Revision as of 19:34, 21 October 2021

his-tag-SpyTag-MnP

Manganese Peroxidase (MnP) is a highly glycosylated lignin peroxidase with heme. This is the version with SpyTag. In order to constructed our multi-enzyme complex, we introduced the SpyTag/SpyCatcher system. We added SpyTag to the N-terminus of each protein through 10 × ELP, which is a oligopeptide linker that does not affect the function of the protein it attaches to, as described in the literature. So this part can be combined with dCas9-SpyCatcher through covalent isopeptide action^[1], thereby being immobilized on dsDNA. His-tag was added to purify the protein. We used BBa_K3853051 to construct the expression system to express and purify the protein.

Biology

Manganese peroxidase (MnP) is a highly glycosylated, heme-containing^[2] lignin peroxidase produced by the white-rot fungus Phanerochaete chrysosporium. MnP catalyzes the hydrogen peroxide-dependent oxidation of Mn²⁺ to Mn³⁺, which is released from the enzyme in complex with oxalate, enabling the oxalate-Mn³⁺ complex to serve as a diffusible redox mediator capable of oxidizing lignin or other refractory chemicals^[2,3] (Fig. 1).

The SpyCatcher/SpyTag system is a bio-coupling technology connected by isopeptide bonds, which is derived from the modification of immunoglobulin like collagen adhesin domain (CnaB2)^[4]. The two can spontaneously form isopeptide bond and bond stably^[5].

Fig. 1 Catalytic cycle of manganese peroxidase.

Usage

The SpyTag was fused to the N-terminus of MnP. The fusion protein could combined with dCas9-SpyCatcher (which is producted by BBa_K3853055 ) for multi-enzyme complex assembly. we obtained the composite part BBa_K3853051(Fig. 2) and transformed the constructed plasmid into Pichia pastoris GS115 to verify its expression. The positive clones were cultivated.

Fig. 2 Gene circuit of SpyTag-MnP.

For MnP itself, fusion with SpyTag will not affect its function, which means that SpyTag-MnP can still exert the powerful ability of degrade pollutants like MnP. In our project, SpyTag-MnP was selected due to its polyethylene (PE) degradation ability and assemblability. It can exert PE degradation ability in the presence of Tween 80 and H₂O₂, and significant changes on PE films can be observed within 10 days. For SpyTag-MnP itself, assembled with dCas9-SpyCather will not affect its function neither, which means that it could be assembled on double-stranded DNA together with other auxiliary enzymes like H₂O₂ producing enzyme and hydrophobin. This indicates that we could adjust the spatial distance and ratio of each enzyme by altering the sequence of double-stranded DNA and corresponding single guide RNA. Besides, SpyTag-MnP and its assembly have high organic solvent stability, which is conducive to its industrial application. Both SpyTag-MnP and its assembly could work normally under the condition of pH 5-6 and lower than 37℃. In order to broaden the application of SpyTag-MnP in industry, we had gained some reliable data through molecular modeling and the potential mutants with better stability and activity were obtained. It is of great significance for further research.

Characterization<h1>

1. Identification

After receiving the pPIC9K plasmid carrying BBa_K3853008, it was linearized and electrotransformed into Pichia pastoris strain GS115. Colony PCR (using primer α-factor and 3'AOX1) was used to verify whether the plasmid was successfully transferred into the yeast genome. The experimental results were shown in Fig. 3, and the corresponding band underwent sequencing verification (see File 1).

Fig. 3 Gel electrophoresis result of colony PCR to detect the insertion of BBa_K3853008 into Pichia pastoris strain GS115. Control refers to the wild-type Pichia pastoris strain GS115 without electrotransformation. A target band around 1325 bp should appear to indicate a successful construction, which was shown as marked.

2. Proof of the expression

We used AOX1 promoter to express SpyTag-MnP in Pichia pastoris strain GS115 in our composite part BBa_K3853051. Then we used sulfate salting out method to get purified SpyTag-MnP. We set a series of concentration gradients of ammonium sulfate solution to determine the best salting-out concentration and our target bands were observed through SDS-PAGE (Fig. 4).

