Part:BBa_K5088000

Protein tyrosine kinase - Promoter+5'UTR from T. kok-saghyz

Background

Motivation

Figure 1: Graphical abstract - from dandelion to natural rubber.

With our project Tarakate, we aim to explore the potential of the Russian dandelion (Taraxacum kok-saghyz) as a sustainable source of natural rubber. This plant, native to Kazakhstan, is unique for its ability to produce significant amounts of high-quality latex in its roots—a trait found in only a few species worldwide (1). Natural rubber is vital due to the global demand of roughly 15 million tons annually (2) and its application in more than 50,000 products (3), ranging from tires to medical supplies. As demand continues to rise, the limitations of traditional rubber sources, such as the rubber tree (Hevea brasiliensis), have become increasingly apparent. The production of rubber from H. brasiliensis has led to significant environmental and economic challenges, including deforestation of approximately four million hectares of rainforest, labor exploitation, and vulnerability to diseases like South American leaf blight (4, 5, 6). These issues, coupled with the geographic constraints of rubber tree cultivation—primarily restricted to tropical regions—underscore the urgent need for alternative rubber sources that can be cultivated in diverse climates and offer greater sustainability.

To harness the potential of T. kok-saghyz as a sustainable source of natural rubber, engineering priorities include optimizing biomass production and morphological traits, such as root architecture and seed size, to maximize yield, improve harvesting efficiency, and enhance overall agricultural practices for easier handling and processing. Besides targeting natural rubber production, there is also a focus on increasing the production of other valuable products like inulin (7), which can be used for the production of biofuel, and various bioactive compounds with potential applications in multiple industries (8). By developing alternative rubber sources like T. kok-saghyz, we aim to mitigate the environmental and economic impacts associated with traditional rubber production and contribute to a more sustainable future.

Lack of Endogenous Regulatory Parts

To successfully implement these engineering efforts, it is essential to have a set of well-characterized regulatory parts, including promoters, 5’ untranslated regions (UTRs), 3’ UTRs, and other genetic sequences that provide precise control over gene expression. These standardized components are fundamental for achieving reliable and predictable outcomes in any synthetic biology project.

With this in mind, we set out to evaluate the current repertoire of regulatory parts used in T. kok-saghyz (TKS). Our review revealed that most constructs developed for TKS rely heavily on a limited selection of regulatory elements, such as the Cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (NOS) terminator.

Part Type	Part Name	Origin	References
Promoter + 5’UTR	35S	Cauliflower mosaic virus	(9, 10, 11, 12, 13, 14)
Promoter + 5’UTR	NOS	Agrobacterium tumefaciens	(9)
Promoter + 5’UTR	PEP16	Hevea brasiliensis	(15)
Promoter + 5’UTR	Ubiquitin4-2	Petroselinum crispum	(16)
Promoter	UBQ1	Arabidopsis thaliana	(10)
Promoter	U6-26	Arabidopsis thaliana	(10)
Promoter	CPTL1	Taraxacum kok-saghyz	(17)
3'UTR	35S	Cauliflower mosaic virus	(9, 10, 11, 12, 13, 14)
3'UTR	NOS	Agrobacterium tumefaciens	(9)
3'UTR	UBQ1	Arabidopssi thaliana	(10)
3'UTR	3A	Pisum sativum	(16)

Table 1: Regulatory elements identified in T. kok-saghyz literature.

To overcome this challenge, our project this year focused on developing a broader collection of regulatory parts specifically tailored for TKS. By increasing the diversity of genetic components, we aim to support more ambitious synthetic biology projects in this promising plant.

