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WO-2026092968-A1 - PLASTIC DEGRADING FUSION PROTEINS, METHODS AND USES THEREOF

WO2026092968A1WO 2026092968 A1WO2026092968 A1WO 2026092968A1WO-2026092968-A1

Abstract

The present invention relates to fusion proteins capable of degrading plastic polymers. The fusion proteins comprise two or more serin hydrolase domains and comprise the necessary features to efficiently and cost effectively be used in degradation of plastic polymers. Provided herein are also nucleic acids and nucleic acid vectors encoding such fusion proteins and cells comprising the same. Provided herein are also methods of producing such fusion proteins, and methods and uses of degrading plastic polymers using the fusion proteins provided herein.

Inventors

  • HATTI KAUL, RAJNI
  • ISMAIL, Mohamed Ismail Fouad
  • ABOUHMAD, Adel Elsayed Attia

Dates

Publication Date
20260507
Application Date
20251007
Priority Date
20241007

Claims (20)

  1. CLAIMS 1. A fusion protein capable of degrading a plastic polymer, wherein the fusion protein comprises two or more serine hydrolase domains coupled by a linker comprising: (i) an (EAAAK)n motif wherein n is an integer greater than or equal to 2; or (ii) an amino acid sequence according to any one of SEQ ID NO: 69, 59, 60, 62, 66, or 70.
  2. 2. The fusion protein of claim 1, wherein the fusion protein is catalytically active after at least 72 hours at a temperature of at least 60°C, optionally wherein the fusion protein is catalytically active after at least 96 hours at a temperature of at least 60°C
  3. 3. The fusion protein of claim 1 or 2, further comprising one or more polymer binding domain(s).
  4. 4. The fusion protein of any of the preceding claims, wherein all serine hydrolase domains are catalytically active.
  5. 5. The fusion protein of any of the preceding claims comprising two serine hydrolase domains.
  6. 6. The fusion protein of any of the preceding claims, wherein at least one serine hydrolase domain is an esterase, a lipase or a protease, or a catalytically active portion thereof.
  7. 7. The fusion protein of claim 6, wherein at least one esterase is selected from the group consisting of a PETase, a MHETase, and a cutinase, or a catalytically active portion thereof.
  8. 8. The fusion protein of any of the preceding claims, wherein at least one serine hydrolase domain comprises a cutinase or a PETase, or a catalytically active portion thereof.
  9. 9. The fusion protein of claim 6, wherein at least one protease is proteinase K, or a catalytically active portion thereof.
  10. 10. The fusion protein of any one of claims 6 or 7, wherein at least one cutinase comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 11, a variant, fragment or derivative thereof, or an amino acid having at least 75%, 80%, 85%, 90%, 95%, 96%, 98% or 99% sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 11.
  11. 11. The fusion protein of claims 7 or 8, wherein at least one PETase comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 12, 13 or 14, a variant, fragment or derivative thereof, or an amino acid having at least 75%, 80%, 85%, 90%, 95%, 96%, 98% or 99% sequence identity to any one of SEQ ID NO: 12, 13 or 14.
  12. 12. The fusion protein of claim 6, wherein at least one lipase comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 9 or 10, a variant, fragment or derivative thereof, or an amino acid having at least 75%, 80%, 85%, 90%, 95%, 96%, 98% or 99% sequence identity to any one of SEQ ID NO: 9 or 10.
  13. 13. The fusion protein of any of the preceding claims, wherein the amino acid sequence of two or more serine hydrolase domains is the same.
  14. 14. The fusion protein of the preceding claims, wherein the amino acid sequences of at least two serine hydrolase domains comprise no more than about 95% sequence identity, such as no more than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% sequence identity.
  15. 15. The fusion protein of claim 2-14, wherein the one or more polymer binding domains are selected from the group consisting of a hydrophobin, a cellulose binding domain, a carbohydrate binding module, an anchor peptide, an anchor linker peptide, PET binding domain and PHA binding domains.
  16. 16. The fusion protein of any one of claims 2-15, wherein the one or more polymer binding domains are capable of binding one or more plastic polymer(s), optionally wherein the one or more plastic polymer(s) are selected from the group consisting of PET, PTT, PBT, PEF, PLA, PCL, PBAT and MHET.
  17. 17. The fusion protein of any one of claims 2-16, wherein the one or more polymer binding domain comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 85-96 and/or SEQ ID NO: 112-121, a variant, fragment or derivative thereof, or an amino acid having at least 75%, 80%, 85%, 90%, 95%, 96%, 98% or 99% sequence identity to any one of SEQ ID NO: SEQ ID NO: 85-96 and/or SEQ ID NO: 112-121.
  18. 18. The fusion protein of any of the preceding claims, wherein at least one serine hydrolase domain and at least one polymer binding domain are coupled by a linker.
  19. 19. The fusion protein of any of the preceding claims, wherein at least one linker is a polypeptide linker.
  20. 20. The fusion protein of claim 19, wherein the linker is selected from the group consisting of a polyglycine linker, a polyalanine linker, an alanine-glycine linker, a glycine-serine linker, a histidine linker, threonine-serine linker, a glutamine linker, an asparagine linker.