For assaying the mRNA expression of SpyTag-MnP, qPCRs were performed. We did data analysis using a variation of the Livak method. To determine the relative expression of SpyTag-MnP vs. reference gene ACT1, total RNA was prepared from an equal volum of yeast solution. In Fig. 5, SpyTag-MnP reached its peak to a fold difference of 0.21 after 2% methanol inducing for 36 h, and kept stable after 48 h. The C_T values for the SpyTag-MnP and the reference gene ACT1 were then used to calculate the fold difference with the following equation:

Fig. 4 SDS-PAGE analysis of SpyTag-MnP after ammonium sulfide salting out. Lane 1: SpyTag-MnP salting out with 40% (NH₄)₂SO₄ ; Lane 2: SpyTag-MnP salting out with 50% (NH₄)₂SO₄ ; Lane 3: SpyTag-MnP salting out with 60% (NH₄)₂SO₄ ; Lane 4: SpyTag-MnP salting out with 70% (NH₄)₂SO₄ . Control refers to the supernatant of wild-type Pichia pastoris strain GS115 without plasmid transfer. We finally chose 60% ammonium sulfate as our salting out concentration.

Fig. 5 qPCR results of SpyTag-MnP using the relative quantitative method. Microbial samples were taken every 24 hours and ACT1 was applied as the reference gene. This result indicated that the SpyTag-MnP expression reached the maximum after 72 h of fermentation in buffered complex methanol medium (BMMY) medium.

3. MnP activity

MnP activity of SpyTag-MnP was measured by monitoring the oxidation of 2,6-dimethyloxyphenol (2,6-DMP) at 469 nm^[6]. H₂O₂ concentration were determined using ε₂₄₀ = 43.6 M^-1 cm^-1. The reaction mixtures contained 0.4 mM MnSO₄, 50 mM sodium malonate (pH 4.5), and 1 mM 2, 6-DMP. For a 96-well plate, 140 μl of the above reaction mixtures and 20 μl enzyme solution were mixed uniformly in advance and then 40 μl 0.1 mM H₂O₂ were added to initiate reaction. The concentration of 2, 6-DMP's oxidation products, 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone, were determined using ε₄₆₉ = 49.6 mM^-1 cm^-1. One unit (U) of MnP activity is defined as the amount of enzyme required to convert 1 μM 2, 6-DMP to 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone in 1 minute.

As shown in Fig. 6A, the absorbance of the reaction system with SpyTag-MnP continued to rise within 1 min, while the absorbance of the control group (without enzyme) did not change. Through UV-visible spectrum of the reaction system after 1 min, the characteristic absorption at 469 nm was observed (Fig. 6B). Besides, SpyTag-MnP showed the same characteristic as MnP and the enzyme activity of the former exhibited a slightly higher profile than the latter, which means that SpyTag-MnP retained the functions of the original MnP and had the potential to surpass the latter (Fig. 6).

Fig. 6 The detection of 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone. Control group refers to the reaction system without enzyme. A: The absorbance change at 469 nm in the reaction system within 1 min. B: UV-visible spectrum of the reaction system after 1 min.

4. Thermolstability of SpyTag-MnP

To evaluate whether SpyTag-MnP had sufficient thermal stability to support our application, the purified SpyTag-MnP were incubated in 20 mM sodium malonate buffer (pH 5.5)^[7] with 100 mM NaCl at different temperature for 6 h and the residual enzyme activity were measured and calculated every 2 h. The relative enzyme activity under different temperatures were calculated with the following equation:

As shown in Fig. 7, relative enzyme activity of SpyTag-MnP under different incubation temperature displayed distinct characteristics. The enzyme remained steady when the temperature was below 37℃. However, when the temperature exceeded 50℃, a sharp decline of enzyme activity within 2 h could be observed, and it gradually decreased in the following 4 hours. This result indicated that SpyTag-MnP should better be used lower than 37℃.

Fig. 7 Effect of temperature on the stability of SpyTag-MnP. The initial MnP activity before incubation was set as 100%.

5. pH stability of SpyTag-MnP

To evaluate whether SpyTag-MnP had sufficient pH stability to support our application, the purified SpyTag-MnP were incubated in 20 mM sodium malonate buffer with 100 mM NaCl under pH 3-7 for 12 h at room temperature^[7]. The relative enzyme activity at different pH conditions were calculated with the following equation:

As shown in Fig. 8, SpyTag-MnP played a poorer performance at low pH range (pH 3-4) and showed higher stability at high pH range (pH 5-6), which indicated that SpyTag-MnP would perform better under higher pH conditions (pH 5-6).

Fig. 8 Effect of pH on the stability of SpyTag-MnP after 12 h incubation. The initial MnP activity before incubation was set as 100%.

