Design
In 2016, Yoshida et al. [1] reported the discovery of the bacterium, Ideonella sakaiensis 201-F6, which develops a two-enzyme system to deconstruct PET plastic to TPA and EG molecules, which could be effectively catabolized as a carbon and energy sources for cells. Further research of this two enzymes system reveals that enzyme PETase is a cutinase-like serine hydrolase that attacks the PET plastic polymer with the highest efficiency in mild temperature among all previously discovered PET degradation enzymes. The metagenome-derived leaf-branch compost cutinase (LCC) shows higher activity on low crystallinity PET film degradation [2]. However, LCC is still not generally applicable due to the cost and high applied optimum temperature around 72 °C [2], which highly limit its further application in engineered bacteria bio-degradation system. Furthermore, the percentage of PET degradation intermediates MHET generated via LCC were near to 60% [3], which could not be removed by MHETase effectively due to the possible loss of its enzymatic activity at LCC applied temperature. Compared to LCC, the MHET removing efficiency in PETase and MHETase coexisting system was near to 100% [4]. LCC reveals near 5.5 times and 4 times lower enzymatic activity to PET film and high crystallinity PET substrate at 30 °C compared to PETase [1] [5]. PETase was considerably more active against PET film at low temperatures than other PET degradation enzymes including TfH, LCC, and FsC [1]. And more efforts of PETase protein rational evolution are made and these works largely improved enzyme PETase thermostability with a Tm value that was increased by 8.81 °C and reacting activity by around 14-fold at 40 °C [5], whose activity is even higher than LCC and BhrPETase isolated from bacterium HR29 [3][5]. Furthermore, in a latter research, highly synergistic relationship between PETase and MHETase was discovered in the conversion of amorphous PET film to monomers [4], which effectively accelerate PET degradation. However, this two enzymes system may be still limited due to enzymatic loss caused by protein fusion, inhibition effects and diffusion of intermediates. And methods of rational evolution of protein may not be applicable to further improve the overall turnover rate [6]. In detail, the MHET molecules produced via PET degradation may be competitive to PETase active sites, which was revealed via calculation results shown in this work. Despite of this, MHET molecules diffusion in space may also be a problem for MHETase remove MHET in time.
Figure 1: PET degradation pathway
Therefore, in this work, we designed an intricate multidomain protein scaffold composed of short peptide tags of RIAD [8] and RIDD [9], enzymes of PETase and MHETase and protein hydrophobin4 [8], in which the enzymes constructed in near positions to each other may work with highly synergistic relationship. All proteins involved have unique functions in this system. PETase and MHETase are two enzymes involved deconstructing polymer PET plastic to MHET and MHET to TPA molecules, respectively. And hydrophobin4, a small fungal protein, possess positive effects on altering the physicochemical properties of PET surfaces and enzyme aggregation enhancement when it was fused with PET degradation enzyme cutinase [10]. Here, hydrophobin4 are involved in our designed system with possible functions of adhering to PET polymers and altering the physicochemical properties of PET for degradation improvement [11]. The peptides of RIDD and RIAD originated from cAMP-dependent protein kinase (PKA) and the A kinase-anchoring proteins (AKAPs), respectively. The RIAD peptide is capable of specifically binds to the RIDD dimer with strong affinity [12]. The following two features make them ideal protein binding modular for our system assembly: (1) the tiny size (44 and 18 amino acids, respectively), which minimizes the disturbances to the structure and activity of the enzymes when fused with these peptides, (2) the strong binding affinity (with a KD of 1.2 nM between RIDD dimer and RIAD peptide demonstrated in our colleagues' previous work [12]) to ensure the stability of the whole enzyme complexes in the environment.
Figure 2: Schematic diagram of this system[12] (The assembly of tri-enzyme units. E1, E2: enzymes; green and blue structure: RIDD dimer; black line: linker; pink structure: RIAD; one orange circle: cysteine; two orange circles: disulfide bond)
Figure 3: Three-protein complex assembly schematic diagram.
In summary, we plan to construct a three-enzyme complex of RIDD-PETase, RIDD-MHETase and RIAD-hydrophobin4 that is combined with RIDD-RIDD-RIAD (Figure 3), and use this complex to degrade PET plastic effectively.
Figure 4: Plasmid construction stagey