Learning about PET's enzymes

Currently, the accumulation of PET waste is steadily increasing and poses a growing threat to ecosystems worldwide. It's estimated that naturally occurring microorganisms take hundreds of years to fully degrade PET plastics. Developing engineered microorganisms capable of breaking down PET into vanillic acid (VA) offers a promising solution, as it transforms plastic waste into a valuable molecule used across various industrial sectors. This approach not only provides a sustainable method for dealing with plastic waste but also supports the concept of a circular economy.
Recently, it was discovered that the bacterium Ideonella sakaiensis is capable of degrading PET plastic into its monomeric components: terephthalic acid (TPA) and ethylene glycol (EG). Japanese scientists were the first to make this breakthrough and to identify the enzymes responsible for the transformation. They showed that two major enzymes PETase and MHETase work synergistically to carry out the following reaction:

Figure 1: PET degradation by PETase and MHETase

Additionally, the article Characterization and Engineering of a Two-Enzyme System for Plastics Depolymerization demonstrated that the PETase-MHETase fusion protein has greater efficiency in depolymerizing PET into TPA and EG compared to using the two enzymes separately.

Circular Economy: Transforming PET into Vanillic Acid

Our team aims to contribute to the circular economy by converting PET, not just into by-products, but into a molecule of economic interest. After analysing all the potential molecules that can be produced from PET and conducting a market study, we identified vanillic acid (VA) as an ideal candidate. VA is relatively easy to produce from PET, and more importantly, it holds significant economic value, as it is utilised across various industries.
In order to convert PET into VA, a series of biochemical reactions is required:
PET -> TPA -> PCA -> VA
In the article Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6, it was shown that the enzymes encoded by the tphRICIA2IA3IBIA1I gene cluster are essential for transforming TPA into protocatechuic acid (PCA) through the following reactions:

Figure 2: Conversion of TPA to PCA by Comamonas sp. strain E6. The following gene products are involved in the conversion of TPA to PCA: TphA2I and TphA2II, oxygenase large subunit of TPADO; TphA3I and TphA3II, oxygenase small subunit of TPADO; TphA1I and TphA1II, reductase component of TPADO; and TphBI and TphBII, DCD dehydrogenase,Courtesy of Hirofumi Hara

In fact, the essential enzymes for this transformation are TphA1,A2,A3 ( also noted sometimes as TphAa,Ab,Ac) and TphB.

Transformation of PCA into Vanillic Acid (VA)

In the article Biological Valorization of Poly(ethylene terephthalate) Monomers for Upcycling Waste PET, the authors identified a Homo sapiens O-methyltransferase (HsOMT) enzyme as the most efficient protein for converting protocatechuic acid (PCA) into vanillic acid (VA). Ana Rita Brochado et al. also reported that HsOMT plays a crucial role in vanillin production and that overexpressing this enzyme in a strain with model-guided modifications can significantly increase the final product yield.
Essentially, this enzyme catalyses the methylation reaction, where a methyl group is added to the hydroxyl group on the PCA molecule, converting it into vanillic acid.

Figure 3: Reaction cascade to produce Vanillic Acid from TPA and PCA, with the main enzymes TphAabc and TphB



References

  1. Sasoh, Mikio & Masai, Eiji & Ishibashi, Satoko & Hara, Hirofumi & Kamimura, Naofumi & Miyauchi, Keisuke & Fukuda, Masao. (2006). Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6. Applied and environmental microbiology. 72. 1825-32. 10.1128/AEM.72.3.1825-1832.2006.
  2. Duan, Shuyan & Zhang, Nan & Chao, Tianzhu & Wu, Yaoyao & Wang, Mengying. (2023). The structural and molecular mechanisms of type II PETases: a mini review. Biotechnology Letters. 45. 1-15. 10.1007/s10529-023-03418-3.