Part:BBa_K4624004

Mature lcc2 laccase from Trametes versicolor

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 710
  Illegal BglII site found at 1142
  Illegal XhoI site found at 742
  Illegal XhoI site found at 1375
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1228
  Illegal NgoMIV site found at 1381
  Illegal AgeI site found at 109
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 174
  

Usage and Biology

It is well known that white-rot basidiomycetes, such as Trametes versicolor, are the most efficient lignin degraders ^[1] and have a great potential for the bioremediation of hazardous wastes (e.g. polycyclic aromatic hydrocarbons, herbicides and synthetic dyestuffs) ^[2]. This ability is due to a system consisting of extracellular ligninolytic enzymes, such as laccase, lignin peroxidase, and manganese-dependent peroxidase ^[3]. Laccase, is a multicopper oxidoreductase, capable of performing non-specific oxidation of a broad range of aromatic substrates with a concomitant four-electron reduction of molecular oxygen to water, offering an advantage over peroxidases that require costly hydrogen peroxide as a co-substrate ^[4]. Taking into account all of the above, iGEM Thessaly 2023 team developed a laccase secretion system in E. coli BL21 (DE3) ^[5] for the degradation of the polymeric, lignin-like phenolic compounds found in Olive oil mill wastewater (e.g. tannins, lignans and catechol melaninic polymers) ^[6], a weakly bio-degradable fraction mainly responsible for the coloration of the by-product ^[7].

Experimental Design and Results

The secretion of the functional laccase from E. coli is essential for our system since it would mean that the poly-phenolic content of Olive oil mill wastewater would be degraded to the maximum. For this reason, the upstream coding sequence of the mature Trametes versicolor lcc2 laccase was fused with four different signal peptides, including its native signal peptide (BBa_K4624400), and then the whole coding sequence was placed under the control of the well-known inducible system AraC/P_BAD (Fig. 1). Finally, to prove the functionality of the secreted laccases we conducted the ABTS assay for the calculation of the enzyme activity.

Figure 1: Schematic representation of the standard level 1 (alpha) construct of the laccase carrying one of the signal peptides (Na.SP, NSP4, PelB and MalE).

At first, the sequences for the signal peptides and the laccase were domesticated to the Golden Braid standards ^[8]. The domestication involved removing internal restriction sites that were not compatible with the GoldenBraid cloning method and the addition of appropriate 3' and 5' 4-nt overhangs (GoldenBraid Domesticator tool). Once designed, the domesticated sequences were ordered to be synthesized by IDT.

Following the standard protocol for digestion-ligation reaction, the domesticated sequences were inserted into a pUPD2 part domestication vector to create level 0 constructs, which were verified through restriction-digestion reaction and gel electrophoresis (Fig. 2, 3 and 4). The resulting level 0 constructs were combined with the ones of the AraC/P_BAD (BBa_I0500)and B0015 double terminator (BBa_J428092)for the creation of the complete level 1 (alpha) constructs (Fig. 1). The inserts were once again confirmed through restriction-digestion reactions and electrophoresis (Fig. 5).

Figure 2: Diagnostic digestion of (1) pUPD2_NSP4, (2) pUPD2_PelB and (3) pUPD2_Na.SP with EcoRV and NotI, expected bands (bp): (1) 1143 903 and 122, (2) 1143, 903 and 128 and (3) 1143, 903 and 125. Lane 4: pUPD2 (no insert).

Figure 3: Diagnostic digestion of pUPD2_MalE with EcoRI and EcoRV, expected bands (bp): 1290 and 896. Lane 2: pUPD2 (no insert).

Figure 4: Diagnostic digestion of pUPD2_laccase(B4+B5) with EcoRV and NotI, expected bands (bp): 1563, 1143 and 903. Lane 2: pUPD2 (no insert).

Figure 5: Diagnostic digestion of (1) pDGB3α1_pBAD-Na.SP-laccase-rrnB T1/T7TE, (2) pDGB3α1_pBAD-NSP4-laccase-rrnB T1/T7TE, (3) pDGB3α1_pBAD-PelB-laccase-rrnB T1/T7TE and (4) pDGB3α1_pBAD-MalE-laccase-rrnB T1/T7TE with EcoRV and BsaHI, expected bands (bp): (1) 2327, 1628, 1291, 1290, 807, 610, 554, 423, 185, 163, 81 and 58, (2) 2327, 1628, 1291, 1290, 807, 607, 554, 423, 185, 163, 81 and 58 (3) 2327, 1628, 1291, 1290, 807, 613, 554, 423, 185, 163, 81 and 58 and (4) 2327, 1628, 1291, 1290, 807, 625, 554, 423, 185, 163, 81 and 58. Lane 5: pDGB3α1 (no insert).

Having all 4 constructs ready, we were able to begin with the evaluation of laccase activity. For this experiment, we followed the ABTS assay designed to assess laccase activity in the culture of the white-rot fungus Trametes versicolor. The basis of this assay involves the oxidation of the non phenolic dye ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by laccase to the radical cation ABTS*+, a soluble chromogen that is green in color and can be determined spectrophotometrically at 425 nm ^[9].