Political & Regulatory Aspect

To ensure that engineered plants can be utilized, it is crucial that they adhere to the regulatory standards of the countries in which they will be deployed. In the European Union (EU), regulations surrounding genetically modified organisms (GMOs) are particularly strict and have sparked intense debate. However, a relaxation of these stringent regulations may be on the horizon.
The EU Parliament recently adopted its position for negotiations with member states regarding the proposal on New Genomic Techniques (NGTs). Currently, all plants developed using NGTs are regulated under the same framework as GMOs. However, Members of the European Parliament support the creation of two distinct categories with corresponding regulatory frameworks for NGT plants. NGT plants deemed equivalent to conventionally bred plants (NGT 1) would be exempt from the GMO legislation, while NGT plants that do not meet this criterion (NGT 2) would still be subject to more stringent regulations.

If this proposal is enacted into European law, engineered plants intended for use within the EU must meet the criteria for NGT 1 status. To achieve this, it is crucial to prioritize the use of endogenous regulatory elements over synthetic ones to fulfill the strict requirements outlined in the proposal.

Plant Synthetic Biology

Gene Structure in Plants

Gene structure in plants, like in other eukaryotes, is characterized by a complex organization of coding and non-coding regions. Unlike the typically continuous coding sequences found in prokaryotes, plant genes consist of exons—both protein-coding and non-coding—separated by introns, which are removed during RNA splicing to produce mature mRNA.

Untranslated regions (UTRs) at both ends of the mRNA also play crucial roles. The 5' UTR influences translation initiation, while the 3' UTR affects mRNA stability and lifespan. These regions enable plants to finely tune protein production in response to environmental and developmental cues.

Promoters, located upstream of the coding sequence, are critical for initiating transcription. These regions contain specific sequences, that regulate when, where, and how much a gene is expressed. The terminator region downstream ensures proper transcription termination and mRNA processing.

In contrast to prokaryotes, where genes are often organized in operons and transcribed as polycistronic mRNA, plant genes are transcribed independently. This complexity, including the roles of introns, UTRs, and promoters, is essential for regulating gene activity in processes like development, differentiation, and environmental response.

Figure 2: Schematic representation of gene expression in plants, illustrating the process from transcription to translation. RNA Polymerase II transcribes the DNA into a primary RNA transcript, which then undergoes processing, including 5' capping, 3' end processing, and polyadenylation. The pre-mRNA is spliced to remove introns and produce mature mRNA, which is exported from the nucleus to the cytoplasm. In the cytoplasm, the mRNA is translated into protein and directed to its subcellular location.

Transcription in Plants

In plants, gene activity is regulated at multiple levels, with transcriptional control being the primary and most studied mode of gene expression regulation. Central to this regulatory landscape are promoter architecture, cis-regulatory elements, and enhancer organization.

The promoter is a region of DNA located upstream of the transcription start site (TSS) and serves as the primary site for transcriptional regulation. The promoter architecture is characterized by a complex arrangement of core promoter elements, proximal promoter regions, and distal regulatory sequences, each playing a distinct role in transcription regulation.

Figure 3: Schematic representation of the gene regulatory region highlighting the promoter structure. The promoter spans approximately 2 kb and is divided into three main regions: the distal promoter, which may contain enhancer elements; the proximal promoter, which includes transcription factor binding sites; and the core promoter, which is essential for the initiation of transcription. The transcription start site is indicated by the arrow, with the downstream region representing the gene structure, including exons (yellow blocks) and additional regulatory elements.

The core promoter typically spans the region immediately upstream of the TSS and includes elements such as the TATA box, the Initiator (Inr) element, and the downstream promoter element (DPE). The TATA box is often found at a conserved position around 25-30 base pairs upstream of the TSS and is recognized by the TATA-binding protein (TBP), a subunit of the transcription factor IID (TFIID). The Inr element, located at the TSS, can act independently or in conjunction with the TATA box to facilitate the assembly of the pre-initiation complex (PIC). These core elements are crucial for the precise recruitment of RNA polymerase II and the initiation of transcription.

Beyond the core promoter, the proximal promoter region extends up to a few hundred base pairs upstream and contains binding sites for various transcription factors (TFs). These TFs interact with the basal transcription machinery, modulating the frequency and efficiency of transcription initiation. The binding of TFs to these sites can either activate or repress transcription, depending on the nature of the TFs and the context of their binding.

