Contribution

Introduction

Our project focuses on the enzymatic degradation of PLA plastics using MGS0156, Est119, RPA1511, and the PET-degrading TTL enzyme. In the context of contributing to the iGEM community, we present two new enzymes (Est119 and RPA1511) and provide additional documentation to the already existing part BBa_K2302003 (Lpp-OmpA anchor). In particular, we investigate the esterase activity of Est119 and RPA1511 and the surface displacement of these two enzymes. The addition of Lpp-OmpA anchors will remove the need for protein purification. This anchor will display the enzymes on the surface of the bacteria allowing them to be in direct contact with PLA, without having to lyse the cells and execute their catalytic functions, facilitating the breakdown of our target plastic material (Figure 1).

Figure 1. Surface displacement of enzymes on bacteria membrane.

Est119 is an esterase originally identified in the Thermobifida alba strain AHK119 (AB298783). Est119 has been shown to degrade aliphatic-aromatic copolyesters and decrease the size of polymer particles of other biodegradable plastics, with an optimal temperature range of 45-55℃ (Hu et al. 2009). RPA1511 is a carboxyl esterase originally identified in Rhodopseudomonas palustris. RPA1511 has been shown to degrade a variety of polymers, including PLA, with an optimal temperature range of 50-60℃ (Hajighasemi et al. 2016). Due to their potential temperature compatibility with Manitoban composting methods, we are interested in Est119 and RPA1511.

Genetic Construct Design & New Parts Contribution

We obtained sequences of Est119 and RPA1511 from the literature to create 2 new basic parts (BBa_K4949004, Est119, and BBa_K4949006, RPA1511). Our team further modified Est119 and RPA1511 sequences to include the Lpp-OmpA anchor (BBa_2302003) to create 2 new basic parts (BBa_K4949005, Lpp-OmpA-Est, and BBa_K4949007, Lpp-OmpA-RPA1511). These parts enable the characterization of the surface-display mechanism of PLAnet Zero. We also include BBa_K921000 (T7LacO mutant), BBa_B0030 (Strong RBS), and BBa_B0015 (Double Terminator) to optimize expression (Figure 2). As a result, we contribute 4 new composite parts: BBa_K4949008, BBa_K4949009, BBa_K4949010, and BBa_K4949011.

Figure 2. Genetic construct for surface-displaced enzymes.

Figure 3. Genetic construct for intracellular enzymes.

Documentation Contribution

a) Effect of the presence of endogenous Lpp signal in cells

We constructed Est119 and RPA1511 with the addition of Lpp-OmpA and transformed them into BL21(DE3) and BL21(DE3) ΔLpp E. coli competent cells for protein expression. BL21(DE3) ΔLpp are E. coli strains that were genetically modified to remove their endogenous genes with Lpp signal. By removing endogenous genes with an Lpp signal, there will be less competition with our Lpp-OmpA-enzymes for secretion machinery and thus, more enzymes will be exported and decorated on the outer membrane.

We then performed protein expression on transformed E.coli cells with Isopropyl ß-D-1-thiogalactopyranoside(IPTG) and performed whole-cell activity assays using 4-nitrophenyl octanoate(NPO) and 4-nitrophenyl butyrate (pNOB) as substrates. NPO and pNOB contain ester bonds (See Figure 3). Once the ester bond is cleaved by esterases, the product is 4-nitrophenol (Figure 3), which absorbs light at 410 nm at pH 7.4. High absorbance at 410 nm indicates the release of a high amount of released product.

Figure 4. (Left) 4-nitrophenyl octanoate(NPO). (Center) 4-nitrophenyl butyrate (pNOB). (Right) 4-nitrophenol.

