Part:BBa_K4171024

NPS-tyrP-Ptrc-melA-B0015

Background

This part is for tyrosinase biosynthesis based on BBa_K274001 with production optimization. NPS-tyrP-P_trc-melA-B0015 (BBa_K4171024) is constructed to express MelA (BBa_K274001), the tyrosinase that is essential for melanin production. TyrP (BBa_K2997000) is a transmembrane protein functions as the importer for tyrosine (Tyr). With this composite part constructed, TyrP contributes to the final production of melanin by increasing Tyr consumption.

Fig. 1. Melanin synthesis pathway

Usage

This part is used to produce tyrosinase, an enzyme that catalyzes the reaction from tyrosine to melanin. In our project, we used MelA to produce melanin. After the addition of selenocysteine (Sec) from tRNA^UTuX (BBa_K4171009) and the in vitro pathway, selenomelanin was expected to be produced.

Fig. 2. Selenomelanin synthesis pathway

Characterization

Quantification of TyrP Function

The gene melA from Rhizobium etli was synthesized and placed under the control of the P_trc and double terminator B0015 in vector pSUI, and tyrP was linked to its NPS (BBa_K4171025, BBa_K4171026). BBa_K4171024 was transformed into E. coli DH5α to completed the construction.

Fig. 3. Confirmation of pSUI-NPS-tyrP-P_trc-melA-B0015 (BBa_K4171024) by double digestion. M: Marker; Lane 1: pSUI-NPS-tyrP-P_trc-melA-B0015 (6800 bp); Lane 2: pSUI-NPS-tyrP-P_trc-melA-B0015 without digestion (negative control).

Tyr, the precursor of melanin, serves as an important ingredient in melanin production. With TyrP, the production of melanin was expected to increase due to higher importation of Tyr. To verify this hypothesis, melanin production in both BBa_K274001 and BBa_K4171024 were measured by OD₄₀₀, which proved that TyrP did play an important role in melanin production optimization.

Fig. 4. Relative melanin production of E. coli with and without TyrP.

As the result shows, melanin production increased significantly with the addition of TyrP.

Culturing Condition Adjustments

Besides this improvement, other experiments were also conducted to maximize the production of melanin. First, the bacteria were cultured in various growing conditions to find the best condition for culturing.

Tyrosinase requires the cofactor Cu²⁺ to function properly ^[1]. However, the concentration of cofactor in previous studies for incubation varied. To determine the best concentration of the cofactor, we added Cu²⁺ to the bacteria with MelA overexpression when OD₆₀₀ reached 0.6. The result is shown in Fig. 5 (A).

Furthermore, Since studies have shown that melanin production prefers to occur under 32°C, we compared the efficiency of melanin polymerization under 30°C and 37°C. The result is shown in Fig. 5 (B).

Fig. 5. With the optimization of the Cu²⁺ concentration (A) and temperature (B) for in vivo melanin production in E. coli DH5α.

As Fig. 5 shows, melanin production was better when cultured in 0.5 mM Cu²⁺ and 37°C. WIth the optimization of each parameter, melanin production had been promoted.

Selenomelanin Synthesis and Function Test

Once melanin was prepared, Sec was added and they together polymerized into selenomelanin, which showed the best ability to protect microorganisms from radiation than melanin.

The function of selenomelanin was verified. Selenomelanized bacteria were placed under UV irradiation with the comparison of melanized and non-melanized bacteria as controls to determine if Se coli, the engineered kind of E. coli which produced selenomelanin automatically, was capable to survive under radiation exposure for a longer period.

As Fig. 6 shows, 17.9% of Se coli survived, while only 5% of melanized bacteria and 3.2% of non-melanized bacteria survived respectively. Selenomelanized bacteria (Se coli) is 3.6 times more tolerant to radiation than melanized-bacteria, and 6 times than non-melanized ones.

Fig. 6. Bacteria under exposure to UV-C (A) Survival rate (B) CFU comparison

Reference

[1] Wang Z, Tschirhart T, Schultzhaus Z, et al. Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application. Applied and Environmental Microbiology. 2020;86(5):e02749-19. doi:10.1128/AEM.02749-19

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2786
  Illegal AgeI site found at 338
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2638
  Illegal BsaI.rc site found at 1943