Once all the needed devices were transformed into E. coli BL21 (DE3) cells, liquid cultures were prepared and incubated O/N at 37^oC and 210 rpm. The next day, these cultures were retrieved and used in order to prepare final cultures with the same starting OD₆₀₀ values. Addition of L-arabinose to a final concentration of 100mM was followed and the cultures were incubated at 30^oC and 160 rpm for 6h. So, after 6h incubation we began the procedure by adding 1.2 ml of sodium tartrate 0.1 M (pH of 4.5 adjusted with NaOH addition) in a cuvette. Next, 0.6 ml of the bacterial culture was added, the cuvette was placed in the spectrometer and the absorbance was set to zero. To initiate the reaction, 0.4 ml of ABTS solution 3 mM were added in the cuvette and pipetted three to four times to mix. The instrument was set back to zero again and the timer started. Absorbance measurements at 425 nm were taken every 20 seconds for 2 minutes. The above procedure was repeated 3 times to get as reliable results as possible. The results for each construct are summarized in Fig. 6. Non-transformed E. coli BL21 (DE3) cells were used as negative control.

Figure 6: Absorbance measurements at 425 nm of the 4 constructs designed for laccase activity calculation (Fig. 1). Measurements were taken every 20 sec after the addition of the ABTS reagent for 2 minutes. Non-transformed E. coli BL21 (DE3) cells were used as (-) control.

Once the measurements were collected, we calculated the enzyme activity based on the absorbance changes using the formula:

U / L = (ΔΑ x Vt) / (Δt x ε x Vs)

U enzyme activity umol min^-1 L^-1, ΔΑ (final absorbance - initial absorbance) the maximum absorbance difference measured at 2 min, Vt total reaction volume (ml), Δt elapsed time between two measurements (min), ε molar extinction coefficient (L mol^-1 cm^-1) = 36 L mol^-1 cm^-1, Vs sample volume (ml). The enzyme activity values for each sample are depicted in Fig. 7.

Figure 7: Laccase activity of the four different constructs estimated with ABTS assay, after 6h incubation with 1,5% arabinose. Non-transformed E. coli BL21 (DE3) cells were used as (-) control.

As we can see from the final results (Fig. 7), the NSP4 signal peptide exhibited the best results, compared to the rest signal peptide and the (-) control, indicating the NSP4 as the ideal selection for our system.

References

1. Kirk TK, Farrell RL. Enzymatic "combustion": the microbial degradation of lignin. Annu Rev Microbiol. 1987;41:465-505. doi: 10.1146/annurev.mi.41.100187.002341. PMID: 3318677.

2. Arregui L, Ayala M, Gómez-Gil X, Gutiérrez-Soto G, Hernández-Luna CE, Herrera de Los Santos M, Levin L, Rojo-Domínguez A, Romero-Martínez D, Saparrat MCN, Trujillo-Roldán MA, Valdez-Cruz NA. Laccases: structure, function, and potential application in water bioremediation. Microb Cell Fact. 2019 Nov 14;18(1):200. doi: 10.1186/s12934-019-1248-0. PMID: 31727078; PMCID: PMC6854816.

3. Hatakka, A. (1994). Lignin-modifying enzymes from selected white-rot fungi: Production and role from in lignin degradation. FEMS Microbiology Reviews, 13(2–3), 125–135.

4. Solomon EI, Sundaram UM, Machonkin TE. Multicopper Oxidases and Oxygenases. Chem Rev. 1996 Nov 7;96(7):2563-2606. doi: 10.1021/cr950046o. PMID: 11848837.

5. Mo Y, Lao HI, Au SW, Li IC, Hu J, Yuen HM, Cheong WM, Lo OLI, Seak LCU. Expression, secretion and functional characterization of three laccases in E. coli. Synth Syst Biotechnol. 2021 Dec 7;7(1):474-480. doi: 10.1016/j.synbio.2021.12.002. PMID: 34938906; PMCID: PMC8665402.

6. Shabir S, Ilyas N, Saeed M, Bibi F, Sayyed RZ, Almalki WH. Treatment technologies for olive mill wastewater with impacts on plants. Environ Res. 2023 Jan 1;216(Pt 3):114399. doi: 10.1016/j.envres.2022.114399. Epub 2022 Oct 27. PMID: 36309216.

7. Morillo JA, Antizar-Ladislao B, Monteoliva-Sánchez M, Ramos-Cormenzana A, Russell NJ. Bioremediation and biovalorisation of olive-mill wastes. Appl Microbiol Biotechnol. 2009 Feb;82(1):25-39. doi: 10.1007/s00253-008-1801-y. Epub 2008 Dec 10. PMID: 19082586.

8. Sarrion-Perdigones A, Vazquez-Vilar M, Palací J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013 Jul;162(3):1618-31. doi: 10.1104/pp.113.217661. PMID: 23669743; PMCID: PMC3707536.

9. Johannes C, Majcherczyk A, Hüttermann A. Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds. Appl Microbiol Biotechnol. 1996 Oct;46(3):313-7. doi: 10.1007/s002530050823. PMID: 8933845.