Distal regions, which may be located thousands of base pairs away from the TSS, also contribute to promoter activity. These regions often contain enhancers, silencers, or insulators, which can exert their regulatory effects through structural interaction with the promoter region, bringing TFs and co-regulators into close proximity with the PIC.

Cis-regulatory elements (CREs) are short, non-coding DNA sequences within promoters that serve as binding sites for transcription factors and other regulatory proteins. CREs are pivotal in defining the specificity of gene expression patterns, enabling plants to finely tune their transcriptional responses.

The specificity and complexity of plant transcription are largely dictated by the combinatorial interactions between TFs and their corresponding CREs. These TFs can act as activators or repressors, depending on the context. For instance, light-responsive elements (LREs) within the promoters of photosynthesis-related genes bind to TFs activated by light, thus ensuring that these genes are expressed in response to light stimuli.

Additionally, stress-responsive elements like the dehydration-responsive element (DRE) and heat shock element (HSE) play crucial roles in enabling plants to rapidly activate stress-related genes when facing environmental challenges. These various layers of regulation highlight the intricate mechanisms of plants to fine-tune gene expression in response to both internal and external signals.

Figure 4: Diversity of Enhancer Organization Models. Shown are a variety of enhancer organization models, showcasing the different ways transcription factors (TFs) can interact with cis-regulatory elements (CREs) on DNA. Alternate Complex Formation - Transcription factors can function both as transcriptional activators and repressors, depending on the cofactors. Transcription Factor Collective - TFs bind cooperatively, using both DNA and proteins as scaffolds. Enhanceosome - TFs bind to CREs in a precise order and orientation. Billboard Model - CREs maintain their composition and position, with regulatory output varying based on TF expression and activity.

Post-Transcriptional Regulation

In plants, post-transcriptional modification is essential for fine-tuning gene expression and enabling adaptive responses to environmental changes. One prominent example is alternative splicing, where a single gene generates multiple mRNA isoforms, each capable of being translated into different protein variants. This process significantly expands proteome diversity, offering plants the flexibility to adapt to various conditions. Additionally, the stability and decay of mRNA, influenced by RNA-binding proteins and microRNAs, further contribute to the regulation of gene expression at the post-transcriptional level.

Translation in Plants

After transcription, a precursor mRNA (pre-mRNA) is synthesized and undergoes splicing, 5' capping, and polyadenylation to form mature mRNA, which is then translated into protein by the ribosome.

The 5' untranslated region (UTR), located between the transcription start site (TSS) and the coding sequence (CDS), plays a key role in regulating gene expression. Although it doesn't code for proteins, the 5' UTR influences translation initiation through elements like upstream open reading frames (uORFs), internal ribosome entry sites (IRES), and secondary structures that either enhance or repress translation.

Beyond translation, the 5' UTR also affects mRNA stability, crucial for determining gene expression levels. Its interactions with RNA-binding proteins or microRNAs (miRNAs) can stabilize or degrade mRNA, thus controlling protein production.

In plants, the 5' UTR can respond to environmental signals, with alternative transcription start sites generating mRNA isoforms of varying lengths. These variations impact translation efficiency, mRNA localization, or stability, adding complexity to gene expression regulation.

====Plant SynBio Standards====

Just as in other engineering disciplines, standardization has been crucial to the growth and innovation in synthetic biology. This is particularly evident in iGEM, where standardization of biological parts is a key aspect, enabling teams to share and build upon each other's work.

In the context of plant synthetic biology, the importance of standardization is amplified due to the inherent complexity of engineering multicellular eukaryotes. The introduction of BioBricks standards marked a significant milestone in microbial engineering, and similar principles are now being applied to plant systems to streamline and enhance their development.

A major advancement in this area is the adoption of the Phytobricks standard, which provides a framework for the standardization of DNA parts used in assembling eukaryotic transcriptional units. This standard is rooted in the widely recognized Golden Gate cloning method, which allows for the efficient and precise assembly of multiple DNA components in a single reaction.

