Main Experiment Design

Figure 1 Design process

To improve the expression and degradation efficiency of PET-degrading enzymes, with the goal of efficiently degrading plastics under neutral conditions at 30°C and recycling plastic degradation products to produce high-value bacterial cellulose, SUPERB will focus on the following four directions:

1. Construction of strains with high enzyme expression
   Methylotrophic yeast Pichia pastoris, due to its ease of genetic manipulation, secretion expression, and high-density fermentation, is an important chassis cell in the enzyme production industry. Current reports suggest that PET hydrolase expressed in Pichia pastoris exhibits greater thermal stability, allowing higher PET hydrolysis efficiency over extended reaction times. Therefore, Pichia pastoris was chosen as the host for PETase expression. First, the enzyme-expressing gene was codon-optimized and expressed in Pichia pastoris. In addition, by optimizing the promoters AOX1, AOXm, GAP, and the signal peptides MF and α-signal peptide, we constructed engineered strains using homologous recombination: AOXm-MF-IsPETase^PA, GAP-MF-IsPETase^PA, AOX1-MF-IsPETase^PA, and AOXm-α signal peptide-IsPETase^PA. This allows screening for the best combination to enhance the expression of IsPETase^PA.

2. Rational design of enzymes
   Through literature research, we identified two enzymes with high plastic-degrading capabilities. IsPETase^PA, an IsPETase variant, underwent site-saturation mutagenesis at key sites, resulting in a 24.75-fold increase in activity compared to the wild type at 40°C. Another variant, FAST-PETase-212/277, had two N-chain glycosylation sites removed and was produced in a 30-L fermenter with antibiotic selection and chaperone co-expression, yielding over 3 g/L. By using machine learning, we predicted additional mutation sites to enhance thermal stability, combined with deglycosylation to improve pH tolerance, and validated the best mutated enzymes through molecular docking.

3. Verification of plastic degradation
   The engineered Pichia pastoris strain will be scaled up, and fermentation conditions optimized for efficient expression of the plastic-degrading enzymes. The optimal conditions for plastic degradation will be explored, and the degradation products will be analyzed by HPLC and SEM to verify their concentration and observe changes in the PET film morphology.

4. Recycling of production
   We will explore the production of bacterial cellulose by three strains, select the best bacterial cellulose-producing strain, and conduct co-cultivation experiments. After Pichia pastoris secretes the plastic-degrading enzymes and degrades the plastic, the bacterial cellulose-producing strain will utilize the plastic degradation products to produce bacterial cellulose.

Additionally, SUPERB plans to conduct preliminary explorations in dual-enzyme systems, multi-copy expression, and fusion proteins.
Researchers have found that the synergistic effect of MHETase and IsPETase^PA can enhance degradation capacity. SUPERB will attempt to enhance PET biodegradation using an IsPETase^PA and MHETase dual-enzyme system, as well as a MHETase and FAST-PETase-212/277 dual-enzyme system, to further degrade PET.

Extended Experiment Design

Figure 2 Extended experiment design route

Moreover, multiple copies of the enzyme can increase protein expression. SUPERB aims to increase the copy number of IsPETase^PA and FAST-PETase-212/277 by using the same downstream enzyme restriction sites BglII and BamHI, to boost protein expression.

Furthermore, multienzyme aggregates are membraneless organelles formed by liquid-liquid phase separation of biomolecules. They serve to isolate and concentrate substrates, intermediates, and enzymes within cells, thereby improving biochemical reaction efficiency. Constructing multi-enzyme condensates using interacting peptides can simulate natural multi-enzyme complexes, increase the local concentration and efficiency of chemical reactions, and reduce the occurrence of side reactions. Based on this principle, we propose linking two proteins with short peptides to construct a fusion protein. We selected two interacting polypeptides, SZ1 and SZ2, and inserted them into our plasmid to construct PETase-mCherry-SZ2 and RGG-SZ1, aiming to combine them for better expression, degradation rate, and the desired properties.