nian, Kunzhi Li. (2023) 14-3-3 proteins regulate the HCHO stress response by interacting with AtMDH1 and AtGS1 in tobacco and Arabidopsis. Journal of Hazardous Materials,Volume 458, 132036,

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