Figure 5: Schematic representation of plant genetic parts designed according to the PhytoBrick standard, recommended for iGEM submission under RFC106. The PhytoBrick standard allows modular assembly of genetic constructs, including Promoter, 5' UTR, Coding Sequence (CDS) and 3' UTR. The figure shows key components and different layout options, emphasizing the standardization and flexibility in designing plant synthetic biology parts.

The Phytobricks standard introduces a common syntax, consisting of 12 fusion sites, which facilitates the consistent assembly of genetic parts across different projects and laboratories. This standardization not only simplifies the DNA assembly process but also fosters collaboration within the scientific community, as it enables researchers to build on each other's work using a shared set of tools and protocols.

Furthermore, the Phytobricks standard is designed to be compatible with other widely adopted systems, such as GoldenBraid2.0 and MoClo, which are extensively utilized in plant research. This compatibility ensures that parts created using the Phytobricks standard can be seamlessly integrated into existing projects and work alongside other standardized components. This integration enhances the modularity and scalability of synthetic biology applications in plants, making it easier to develop complex biological systems in a systematic and efficient manner.

Design

In plant bioengineering, the precise modulation of transgene expression is essential for a variety of applications, including the construction of gene circuits, plant metabolic engineering, and enhancing the efficacy of gene editing efforts. Achieving this control is critical for optimizing gene function and ensuring successful outcomes in these complex engineering tasks. One of the most effective approaches to modulating gene expression has been the development of part toolboxes and libraries.

A notable example of the impact of these regulatory elements is seen in Escherichia coli, where the early development of the Anderson promoter library has played a pivotal role. These promoters have been widely adopted in science, particularly in iGEM, where teams have utilized these parts to improve their experimental designs and metabolic engineering efforts. The success of these promoters in microbial systems highlights the potential for similar approaches in plant systems. However, current efforts in plant bioengineering are hindered by the reliance on a limited set of strong promoters. This limitation restricts the ability to undertake more nuanced and refined genetic engineering endeavors in planta, where a broader range of transcriptional control is necessary.

To overcome this limitation, our strategy focused on characterizing a suite of constitutive promoters for dandelion that provide a broad spectrum of transcriptional levels. The goal was to create a versatile toolkit in the Phytobricks standard, that would allow for precise control over gene expression in T. kok-saghyz. This toolkit was designed not only to include promoters that span a wide dynamic range, but also to integrate other critical regulatory elements, such as 3’ UTRs.