6. Organic Solvents Stability of SpyTag-MnP

To evaluate whether SpyTag-MnP could be used in organic solvents, we conducted an organic solvents stability test on SpyTag-MnP. The purified SpyTag-MnP were incubated in methanol and ethanol (10-30%) for 12 h at the room temperature^[7], respectively. The incubation process was held in 20 mM sodium malonate buffer (pH 5.5) with 100 mM NaCl and the residual enzyme activity were measured and calculated after 12 h. The relative enzyme activity of different organic solvent at distinct concentrations were calculated with the following equation:

As shown in Fig. 9, SpyTag-MnP played a good performance in almost all concentration gradient we set both in methanol and ethanol. However, when the concentration of ethanol reached to 30%, its stability declined. This result may reflect that SpyTag-MnP could withstand low-concentration organic solvent environments, while functioning normally. This was conducive to the industrial application of SpyTag-MnP, as both methanol and ethanol were solvents commonly used in industry.

Fig. 9 Effect of different concentrations of different organic solvents on the stability of SpyTag-MnP after 12 h incubation. The MnP activity without adding any organic solvent was set to 100% as the control.

7. Ability of polyethylene degradation

To analysis the polyethylene degradation ability of SpyTag-MnP, corresponding experiments were designed. We cut the PE film from polyethylene gloves to a size of 0.5 cm × 0.5 cm, washed it with 75% ethanol , and dried at 37°C for 2 h. All PE films were divided into two groups: one group were immersed in the degradation system (200 μl), containing 50 mM sodium malonate (pH 6.0), 0.2 mM MnSO₄, 0.1 mM H₂O₂, 150 mM NaCl, 0.1% Tween 80 and 1 U SpyTag-MnP^[8], while the other group didn't treated. Then, the two groups were incubated in a shaker at 37°C and 200 rpm for 10 days. After 10 days, all the PE films were washed with 1% SDS, 0.1 M NaOH, ddH₂O and 75% ethanol successively.

We next used multiple techniques to further verify the PE degradation ability of SpyTag-MnP. First, we used Fourier Transform Infrared (FTIR) imaging to analyze the changes of surface chemical components and functional groups. In SpyTag-MnP treated PE, FTIR spectra showed two distinct peaks (Fig. 10). One peak was observed in the vicinity of 1636 cm⁻¹, indicating carbonyl bonds (-C=O-), while the other peak was observed at a wave number of 3400 cm⁻¹ and was attributed to hydroxyl groups^[9] (Fig. 10). Overall, compared with the untreated PE, our FTIR spectra data suggested an oxidation reaction of PE films happened in SpyTag-MnP treated PE, including the formation of hydroxyl groups and carbonyl bonds.

We then monitored the surface structure changes of the two groups by Scanning Electron Microscopy (SEM). As shown in Fig. 11, obvious fragments appeared in SpyTag-MnP treated PE, while the untreated PE seemed to have no change.

We eventually analyzed molecular weight distribution (MWD) changes using High Temperature Gel Permeation Chromatography (HT-GPC). We found the MWD of SpyTag-MnP treated PE film showed molecular weights decreasing from xxxx Da to 298046 Da (Fig. 12C, D). And not only the peak molecular weight response value had decreased, but the proportion of hydrocarbon chain with a molecular weight of less than 10000 Da had declined from 1.37% to 0.70% after SpyTag-MnP treated (Fig. 12A, B).

In summary, our SpyTag-MnP has good PE degradation ability.

Fig. 10 FTIR analysis of untreated PE films and PE films treated by SpyTag-MnP after 10 days incubation.

Fig. 11 SEM observation of untreated PE films and PE films treated by SpyTag-MnP after 10 days incubation. A-D: SEM observation of PE films treated by SpyTag-MnP after 10 days incubation. E-H: SEM observation of untreated PE films after 10 days.

Fig. 12 HT-GPC analysis of untreated PE films and PE films treated by SpyTag-MnP after 10 days incubation. A: HT-GPC spectrum of untreated PE. B: HT-GPC spectrum of SpyTag-MnP treated PE. C: HT-GPC calculus curve graph of untreated PE. D: HT-GPC calculus curve graph of SpyTag-MnP treated PE.

8. Assemblability of SpyTag-MnP

For assembling the dCas9-SpyCather/SpyTag-MnP complex, SpyTag-MnP was mixed with dCas9-SpyCather in a ratio of 1 : 1 and allowed to conjugate for 1 h at 37℃^[10]. As shown in Fig. 13, the band of the complex appeared, which was higher than that of dCas9-SpyCather and the original SpyTag-MnP band had disappeared. Then we compared the difference in MnP activity between SpyTag-MnP and the complex, and, as shown in Fig. 14, there was no significant change. This result suggested that the assembly of SpyTag-MnP and dCas9-SpyCather will not affect the enzyme activity.