We perform whole-cell catalysis by mixing cells expressing Lpp-OmpA-Enzyme constructs with the substrate (either NPO or pNOB) in PBS (0.137 M NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.4). Cell suspensions used in whole cell-catalysis experiments are normalized to have OD600 of 1.0 in 1 mL (i.e. all samples contain ~109 cells/mL). We also prepare control cases by diluting substrates in PBS. These reactions are then incubated at room temperature. After a set interval (every 5 minutes), we aliquot 100 µL of the mixture into a clean tube, centrifuge to pellet all cells, and then sample 2 µL using a nanodrop. We measure absorbance at 410 nm to detect the presence of 4-nitrophenyl, a product of esterase activity and at 600 nm to detect the presence of cells in the supernatant that can cause light scattering. When our absorbance is above 1, we dilute our samples until the absorbance is below 1 for measurement and then, calculate the final value, taking the dilution factor into account. Our hypothesis is if our cells are decorated with enzymes on the outer membrane, there would be a high absorbance of 410 nm, indicating the presence of a product, and a low absorbance of 600 nm, indicating no cells are in solution to scatter light at 410 nm.

Figure 5. Whole-cell catalytic activity assay with cells decorated with enzymes on outer membrane. Samples are measured absorbance at 410 nm using a Nanodrop. Top panel: RPA1511 enzyme. (Left): NPO substrate. (Right): pNOB substrate. Bottom panel: Est119 enzyme. (Left): NPO substrate. (Right): pNOB substrate.

Figure 5 shows that in all cases (both Lpp-OmpA-Est119 (BBa_K4949005) and Lpp-OmpA-RPA1511 (BBa_K4949007)), the absorbance at 410 nm increases for treatment reactions with cells decorated with enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This is an indication that enzymes, which are anchored on the outer cell membrane, catalyze the cleavage of substrate into 4-nitrophenol. The rate of increase in absorbance at 410 nm is higher for pNOB substrate than NPO substrate in both enzymes. Furthermore, regardless of substrate, there is more released product when Lpp-OmpA-Est119 is used, which is indicated by higher overall absorbance at 410 nm. Finally, there is no significant difference between two different cell lines (BL21(DE3) and BL21(DE3) ΔLpp, except when Lpp-OmpA-RPA1511 is used to cleave NPO substrate. This indicates the competition between lpp-OmpA-Enzyme and endogenous genes with Lpp secretion signal for secretion machinery in cells is likely to be not the rate-determining step. To test the significance of the presence of Lpp signal in our gene construct and to further demonstrate successful decoration of enzymes on the outer membrane, we also compare cells with Lpp-OmpA-Enzyme and Enzyme constructs.

b)Effect of the presence of Lpp signal on enzyme construct in cells

We transformed Lpp-OmpA-Est119 (BBa_K4949009), Lpp-OmpA-RPA1511 (BBa_K4949011), Est119 (BBa_K4949008), and RPA1511 (BBa_K4949010) into BL21(DE3) competent cells for protein expression and characterization (as described above). Unlike BBa_K4949009 and BBa_K4949011, since BBa_K4949008 and BBa_K4949010 do not have Lpp-OmpA secretion signal and anchor, we expect the enzymes to be expressed and retained intracellularly. We do not expect intracellular enzymes to come into contact with the substrate and thus, cells expressing enzymes intracellularly are not expected to have esterase activity thus, absorbance at 410 nm is not expected.

Figure 6. Whole-cell catalytic activity assay with cells decorated with enzymes on the outer membrane or cells with intracellular enzymes. Samples are measured absorbance at 410 nm using a Nanodrop. Top panel: Est119 enzyme. (Left): NPO substrate. (Right): pNOB substrate. Bottom panel: RPA1511 enzyme. (Left): NPO substrate. (Right): pNOB substrate.

Figure 6 supports a functional Lpp-OmpA-Est119 and Lpp-OmpA-RPA1511 surface display mechanism. As observed before, the absorbance at 410 nm increases for treatment reactions with cells containing enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This indicates that our whole cell catalysis approach works. In all treatment cases, the absorbance at 410 nm for treatment cases containing cells with Lpp-OmpA-Enzymes increases faster than cases containing cells with Enzymes. These observations suggest two conclusions:

1. Cells with Lpp-OmpA-Enzymes have enzymes anchoring on the outer membrane and thus can readily access substrates, which enhance catalysis rate and result in a higher rate of increase in absorbance at 410 nm.