Genetic Context of the Part

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The Dandelion Toolbox

Overview

Part Identifier	Part Type	Nickname	Part Description
BBa_K5088000	Promoter + 5'UTR	P_PTI1	Protein tyrosine kinase - Promoter+5'UTR from T. kok-saghyz
BBa_K5088001	Promoter + 5'UTR	P_RPL28	Large subunit ribosomal protein L28e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088002	Promoter + 5'UTR	P_GSK3B	Glycogen synthase kinase 3 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088003	Promoter + 5'UTR	P_MGRN1	E3 ubiquitin-protein ligase - Promoter+5'UTR from T. kok-saghyz
BBa_K5088004	Promoter + 5'UTR	P_betB	Betaine-aldehyde dehydrogenase - Promoter+5'UTR from T. kok-saghyz
BBa_K5088005	Promoter + 5'UTR	P_pgm	Phosphoglucomutase - Promoter+5'UTR from T. kok-saghyz
BBa_K5088006	Promoter + 5'UTR	P_FKBP4_5	FK506-binding protein 4/5 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088007	Promoter + 5'UTR	P_CLTC	Clathrin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088008	Promoter + 5'UTR	P_RPL31	Large subunit ribosomal protein L31e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088009	Promoter + 5'UTR	P_CUL1	Cullin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088010	Promoter + 5'UTR	P_VPS4	Vacuolar protein-sorting-associated protein 4 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088011	Promoter + 5'UTR	P_EIF2S3	Translation initiation factor 2 subunit 3 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088012	Promoter + 5'UTR	P_Tubulin	Tubulin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088013	Promoter + 5'UTR	P_EIF5A	Translation initiation factor 5A - Promoter+5'UTR from T. kok-saghyz
BBa_K5088050	Inducible Promoter + 5'UTR	P_HSP12.6	HSP12.6 - Heat inducible promoter+5'UTR from T. koksaghyz
BBa_K5088051	Inducible Promoter + 5'UTR	P_HSP23.5	HSP23.5 - Heat inducible promoter+5'UTR from T. koksaghyz
BBa_K5088102	3'UTR	T_PTI1	Protein tyrosine kinase - 3'UTR from T. kok-saghyz
BBa_K5088103	3'UTR	T_RPL28	Large subunit ribosomal protein L28e - 3'UTR from T. kok-saghyz
BBa_K5088104	3'UTR	T_EPS15	Epidermal growth factor receptor substrate 15 - 3'UTR from T. kok-saghyz
BBa_K5088105	3'UTR	T_GSK3B	Glycogen synthase kinase 3 - 3'UTR from T. kok-saghyz
BBa_K5088106	3'UTR	T_MGRN1	E3 ubiquitin-protein ligase - 3'UTR from T. kok-saghyz
BBa_K5088107	3'UTR	T_RPL35A	Large subunit ribosomal protein L35Ae - 3'UTR from T. kok-saghyz
BBa_K5088108	3'UTR	T_betB	Betaine-aldehyde dehydrogenase - 3'UTR from T. kok-saghyz
BBa_K5088109	3'UTR	T_pgm	Phosphoglucomutase - 3'UTR from T. kok-saghyz
BBa_K5088110	3'UTR	T_ATP-synt	ATPase subunit gamma - 3'UTR from T. kok-saghyz
BBa_K5088111	3'UTR	T_EIF3B	Translation initiation factor 3 subunit B - 3'UTR from T. kok-saghyz
BBa_K5088112	3'UTR	T_RPL31	Large subunit ribosomal protein L31e - 3'UTR from T. kok-saghyz
BBa_K5088113	3'UTR	T_TM9SF2_4	Transmembrane 9 superfamily member 2/4 - 3'UTR from T. kok-saghyz
BBa_K5088114	3'UTR	T_CUL1	Cullin - 3'UTR from T. kok-saghyz
BBa_K5088115	3'UTR	T_PSMB6	20S proteasome subunit beta 1 - 3'UTR from T. kok-saghyz
BBa_K5088116	3'UTR	T_RPSA	Small subunit ribosomal protein SAe - 3'UTR from T. kok-saghyz
BBa_K5088117	3'UTR	T_VPS4	Vacuolar protein-sorting-associated protein 4 - 3'UTR from T. kok-saghyz
BBa_K5088118	3'UTR	T_EIF2S3	Translation initiation factor 2 subunit 3 - 3'UTR from T. kok-saghyz

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References

[1] J. B. van Beilen, Y. Poirier, Establishment of new crops for the production of natural rubber. Trends Biotechnol. 25, 522–529 (2007).

[2] MRC, Malaysian Rubber Council (MRC), MRC Official Website. https://www.myrubbercouncil.com/.

[3] Cherian, S., Ryu, S. B., & Cornish, K. (2019). Natural rubber biosynthesis in plants, the rubber transferase complex, and metabolic engineering progress and prospects. In Plant Biotechnology Journal (Vol. 17, Issue 11, pp. 2041–2061). Wiley. https://doi.org/10.1111/pbi.13181

[4] R. Lieberei, South American Leaf Blight of the Rubber Tree (Hevea spp.): New Steps in Plant Domestication using Physiological Features and Molecular Markers. Ann. Bot. 100, 1125–1142 (2007).

