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Metabolic pathways

After reading numerous articles, we chose four pigments, melanin, indigo, dopaxanthin, and indoline-betacyanin, which are black, blue, yellow, and pink-reddish, respectively. Since they resemble the makeup of a color printer (CYMK), we envisaged mixing these pigments together to achieve a wider color range. The following are these pigments' metabolic pathways.

Melanin

L-Tyrosine oxidizes to form L_DOPA, which then transforms into dopaquinone (DQ). DQ undergoes a complicated process and finally polymerizes into eumelanin, the type of melanin present in black hair. All three steps can be catalyzed by tyrosinase (Wang et al., 2019).

Indigo

Tryptophan first transforms into indole in a reaction catalyzed by trytophanase. Next, catalyzed by FMO, indole changes to indoxyl. Indoxyl spontaneously oxidizes to form indigo ("Team:Berkeley/Project/Introduction - 2013.igem.org", 2020).

Betalains (Dopaxanthin and Indoline-betacyanin)

When L-DOPA is added as the substrate, DOPA 4,5-extradiol dioxygenase (4,5-DODA) catalyzes its transformation into 4,5-seco-DOPA, which will spontaneously cyclize to form betalamic acid. When different precursors are added, betalamic acid forms different betalains (Guerrero‐Rubio, López‐Llorca, Henarejos‐Escudero, García‐Carmona & Gandía‐Herrero, 2019). Considering the availability of these precursors and the color combinations, we choose dopaxanthin (synthesized from L-DOPA) and indoline-betacyanin (synthesized from indoline) as our pigments of interest.

Faster synthesis of pigments precursors by Vibrio natriegens

Previous studies have indicated that Vibrio natriegens can double within less than 10 minutes under optimal growth conditions (Eagon, 1962). Due to its fast growth rate and being non-pathogenic, V. natriegens has the potential to be used to accelerate genetic engineering, molecular biology research, and industrial production.

Tschirhart et al. (2019) tried different combinations of antibiotic resistance markers, promoters, ribosome targets, reporter genes, terminators, decomposition markers, replication start sequences, and plasmid skeletons in V. natriegens. They showed that the plasmid copy number, plasmid maintenance rate (in absence of antibiotics), and expression yield of V. natriegens were similar to that of E. coli when other factors were kept constant. Therefore, V. natriegens may be tested as an alternative to E. coli, with the hope of improving the rate of production of our pigments in interest.

Potential Application of Laccase in Hair-Dyeing

Inspired by the principle of synthetic hair dyes, which combine pigment precursors with oxidants, we proposed combining bacterial laccase, a milder oxidase, with natural pigment precursors to dye hair.

Synthetic hair dyes are mainly composed of oxidants and pigment precursors. When the two are mixed and applied to the hair, under suitable conditions, the pigment precursor is oxidized and polymerized to form the pigment that gives the hair the targeted color. Laccase may be an alternative to the commonly-used hydrogen peroxide to oxidize the pigment precursors.

Laccase has been shown to catalyze the oxidation of a variety of pigment precursors to form a variety of colors, including red, orange, yellow, and black (Jeon et al., 2010). Among them, gallic acid was mixed with syringic acid to form brown after oxidization, catechin was mixed with catechol to form black, and ferulic acid was mixed with syringic acid to form wine red (Jeon et al., 2010). Since pigment precursors are smaller molecules, we hypothesized that this method will produce better results than directly using the pigments.

Bacterial laccase from Bacillus sp. has been heterogeneously synthesized with E. coli (Mohammadian, Fathi-Roudsari, Mollania, Badoei-Dalfard & Khajeh, 2010). The genetic sequence coding for laccase from Bacillus sp. is open-source in the NCBI database.

Dopamine is a common pigment precursor. When oxidized, it can polymerize to form a black color. Therefore, we proposed adding biosynthesized dopamine to the list of natural pigment precursors. In terms of the metabolic pathway, tyrosine is first oxidized to L-DOPA through catalyzation of HpaB and HpaC enzymes. L-DOPA is further oxidized to dopamine when catalyzed by DDC (Das, Verma & Mukherjee, 2017).

References

1. Wang, Z., Tschirhart, T., Schultzhaus, Z., Kelly, E., Chen, A., & Oh, E. et al. (2019). Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application. Applied And Environmental Microbiology, 86(5). doi: 10.1128/aem.02749-19

2. Team:Berkeley/Project/Introduction - 2013.igem.org. (2020). Retrieved June 2020, from http://2013.igem.org/Team:Berkeley/Project/Introduction

3. Guerrero‐Rubio, M., López‐Llorca, R., Henarejos‐Escudero, P., García‐Carmona, F., & Gandía‐Herrero, F. (2019). Scaled‐up biotechnological production of individual betalains in a microbial system. Microbial Biotechnology, 12(5), 993-1002. doi: 10.1111/1751-7915.13452

4. Eagon, R. (1962). PSEUDOMONAS NATRIEGENS, A MARINE BACTERIUM WITH A GENERATION TIME OF LESS THAN 10 MINUTES. Journal Of Bacteriology, 83(4), 736-737. doi: 10.1128/jb.83.4.736-737.1962

5. Tschirhart, T., Shukla, V., Kelly, E., Schultzhaus, Z., NewRingeisen, E., & Erickson, J. et al. (2019). Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens. ACS Synthetic Biology, 8(9), 2069-2079. doi: 10.1021/acssynbio.9b00176

6. Jeon, J., Kim, E., Murugesan, K., Park, H., Kim, Y., & Kwon, J. et al. (2010). Laccase-catalysed polymeric dye synthesis from plant-derived phenols for potential application in hair dyeing: Enzymatic colourations driven by homo- or hetero-polymer synthesis. Microbial Biotechnology, 3(3), 324-335. doi: 10.1111/j.1751-7915.2009.00153.x

7. Mohammadian, M., Fathi-Roudsari, M., Mollania, N., Badoei-Dalfard, A., & Khajeh, K. (2010). Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization. Journal Of Industrial Microbiology & Biotechnology, 37(8), 863-869. doi: 10.1007/s10295-010-0734-5

8. Das, A., Verma, A., & Mukherjee, K. (2017). Synthesis of dopamine in E. coli using plasmid-based expression system and its marked effect on host growth profiles. Preparative Biochemistry & Biotechnology, 47(8), 754-760. doi: 10.1080/10826068.2017.1320291

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