Part:BBa_K3633006

Coding sequence for 4,5-DODA enzyme

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

Successful production in E.coli BL21(DE3)(T7 promoter)

The team members constructed the plasmid T7-4,5-DODA using Gibson Assembly. They added 0.1 mM IPTG for induction. Again, after 20 h culture at 37℃, 220 rpm, we centrifuged the cells, discarded the LB medium, and resuspended the cell pellet in sterilized water. They then cultured the cells at 20℃, 120 rpm for 102 h, and acquired expected results. They did not test the production in Vibrio natriegens because the bacterial strain we acquired (ATCC 14048) does not have the T7 promoter.

Fig 2. Production of dopaxanthin and indoline-betacyanin in E. coli BL21(DE3) after induction. (A) Production of dopaxanthin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is the absorbance of the bacterial solution at 415 nm. (B) Production of indoline-betacyanin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is the absorbance of the bacterial solution at 525 nm. (C) Production of dopaxanthin from 0-102 h. (D) Production of indoline-betacyanin from 0-102 h.

Detecting the products of interest with HPLC-MS

We were honored to have the opportunity to use an HPLC-MS machine from Shimadzu Enterprise Management (China) Co., Ltd. to isolate the substances in our fermentation broth and detect the pigments we targeted for. We measured the accurate molecular weights of dopaxanthin and indoline-betacyanin and determined that we had synthesized the correct products.

Fig 3. Mass spectrum results of dopaxanthin and indoline-betacyanin.

Hair dye using Indoline-Betacyanin produced by engineering E.coli BL21(DE3)(T7 promoter)

We first incubated the hair in pH=9 Ca(OH)2 at 50℃ for 40 min. Next, we added the indoline-betacyanin bacterial solution and kept at 50℃ for another 40 min. We successfully dyed hair with our synthesized pigment. Notably, the hair color dyed with synthesized indoline-betacyanin was darker than that dyed with standard betacyanins. It is hypothesized that dopaxanthin failed because the color of the bleached hair was too similar to dopaxanthin.

Fig 3. Hair dye results with synthesized pigments including indoline-betacyanin hair dye which is successful, with positive and blank controls.

Sequence & Features

Assembly Compatibility:

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