Description

PLASTIC DEGRADING FUSION PROTEINS, METHODS AND USES THEREOF FIELD The present invention is in the field of fusion proteins, in particular those for use in plastic and/or plastic polymer degradation. BACKGROUND Plastics are versatile materials affecting almost all aspects of modern human life, addressing all kinds of needs of health, energy, clothing, packaging, new technologies, construction, housing, transport and communication. The annual production of plastics has significantly increased globally since the early 2000s, with expectation to reach 445.25 million metric tons by 2025 (1). In the past decade, plastic production has increased by more than 35% and is expected to grow to 700 Mt in 2030, representing 80 kg of plastics per human being (2). The increased global consumption and production of plastics had come with a cost. Currently, plastic production accounts for 6% of the global oil consumption and will reach 20% by 2050. Additionally, plastics life cycle has a great impact on greenhouse gas emissions, in 2019, plastics were responsible for 1.8 billion tons of CO2 equivalent emissions, which account for 3.4% of the global emissions (3, 4). As a result of the poor plastic waste management, there have been increasing concerns about the negative impact of plastic pollution on ecosystems and human health. Since its commercial application, around 8,300 million tons of plastics have been produced, generating 6,300 million tons of plastic waste. The majority (79%) of this waste has been landfilled, 12% has been incinerated and only 9% has been recycled (5). The majority of this waste is in the form of microplastics (particles and fibers ≈ 5 mm size) which are a ubiquitous pollutant in aquatic and terrestrial environments resulting in acute and chronic toxicity in animals, plants, and microbes as a result of landfill disposal (6, 7). On the other hand, plastic waste incineration results in harmful gas emissions such as methane, carbon monoxide and polyaromatic hydrocarbons, which impose the global carbon cycle exaggerating the greenhouse emissions (8). Considering the impact of plastics on the environment and living organisms, the European Commission communicated “The European Strategy for Plastics in a Circular Economy” combined with the promotion of various other initiatives (the Circular Plastic Alliance, etc.) recommending improved design to facilitate reuse and recycling, and decoupling production from fossil resources. Furthermore, a global plastics treaty involving 175 countries will be established by 2024, signifying a pivotal opportunity to end plastic pollution (9). Polyesters are a broad class of thermoplastic polymers that, depending on their monomer compositions can either be recyclable or biodegradable. Polyethylene terephthalate (PET) is the most popular fossil-based synthetic polymer comprising 18% of the global polymer production (10). It is the most common polymer in the textiles and packaging industries due to its diverse properties such as high chemical, mechanical and thermal resistance, low cost, high transparency, excellent gas-barrier properties (2). PET is industrially synthesized via heating purified terephthalic acid (TPA) with excess ethylene glycol (EG) that results in bis (2-hydroxyethyl) terephthalate (BHET). BHET is then pre-polymerized followed by subsequent melting condensation or solid-state polymerization to yield low molecular weight PET or high molecular weight PET, respectively (11). Enzymatic recycling of post-consumer PET (pc-PET) wastes and the subsequent utilization of the released monomers is considered a sustainable and economically feasible process implementing the circular plastic economy concept and mitigating plastic environmental concerns (12). Unlike chemical degradation of PET that requires high pressure, elevated temperatures and production of toxic gases, bio-based plastic depolymerization via enzymatic processes is more sustainable and environmentally friendly alternative as it can be run at mild pH, moderate temperatures without any hazardous chemicals (13). Moreover, the specificity of the enzymes towards the ester bonds in the PET allows its direct depolymerization from blended (mixed) plastic waste without pre-sorting (14). Since the first report of the cutinase TfH isolated from the actinomycete Thermobifida fusca being a PET hydrolase, research is ongoing to find the best PET hydrolase with the highest activity and stability. Engineering of PET hydrolases is crucial to make the enzymatic recycling process feasible and competitive. Different aspects have been employed to engineer PETases to create efficient enzymes with new properties including enhanced soluble protein expression, improved thermostability, higher reaction turnover rates with versatile substrates. Increased enzymatic thermostability is important for PETase activity, since ideally the enzyme should work around the glass transition (Tg) of the PET which is between 60- 70 °C, and th