Part 1 Screening of chassis cells and construction of recobinant strain for PET hydrolase

1.1 Construction of expression plasmids.

For expression PET hydrolase in bacteria or yeast. Genes encoding the IsPETase^PA and FAST-PETase-212/277 were synthesized according to codon-optimized method for Pichia pastoris or E. coli. The basic parts are BBa_K5249002 (IsPETase^PA for E.coli), BBa_K5249003 (FAST-PETase-212/277 for Pichia pastoris). Basic part BBa_K5249012 with mCherry and 6×His coding sequence at the C-terminus was firstly cloned scarlessly into plasmid pET28a using T7 promoter with kanamycin resistance. In addition, basic part . BBa_K5249000 was cloned into vector pHKA plasmid. The PCR products were digested with DpnI to remove the template DNA, and then transformed into E. coli Top 10 cells with selection for resistance to kanamycin (50 mg/mL). All constructed plasmids were validated by PCR and DNA sequencing.

1.2 Expression of PET hydrolase

All expression vectors were transformed into BL21 (DE3) or GS115 cells via heat shock or electroporation according to the protocol. E. coli expression cultures were cultivated at 37℃ in 2 L shake flasks in a culture volume of 600 mL lysogenic broth medium supplemented with 50 µg/mL Kanamycin; cultures were induced at an optical density of 0.3-0.5 at 600 nm via the addition of IPTG (1 mM final concentration) and subsequently incubated for 20 h at 16℃ shaking at 250 rpm. For yeast production, single colonies were inoculated in 5 mL YPD and cultured at 30℃ for 24 h prior to induction by BMMY at 30℃ for 120 h. The protein expression in culture supernatant was examined by SDS-PAGE or BCA kit, and the highest clones were chosen for further experiments.

Part 2 Rational design for enhanced PET Hydrolase activity

2.1 MutCompute Predictions

MutCompute predictions were obtained by running IsPETase^PA (PDB ID: 8J17) and FAST-PETase-212/277 (PDB ID: 7SH6) at https://mutcompute.com. Residues were filtered by sorting by the probability assigned to the wild type amino acid. We filtered 56 and 52 residues from IsPETase^PA and FAST-PETase-212/277 crystal structures, respectively. From these filtered residues, we prioritized our experimental mutagenesis by selecting the 10 residues from each crystal structure with the highest log2 fold change between the predicted amino acid and wild type amino acid probabilities. Prior to experimentation, selected residues were visualized with Mutcompute-View, which is built on top of NGLViewer https://github.com/nglviewer/ngl, to ensure three things: 1) the prediction was chemically sound and not due to a crystal or model artifact, 2) we were avoiding the active site and binding pocket and 3) avoiding epistatic interactions between predictions and instead targeting predicted “instability hotspots”. MutCompute-View has been made publicly available at https://mutcompute.com/view.

2.2 Molecular docking simulations

Molecular docking simulations were performed using the AutoDock molecular docking software. Input files were prepared using the AutoDock tools desktop application. Molecular docking was performed to generate geometries for the enzyme-substrate complex for both the HEMT and PET ligands. The coordinate structure of the enzyme was obtained from the protein data bank (PDB ID: 8J17 and 7SH6). By comparing the binding free energies of the PET and HEMT before and after the mutation, RMSD values and the number and length of hydrogen bonds, the rationality and validity of machine learning can be verified.

2.3 Glycosylation site analyses

N-glycosylation site analyses were carried out using the NetNGlyc 1.0 Server http://www.cbs.dtu.dk/services/NetNGlyc/; accessed on 20 December 2021). We predicted that both IsPETase^PA and FAST-PETase-212/277 had 10 N-glycosylation sites, respectively. O-glycosylation site analyses were carried out using the NetNGlyc 1.0 Server (NetNGlyc 1.0 - DTU Health Tech - Bioinformatic Services). We predicted that IsPETase^PA and FAST-PETase-212/277 had 56 and 57 O-glycosylation sites, respectively. According to MutCompute Predictions and Molecular docking simulations, 4 mutagenesis sites from IsPETase^PA and 5 mutagenesis sites from FAST-PETase-212/277 were chosen.

Part 3 Verification of plastic degradation conditions

3.1 Plastic degradation experiments of IsPETase^PA and its mutants

Under dynamic conditions at 30°C (200 rpm/min), a pH gradient of 6.0, 7.0, 8.0, 9.0, and 10.0 is set in 96-well plates to conduct PET degradation experiments using IsPETase^PA mutants (mutation sites at positions 29, 59, 122, and 183). The total volume is 200 μL, and the protein concentration in the system (enzyme solution + buffer) is controlled at 50 μg/200 μL to degrade PET discs with a diameter of 4.5 mm. Without replacing the enzyme solution, plastic degradation by each enzyme over 120 hours is observed through color changes.

