Coding

Part:BBa_K5218005

Designed by: Amy Fu, Juliette, Lichuan Chen, Xiaojuan Wang   Group: iGEM24_BGI-MammothEdu-South   (2024-08-24)


RtTAL

Tyrosine ammonia lyase (TAL) CDS from Rhodotorula toruloides with codon optimization.

Base Pairs：2148 bp

Function：Tyrosine ammonia-lyase (TAL) that catalyzes the formation of p-coumaric acid from tyrosine.

Figure 1. TAL plays a role in the pathway of caffeic acid biosynthesis.
Figure adopted from L. Liu et al, 2019 [1]

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1015
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 271
    Illegal BglII site found at 304
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1015
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1015
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

Ammonia-lyases catalyze the deamination of amino acids. Members of this family include histidine ammonia lyase (HAL) that converts histidine to urocanic acid, phenylalanine ammonia lyase (PAL) that converts phenylalanine to cinnamate (CA), and tyrosine ammonia lyase (TAL) that converts tyrosine to p-coumaric acid. p-coumaric acid is an important precursor in many metabolic pathways, one of which is the production of caffeic acid with the catalysis of coumarate 3-hydroxylase (C3H).

PALs, as key rate-limiting enzymes, are commonly found in plants, algae, fungi and bacteria[2]. The presence of TAL enzymes have been reported in a few microorganisms. TAL from Rhodosporidium toruloides was demonstrated to synthesize p-coumaric acid from L-tyrosine and obtained a yield of 117.5 mg/L [1]. TAL from Rhodobacter capsulatus has 32% identity with plant PAL sequence of Pinus taeda[3]. iGEM13_Uppsala team registered a TAL gene from rhodobacter sphaeroides (Part:BBa_K1033000) and characterized the enzyme with spectrophotometry and chromatography.

Rhodotorula toruloides is a species of oleaginous yeast. It is a red basidiomycete isolated from the wood pulp of conifers and naturally accumulates carotenoids, neutral lipids, and enzymes relevant to the chemical and pharmaceutical industries (Wikipedia).

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 Fungal Bioluminescence Pathway (FBP) via eGFP reporter system (pS1300-GFP plasmid). The RtTAL protein ID is CDR39392.1 (NCBI). Team BGI-MammothEdu-South retrieved the RtTAL CDS from published article[1], conducted codon optimization for plant system and obtained the sequence through De novo DNA synthesis.

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

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

Figure 4: Cloning of RtTAL CDS into pS1300-GFP expression vector.
A. RtTAL CDS PCR product on agarose gel electrophoresis. Lane M, 1kb plus DNA ladder; lane1, RtTAL CDS
B. Single colonies of pS1300-RtTAL-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 RtTAL in FBP.

The pS1300-RtTAL-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 RtTAL-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.

he pS1300-RtTAL-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-RtTAL-GFP lines were much more brighter compared to the control lines.

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

A. Picture of control and pS1300-RtTAL-GFP lines in the light. Control:FBP-22 only; vector control: FBP-22 transformed with pS1300-GFP.
B. Picture of control and pS1300-RtTAL-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 RtTAL 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 sets of 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]


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 10. 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 11. 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 12. 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 13. Promoter candidates negatively responded to HCHO stress in dissected leaf GUS staining.


Third DBTL Circle: Creating a Controlled Bioluminescence Intensity Regulation Module

Since RtTAL 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 detection. For this reason, team BGI-MammothEdu-South 2024 generated a new construct, p1300-GRF3-RtTAL-GFP, in which we used pS1300-GFP backbone, replaced super promoter with a formaldehyde-responsive promoter, pGRF3[6] to regulate the expression of RtTAL. We then transformed the construct into Agrobacterium GV3101 strain followed by infiltration to generate transient expression lines.

Figure 14. p1300-pGRF3-RtTAL-GFP plasmid map.

Upon 2mM HCHO treatment for 36 hours, the bioluminescence intensity was repressed in pS1300-GFP control lines compared to water treatment, whereas the pS1300-RtTAL-GFP and p1300-pGRF3-RtTAL-GFP lines could compensate the repression. The results showed that RtTAL could stably enhance the bioluminescence of plants, and GRF3 has some level of responsiveness to HCHO. The enhancement of light intensity by GRF3 promoter is limited though. It is worth memtioning that the batch of tobacco materials for this experiment went through significant injury stress in the process of injection, which may affected the bioluminescence analysis and interpretation. Unfortunately, due to limited time, we did not conduct a second test on new batch of plants.

Figure 15. pGRF3-RtTAL in response to HCHO.

Reference

1.Liu, Langqing, Liu, Hong, Zhang Wei, Yao, Mingdong, Li, Bingzhi, Liu, Duo, Yuan Yingjin. (2019) Engineering the biosynthesis of caffeic acid in saccharomyces cerevisiae with heterologous enzyme combinations. Engineering, 5(2), 9

2.Zhang Fulin, Wang Juan, Li Xianguo, Zhang Jun, Liu Yuxiang , Chen Yijia, Yu Qinghui, Li Ning (2023) Genome-wide identification and expression analyses of phenylalanine ammonia-lyase gene family members from tomato (Solanum lycopersicum) reveal their role in root-knot nematode infection. Frontiers in Plant Science, 14:1204990

3.Kyndt J.A., Meyer T.E., Cusanovich M.A. and Van Beeumen J.J. (2002) Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein, FEBS Letters, 512

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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