2. Cells with Enzymes without anchors can also catalyze cleavage of substrate, unlike our expectation. This suggests that substrates can cross cell membranes to reach enzymes. As cells are pellet before measurement, it also suggests that the product can then cross the membrane again and diffuse into the solution. This hypothesis is further supported by the general higher rate of increase of absorbance at 410 nm when pNOB is used instead of NPO. pNOB has a shorter carbon chain (4) in comparison to NPO (8) and thus, is expected to cross the membrane more readily.

Furthermore, when comparing treatment cases with Est119 and RPA1511 using pNOB as substrate, we observe a much more distinct difference between Lpp-OmpA-Est119 and Est119 in comparison to Lpp-OmpA-RPA1511 and RPA1511. This observation is possibly due to a much higher catalytic rate of cells with Est119 in comparison to cells with RPA1511. When pNOB is used, while samples from case with cells containing intracellular Est119 has average absorbance of 1.56, samples from case with cells containing intracellular RPA1511 has average absorbance of 0.49. The superior catalytic rate of cells with Est119 to cells with RPA1511 is further amplified when two enzymes are decorated on the outer membrane as reaction is not affected by the diffusion of the substrate. The same trend can also be observed when NPO is used as a substrate. Additionally, Est119 and RPA1511 are observed to be less efficient in cleaving NPO.

Conclusion

Our result demonstrates Est119 and RPA1511 activity towards pNOB and NPO. Furthermore, we demonstrate that the surface displacement approach using an existing part (BBa_K2302003) is successful and thus, our enzymes are placed in the outer membrane. Our surface displacement approach can enhance the whole-cell catalysis rate by eliminating substrate diffusion through cell membrane.

Future Work

Our work demonstrates an apparent better catalytic rate of whole cells with Est119 in comparison to whole cells with RPA1511. We propose two possible explanations:

1. Est119 is more readily and easily expressed and thus, each individual cell contains a high concentration of Est119 while that is not the same for RPA1511.

2. Est119, by itself, has better specificity constant (kcat/KM). Further research is needed to characterize these enzymes and measure protein concentration in cells.

Usefulness for Future iGEM Team

In total, we deposited 4 new basic parts and 4 new composite parts to the iGEM Registry. We also provide more documentation for already existing parts (BBa_K2302003). Our composite parts provide a framework for future teams to apply our surface displacement approach for their project. Specifically, our contribution can be useful for future iGEM teams in two ways:

1. Our new enzymes Est119 and RPA1511 are new esterase enzymes that can be potentially used for PLA degradation. It should be noted that these esterases should not be limited to just degrading PLA. Future teams who wish to degrade ester-based polymers (polymers in which monomeric subunits are connected by ester bonds) can experiment and use our parts. Furthermore, these esterases can also be used in applications other than plastic degradation such as catalysis and production of a specific product.

2. Our experimental design has the potential to be useful for future researchers conducting similar experiments and for companies interested in plastic degradation. The use of Lpp-OmpA as a potential secretion signal and anchor can enhance enzyme catalysis and improve plastic degradation, making it a valuable approach for addressing plastic pollution. Notably, the surface displacement of the enzyme is not uniquely beneficial to just our project. Future iGEM teams can explore part BBa_K2302003 for different applications such as displacement of enzymes on the outer membrane for whole catalysis assay or enzyme cascade for production of bio-based materials. The surface displacement of enzymes eliminate the need for substrate diffusion through the membrane and thus, this technique is especially useful when reactants are known to not cross the membrane readily.

References

Hajighasemi, M., Tchigvintsev, A., Nocek, B., Flick, R., Popovic, A., Hai, T., Khusnutdinova, A. N., Brown, G., Xu, X., Cui, H., Anstett, J., Chernikova, T. N., Brüls, T., Le Paslier, D., Yakimov, M. M., Joachimiak, A., Golyshina, O. V., Savchenko, A., Golyshin, P. N., Edwards, E. A., … Yakunin, A. F. (2018). Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters. Environmental science & technology, 52(21), 12388–12401. https://doi.org/10.1021/acs.est.8b04252

Hu, X., Thumarat, U., Zhang, X., Tang, M., & Kawai, F. (2010). Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119. Applied microbiology and biotechnology, 87(2), 771–779. https://doi.org/10.1007/s00253-010-2555-x