Part:BBa_K3633011

A composite part to express 4,5-DODA, responsible for production of betalains

Description

Fig 1. Betalains Synthesis Pathway

Betalains are water-soluble nitrogen-containing pigments that are subdivided into red-violet betacyanins and yellow-orange betaxanthins. Due to glycosylation and acylation, betalains exhibit a huge structural diversity. Betanin (betanidin-5-O--glucoside) is the most common betacyanin in the plant kingdom.

The biosynthesis of betalains in plants excludes that of anthocyanins. During the biosynthesis of betalains in the cytoplasm, three enzymes are involved: Tyrosinase, 4,5-DOPA-extradioldioxygenase, and betanidin-glucosyltransferase. The amino acid L-tyrosine, which is enzymatically formed over the shikimate pathway from arogenic acid, is the precursor for L-DOPA's biosynthesis. Tyrosine is hydroxylated through the enzyme tyrosinase to DOPA (I) formed to betalamic acid or cyclo-DOPA. The biosynthesis of betalamic acid, which is the basic structure of betalains as follows: 4,5-DOPA-extradiol dioxygenase opens the cyclic ring of L-DOPA between carbons 4 and 5, thus producing 4,5-seco-DOPA (II). This intermediate product occurs naturally. Due to spontaneous intramolecular condensation between the amine and the aldehyde groups, 4,5-seco-DOPA betalamic acid is formed.

To produce the betalains, Shanghai_SFLS_SPBS built the biobrick with 4,5-DODA and two kinds of promoters and added the substrate L-Dopa and 0.1mM IPTG to induce the promoter. The 4,5-seco-DOPA will spontaneously convert into Betalamic acid with the help of ascorbic acid (Vitamin C). And Dopaxanthin/Indoline-Betacyanin will be subsequently synthesized by adding the substrate of L-DOPA/Indoline. The 4,5-DODA was successfully expressed in E.coli BL21(DE3). The two kinds of pigments were produced, and the hair-dye process with indoline-betacyanin was successful.

Experiments & Results

Although the enzyme was successfully expressed, the betalains were oxidized to form black solutions.

Sequence & Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. M. Guerrero‐Rubio, R. López‐Llorca, P. Henarejos‐Escudero, F. García‐Carmona and F. Gandía‐Herrero, "Scaled‐up biotechnological production of individual betalains in a microbial system", Microbial Biotechnology, vol. 12, no. 5, pp. 993-1002, 2019. Available: 10.1111/1751-7915.13452.

2. "Betalaine", De.wikipedia.org, 2020. [Online]. Available: https://de.wikipedia.org/wiki/Betalaine. [Accessed: Jun-2020].

3. G. Polturak and A. Aharoni, "“La Vie en Rose”: Biosynthesis, Sources, and Applications of Betalain Pigments", Molecular Plant, vol. 11, no. 1, pp. 7-22, 2018. Available: 10.1016/j.molp.2017.10.008.

4. P. Grewal, C. Modavi, Z. Russ, N. Harris and J. Dueber, "Bioproduction of a betalain color palette in Saccharomyces cerevisiae", Metabolic Engineering, vol. 45, pp. 180-188, 2018. Available: 10.1016/j.ymben.2017.12.008.