Figure 1

Under dynamic conditions at 40°C (200 rpm/min), a pH gradient of 6.0, 7.0, 8.0, 9.0, and 10.0 is set in 96-well plates to conduct PET degradation experiments using IsPETase^PA and its mutants (mutation sites at positions S29A, T59S, T122P, and N183A). The total volume is 200 μL, and the protein concentration in the system (enzyme solution + buffer) is controlled at 50 μg/200 μL to degrade PET discs with a diameter of 4.5 mm. The enzyme solution is replaced every 24 hours, and the degradation of plastic by each enzyme over 120 hours is analyzed by HPLC and SEM.
Under static conditions at 40°C, a pH gradient of 6.0, 7.0, 8.0, 9.0, and 10.0 is set in 96-well plates to conduct PET degradation experiments using IsPETase^PA and its mutants (mutation sites at positions S29A, T59S, T122P, and N183A). The total volume is 200 μL, and the protein concentration in the system (enzyme solution + buffer) is controlled at 50 μg/200 μL to degrade PET discs with a diameter of 4.5 mm. The enzyme solution is replaced every 24 hours, and the degradation of plastic by each enzyme over 120 hours is analyzed by HPLC and SEM.
Under static conditions at 30°C, 40°C, and 50°C, a pH gradient of 6.0, 7.0, 8.0, 9.0, and 10.0 is set in 96-well plates to conduct PET degradation experiments using IsPETase^PA and its mutant (mutation site at position T122P) . The total volume is 200 μL, and the protein concentration in the system (enzyme solution + buffer) is controlled at 50 μg/200 μL to degrade PET discs with a diameter of 4.5 mm. The degradation of plastic by each enzyme over 24 hours is analyzed by HPLC and SEM.
Under static conditions at 30°C, a pH of 9.0 is set in 96-well plates to conduct PET degradation experiments using IsPETase^PA mutants (mutation sites at positions T122P and N183A). The total volume is 200 μL, and the protein concentration in the system (enzyme solution + buffer) is controlled at 50 μg/200 μL to degrade PET discs with a diameter of 4.5 mm. The enzyme solution is replaced every 24 hours, and the degradation of plastic by each enzyme over 120 hours is analyzed by HPLC and SEM.
Under dynamic conditions at 30°C, a pH of 9.0 is set in 96-well plates to conduct PET degradation experiments using IsPETase^PA mutants (mutation sites at positions T122P and N183A). The total volume is 200 μL, and the protein concentration in the system (enzyme solution + buffer) is controlled at 50 μg/200 μL to degrade PET discs with a diameter of 4.5 mm. The enzyme solution is replaced every 24 hours, and the degradation of plastic by each enzyme over 120 hours is analyzed by HPLC and SEM.

Part 4 Product recycling and upgrading

4.1 Strain Screening

Terephthalic acid and ethylene glycol are known to significantly increase the production of bacterial cellulose. Therefore SUPERB found three strains with this ability through the literature, experimentally evaluated the ability of each strain to produce bacterial cellulose, and finally chose the best strain for mixed culture with Picrosporum to convert PET degradation products into bacterial cellulose.

DSM 2004

The HS liquid medium was set up with a pH gradient (3.0, 5.0, 6.0, 7.0), after which it was inoculated separately with DSM 2004 for static incubation, and the status of bacterial cellulose film-forming was continuously observed over a period of fifteen days.

CCTCC AB2012861

CCTCC AB2012861 was inoculated into HS pH 5.45 liquid medium for static incubation, and the bacterial cellulose film-forming status was continuously observed for fifteen days.

BC 2R

BC 2R was inoculated into HS pH 5.0 liquid medium for static incubation and the bacterial cellulose film-forming status was continuously observed over a period of fifteen days.

4.2 Monomicrobial Growth

Pichia pastories GS115 is an industrial strain that can be widely used in enzyme production and is suitable for dynamic cultivation in liquid medium, commonly used mediums are YPD, BMGY, BMMY medium, with the pH value in the range of 5.0-6.0, and the suitable growth temperature is 28-30℃, if the temperature is more than 32℃ it is unfavourable to the protein expression and may lead to the death of the cells.

Komagataeibacter xylinus DSM 2004 is a Gram-negative, aerobic bacteria, the optimal pH range is 5.4-6.34, the optimal growth temperature is 25-30 ℃, commonly used HS liquid medium for culture, pH adjusted to 6.0.

GS115/△FHU/pp2c-mRFP

1)GS115/∆FHU/pp2c-mRFP were inoculated in BMMY medium at pH 6.0 and pH 7.0 for dynamic culture, respectively, and the OD and red fluorescence of GS115/∆FHU/pp2c-mRFP were subsequently measured on different media for five consecutive days.The growth of GS115/△FHU/pp2c-mRFP _was monitored through OD measurements, and the protein expression level of _GS115/△FHU/pp2c-mRFP was observed via red fluorescence.

2)The HS liquid medium was set on a pH gradient (3.0, 5.0, 6.0, 7.0), after which it was inoculated separately with DSM 2004 for static incubation, and the bacterial cellulose film-forming status was observed after fifteen days.

