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.

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.

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.

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]