Part Collection

Parts

Through PLAnet Zero, we attempt to improve polylactic acid (PLA) composting through a combination of wet lab experiments, dry lab models, understanding of the societal aspect of recycling, and education of the general public in combating plastic pollution. As a result of our wet lab experiments and dry lab models, we have obtained 7 basic parts and created 4 composite parts with a theme of enzymes for degrading polylactic acid.

Basic Parts

Composite Parts

The Plan

Our wet lab and dry lab project study enzymatic degradation of PLA using four potential PLA-degradable proteins: Est 119 (BBa_K4949004), GEN0105 (BBa_K4949003), MGS0156 (BBa_K4949002), RPA1511 (BBa_K4949006). We also use one PET-degrading enzyme (TTL) (BBa_K4949001) as a case study. To achieve our goal of degrading PLA for composting, we simultaneously examine 2 different approaches: surface displacement of enzymes on the cell membrane and genetic code engineering to incorporate non-canonical amino acids. Due to the limited time, we divided our enzyme collections into 2 working groups.

1

Surface displacement of Est119 and RPA1511 on cell membranes. We use a part (BBa_K2302003), found in the iGEM registry, which encodes lpp-OmpA for surface displacement. For this objective, we construct our new parts (BBa_K4949004, BBa_K4949005, BBa_K4949006, and BBa_K4949007). We aim to express these parts and then perform whole-cell catalysis assays. We envision cells decorated with esterases will degrade plastics into building blocks for use as carbon sources for biomass and for the synthesis of organic compounds in compost. This is important for future deployment as the purification of enzymes can be expensive and drive up the cost of implementation. Furthermore, this work provides a basis for a versatile organism that can produce economically valuable biomaterials from plastic waste.

2

Genetic code engineering to incorporate non-canonical amino acids for improving MGS0156 and GEN0105. The literature reports that the global replacement of Methionine with Norleucine (Nle) is shown to improve TTL’s ability to degrade PET plastic (Haernvall et al. 2022). Hence, we aim to incorporate Nle into TTL and characterize this mutant using enzyme assay and modelling to assess changes to Michaelis Menten parameters (k_cat, k<sbubM, specificity constant) and operating temperatures. We then apply the same principle to incorporate Nle and Methoxinine (Mex) into MGS0156 and GEN0105. While Nle is a very hydrophobic amino acid, Mex is a hydrophilic amino acid. Similar to TTL, we aim to analyze and understand how changing the hydrophobicity can impact the ability of MGS0156 and GEN0105 to degrade PLA. Our work adds to the foundational understanding of incorporating ncAAs for improved esterase activity and provides insight for future efforts to engineer and customize enzymes for specific plastics with desirable properties.

Results

We use BBa_K4949001 as a case study for understanding the impact of the incorporation of non-canonical amino acids on enzyme activity and then apply the lessons learned to engineering BBa_K4949002 for desirable enzymatic properties. Our documentation for parts BBa_K4949001 and BBa_K4949002 demonstrates how the global replacement of methionine with norleucine can change the hydrophobicity of an enzyme and thus, the activity towards two substrates: para-nitrophenyloctanoate (NPO) and para-nitrophenylbutyrate (pNOB). We show that the significant increase in binding of NPO in Norleucine-incorporated TTL relative to native TTL (BBa_K4949001) can be explained by the hydrophobic interaction between Nle residues near the active site of TTL(Nle) variant and the long C8 chain of NPO. This increase in binding of NPO is not observed in Norleucine-incorporated MGS0156 (BBa_K4949002), possibly due to the lack of Nle residue near the active site.

We also observe that the incorporation of Nle into both BBa_K4949001 and BBa_K4949002 does not improve the enzymatic binding of pNOB. Our modelling shows a lack of interaction between Nle residues and pNOB and thus, no increase in binding is observed. These observations suggest that future efforts to incorporate non-canonical amino acids should be carried out with more attention to the position of the residues relative to the active site to promote interaction between non-canonical residues and substrates. This opens up possibilities for engineering and tailoring different esterases toward different substrates and plastic types. It should be noted that the lessons learned from these documentations are applicable to our other esterases (BBa_K4949003, BBa_K4949004, BBa_K4949006) as these enzymes are also viable candidates for Nle incorporation.

Our documentation for parts BBa_K4949004, BBa_K4949005, BBa_K4949006, and BBa_K4949007 demonstrate successful displacement of enzymes on the cell surface. While cells expressing BBa_K4949004 and BBa_K4949006 do not exhibit esterase activities towards NPO and pNOB, cells expressing BBa_K4949005 and BBa_K4949007 exhibit the desired esterase activities towards NPO and pNOB. Our part collection also consists of composite parts (BBa_K4949008 to BBa_K4949011) made up of parts from the Registry of Standard Biological parts. These composite parts consist of parts optimized for the expression of surface-displaced enzymes. Conclusions from these documentations are relevant to parts BBa_K4949001, BBa_K4949002, and BBa_K4949003 as each enzyme we present can be modified to contain lpp-OmpA (BBa_K2302003) to be secreted and anchored on cells’ membranes.

Conclusion & Future Vision

Our part collection consists of a lipase (BBa_K4949001) that is shown to degrade Polyethylene terephthalate (PET) and several esterases (BBa_K4949002 to BBa_K4949007) that can be potentially used for Poly-lactic acid (PLA). Our part collection is harmonious due to the applicability of the conclusion from each work to another. I.e., conclusion and learned principles from documentation of parts BBa_K4949001 and BBa_K4949002 can be applied to parts BBa_K4949004, BBa_K4949005, BBa_K4949006, and BBa_K4949007 or vice versa. More work is required to characterize and optimize working conditions for different enzymes.

In the future, we envision our parts and principles to be used collectively to build surface displacement of synthetic enzymes incorporated with non-canonical amino acids for desirable properties to degrade PLA. Furthermore, our parts can be used collectively and interchangeably in a compost system to degrade multiple plastics (e.g: BBa_K4949001 for PET and BBa_K4949002 for PLA). Possible implementations include a co-culture with different unique cells, each decorated with a unique non-canonical amino-acid-incorporated-variant of a unique esterase, to target different plastics and thus solve the problems of mixing different types of plastics. Furthermore, once our collection of esterases (BBa_K4949002 to BBa_K4949007) is further characterized to determine optimal working conditions for each, they offer different options for a wide range of conditions (such as mesophilic or thermophilic temperatures) to enable composting of plastic waste throughout the year regardless of seasonal fluctuation in temperatures.

Usefulness to the iGEM Community

The iGEM community can benefit from our part collection and build upon it to expand the substrate range through non-canonical amino acid incorporation and enable the degradation of different plastics in which ester bonds link monomeric subunits. Furthermore, future teams can apply the same principles to secrete and anchor desired enzymes on cell surfaces for applications in whole-cell catalysis.

References

1. Haernvall, K., Fladischer, P., Schoeffmann, H., Zitzenbacher, S., Pavkov-Keller, T., Gruber, K., Schick, M., Yamamoto, M., Kuenkel, A., Ribitsch, D., Guebitz, G. M., & Wiltschi, B. (2022). Residue-specific incorporation of the non-canonical amino acid norleucine improves lipase activity on synthetic polyesters. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.769830
2. 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
3. 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