[5] T. S. Suryanarayanan, J. L. Azevedo, From forest to plantation: a brief history of the rubber tree. Indian J. Hist. Sci. 58, 74–78 (2023).

[6] Y. Wang, P. M. Hollingsworth, D. Zhai, C. D. West, J. M. H. Green, H. Chen, K. Hurni, Y. Su, E. Warren-Thomas, J. Xu, A. Ahrends, High-resolution maps show that rubber causes substantial deforestation. Nature 623, 340–346 (2023).

[7] D. A. Ramirez-Cadavid, K. Cornish, F. C. Michel, Taraxacum kok-saghyz (TK): compositional analysis of a feedstock for natural rubber and other bioproducts. Ind. Crops Prod. 107, 624–640 (2017).

[8] S. Piccolella, C. Sirignano, S. Pacifico, E. Fantini, L. Daddiego, P. Facella, L. Lopez, O. T. Scafati, F. Panara, D. Rigano, Beyond natural rubber: Taraxacum kok-saghyz and Taraxacum brevicorniculatum as sources of bioactive compounds. Ind. Crops Prod. 195, 116446 (2023).

[9] J. Collins-Silva, A. T. Nural, A. Skaggs, D. Scott, U. Hathwaik, R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish, D. Shintani, Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism. Phytochemistry 79, 46–56 (2012).

[10] X. Cao, H. Xie, M. Song, J. Lu, P. Ma, B. Huang, M. Wang, Y. Tian, F. Chen, J. Peng, Z. Lang, G. Li, J.-K. Zhu, Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture. The Innovation 4, 100345 (2023).

[11] A. Stolze, A. Wanke, N. van Deenen, R. Geyer, D. Prüfer, C. Schulze Gronover, Development of rubber-enriched dandelion varieties by metabolic engineering of the inulin pathway. Plant Biotechnol. J. 15, 740–753 (2017).

[12] N. van Deenen, K. Unland, D. Prüfer, C. Schulze Gronover, Oxidosqualene Cyclase Knock-Down in Latex of Taraxacum koksaghyz Reduces Triterpenes in Roots and Separated Natural Rubber. Molecules 24, 2703 (2019).

[13] S. M. Wolters, V. A. Benninghaus, K.-U. Roelfs, N. van Deenen, R. M. Twyman, D. Prüfer, C. Schulze Gronover, Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis. Front. Plant Sci. 14 (2023).

[14] V. A. Benninghaus, N. van Deenen, B. Müller, K.-U. Roelfs, I. Lassowskat, I. Finkemeier, D. Prüfer, C. Schulze Gronover, Comparative proteome and metabolome analyses of latex-exuding and non-exuding Taraxacum koksaghyz roots provide insights into laticifer biology. J. Exp. Bot. 71, 1278–1293 (2020).

[15] I. Ganesh, S. C. Choi, S. W. Bae, J.-C. Park, S. B. Ryu, Heterologous activation of the Hevea PEP16 promoter in the rubber-producing laticiferous tissues of Taraxacum kok-saghyz. Sci. Rep. 10, 10844 (2020).

[16] A. Wieghaus, D. Prüfer, C. S. Gronover, Loss of function mutation of the Rapid Alkalinization Factor (RALF1)-like peptide in the dandelion Taraxacum koksaghyz entails a high-biomass taproot phenotype. PLOS ONE 14, e0217454 (2019).

[17] E. Niephaus, B. Müller, N. van Deenen, I. Lassowskat, M. Bonin, I. Finkemeier, D. Prüfer, C. Schulze Gronover, Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis-prenyltransferase-like 1 protein. Plant J. 100, 591–609 (2019).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1262
  Illegal XbaI site found at 184
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1262
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1262
  Illegal BglII site found at 409
  Illegal XhoI site found at 436
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1262
  Illegal XbaI site found at 184
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1262
  Illegal XbaI site found at 184
  Illegal AgeI site found at 1761
  
• 1000
  COMPATIBLE WITH RFC[1000]