Fig. 13 SDS-PAGE showing the conjugation of SpyTag-MnP to dCas9-SpyCatcher. Lane 1: SpyTag-MnP (0.3 μM); Lane 2: dCas9-SpyCatcher (0.3 μM); Lane 3: SpyTag-MnP (0.3 μM) mixed with dCas9-SpyCatcher (0.3 μM). Upon mixing the two components, the upward shift in the band corresponding to dCas9-SpyCatcher as well as the disappearance of the band corresponding to SpyTag-MnP were observed, indicating successful conjugation. Note that the conjugation is unaffected by the SDS-PAGE conditions due to covalent isopeptide bond formation.

Fig. 14 Comparision of MnP activity between SpyTag-MnP and dCas9-SpyCather/SpyTag-MnP complex. Complex refers to the dCas9-SpyCather/SpyTag-MnP complex. p > 0.05.

9. Thermostability of dCas9-SpyCather/SpyTag-MnP complex

We used the above method to test whether assembly would affect thrermal stability of SpyTag-MnP and as shown in Fig. 15, the relative enzyme activity of the complex would decline gradually after 6 h incubation in all temperture we set. However, compared with unassembled SpyTag-MnP, it shown an improve of thermostability at mild temperture (below 60 ℃) (Fig. 16).

Fig. 15 Thermal stability of dCas9-SpyCather/SpyTag-MnP complex. The initial MnP activity before incubation was set as 100%.

Fig. 16 Effect of temperature on the stability of the complex and SpyTag-MnP after 6 h incubation. The initial MnP activity before incubation was set as 100%. The complex refers to dCas9-SpyCather/SpyTag-MnP complex. ^*P < 0.05, ^**P < 0.01.

10. pH stability of dCas9-SpyCather/SpyTag-MnP complex

We used the mentioned method to test whether assembly would affect pH stability of SpyTag-MnP and as shown in Fig. 17, it didn't perform well at low pH range (pH 3-5) as both the complex and SpyTag-MnP had precipitated (Fig. 17), which probably because the 100 mM NaCl was not enough for the complex as its molecular weight became about 5 times than the unassembled SpyTag-MnP. However, it would perform better at high pH range (pH 6-7), although its relative enzyme activity was lower than SpyTag-MnP at pH 7.

Fig. 17 Effect of pH on the stability of the complex and SpyTag-MnP after 12 h incubation. The initial MnP activity before incubation was set as 100%. The complex refers to dCas9-SpyCather/SpyTag-MnP complex. ^*P < 0.05.

11. Organic solvents stability of dCas9-SpyCather/SpyTag-MnP complex

As for organic solvent stability, we would be happy to say that our assembly could tolerate methanol and ethanol while concentration was less than 30% (Fig. 18). This may mean that our assemblies would more adaptable to industrial environments. Besides, its organic solvent stability was significantly higher than the unassembled SpyTag-MnP (Fig. 18).

Fig. 18 Effect of different concentrations of different organic solvents on the stability of the complex and SpyTag-MnP after 12 h incubation. The MnP activity without adding any organic solvent was set to 100% as the control. The complex refers to dCas9-SpyCather/SpyTag-MnP complex. A: The effect of different concentrations of methanol on MnP activity. B: The effect of different concentrations of ethanol on MnP activity. ^*P < 0.05, ^***P < 0.001.

References

[1] Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chemical communications (Cambridge, England) 56, 4950-4953, doi:10.1039/d0cc01174f (2020).

[2] Martínez, A. T. et al. Oxidoreductases on their way to industrial biotransformations. Biotechnol Adv 35, 815-831, doi:10.1016/j.biotechadv.2017.06.003 (2017).

[3] Sáez-Jiménez, V. et al. Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY. J Biol Chem 290, 23201-23213, doi:10.1074/jbc.M115.665919 (2015).

[4] Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697, doi:10.1073/pnas.1115485109 (2012).

[5] Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Current opinion in chemical biology 29, 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).

[6] Wariishi, H., Valli, K. & Gold, M. H. Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. The Journal of biological chemistry 267, 23688-23695 (1992).

[7] Qin, X., Zhang, J., Zhang, X. & Yang, Y. Induction, purification and characterization of a novel manganese peroxidase from Irpex lacteus CD2 and its application in the decolorization of different types of dye. PLoS One 9, e113282, doi:10.1371/journal.pone.0113282 (2014).

[8] Iiyoshi, Tsutsumi & Nishida. Polyethylene degradation by lignin-degrading fungi and malaganese peroxidase. J WOOD SCI 1998,44(3), 222-229 (1998).

[9] Gao, R. & Sun, C. A marine bacterial community capable of degrading poly(ethylene terephthalate) and polyethylene. J Hazard Mater 416, 125928, doi:10.1016/j.jhazmat.2021.125928 (2021).

[10] Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chem Commun (Camb) 56, 4950-4953, doi:10.1039/d0cc01174f (2020).

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]