DSM 2004

DSM 2004 was inoculated in BMMY medium at pH 6.0 and pH 7.0 and HS medium at pH 6.0 and pH 7.0, respectively, for dynamic incubation, and the OD of DSM 2004 on different media was subsequently determined for five consecutive days.

4.3 Co-culture

We prepared four kinds of HS, BMMY, YPS and HS-sucrose culture-medium, and set pH 6.0 and pH 7.0 respectively, in accordance with 1: 1. The same OD ratio (both 0.05) was inoculated with GS115/△FHU/pp2c-mRFP and DSM 2004, and the co-culture conditions of the two bacteria were investigated under different culture methods of static culture and dynamic culture.Subsequently, bacterial cellulose was extracted and weighed to obtain the yield results, further analyzing the impact of various components of the culture media on the bacterial cellulose yield.

By comparing the composition and content of HS and HS-sucrose, we found that when glucose and sucrose were used as carbon sources, glucose could better promote the production of bacterial cellulose than sucrose. However, since glucose would inhibit the expression of AOX1 promoter, sucrose was selected as the carbon source.

To validate the above analysis results, we set up YPS and HS-sucrose media with pH= 6,7 to explore which medium produced more bacterial cellulose at the same pH and carbon source.

4.4 Mixed fermentation

Even though we eventually obtained BMMY-sucrose as the common co-culture medium for the growth of both strains in the experimental study, the yield of sucrose as a carbon source was significantly lower than that of glucose. Furthermore, we found that if the yeast was dynamically cultured in the BMMY medium to express the exogenous protein and degrade PET to generate EG and TPA, and then statically cultured to form a complete bacterial cellulose membrane, carbon source could be added to the medium during the static culture period, without considering the inhibitory effect of glucose on the yeast promoter.

In this experiment, the optimal conditions for mixed culture were explored, and the fermentation experiments were carried out, and the single factor optimization was carried out from the following aspects:

Carbon sources

Sucrose and glucose were used as the main carbon sources to carry out fermentation experiments in 96-well plates. The fermentation system (fermentation broth + carbon source solution + bacterial solution) was prepared with 200 μL as the total volume, the control DSM 2004 OD was 0.1 and the same concentration of carbon source solution, and the bacterial cellulose was weighed after seven days of fermentation.

Set the OD gradients to 0.1, 0.5, and 1.0 for fermentation experiments in a 96-well plate. The fermentation system (fermentation broth + glucose solution + bacterial solution) was prepared with an OD of 0.1, 0.5 and 1.0 with a total volume of 200 μL, respectively, and the bacterial cellulose was weighed after static mixed culture for seven days.

EG+TPA impact

The fermentation broth without adding PET and adding PET for five days for degradation was used as the basic system, and the DSM 2004 OD was 0.5 and the glucose solution of the same concentration was controlled, and the fermentation system (fermentation broth + carbon source solution + bacterial liquid) was prepared, and the bacterial cellulose was weighed after standing and mixed culture for seven days.

Part 5 Extended experiments

5.1 RGG-SZ1 & PETase-mcherry-SZ2

We introduced the plasmids PETase-mcherry-SZ2 and RGG-SZ1 into E.coli TOP10 by heat shock transformation. We transferred E. coli TOP10 into LB medium for activation, and when it grew to the logarithmic growth stage, we transferred it into TB medium with more nutrients, and let it continue to grow and expand in TB. When OD₆₀₀=0.6~0.8, 0.2M IPTG was added to induce expression. To make E. coli TOP10 secrete our PETase, which is then broken by ultrasound, releasing the proteins inside the cell to the outside.

In the process of purification, in order to find out the most suitable eluent conditions, we configured gradient eluents with different imidazole concentrations, followed by protein glue verification, and found that the optimal elution concentration of PET was 200mM (F1), and then we also used BCA kit to determine the protein concentration. Subsequently, we also carried out microscopic examination to verify that the mcherry red fluorescence in it could emit normal light, and RGG-SZ1 and PET-mcherry-SZ2 would obviously agglomerate together after mixing (F2), which proved that our multi-enzyme agglomerate was successfully constructed, and the two enzymes could indeed be joined together.

5.2 Multienzyme system

We took MHETase plasmid as the skeleton, performed enzyme digestion with Bamh I restriction enzyme, linearized the circular plasmid, and then performed enzyme digestion with Bamh I and Bgl II restriction enzyme to obtain FAST-PETase-212/277 and IsPETase^PA fragments. They were then linked with T4 ligase, and finally linearized with BamH I or Bgl II restriction enzymes for banding verification. In addition, plasmids with correct bands were transferred to E. coli TOP 10, andthen obtained single colonies by the dilution coating plate method, performed colony PCR and bacteria with correct bands were selected for expanded culture. After extracting the plasmids, they were linearized, and the linearized plasmids were introduced into Pichia Pastoris GS115 for expression by electric transfer.

5.3 Multi-copy

We took a single copy of the IsPETase^PA skeleton, digested it with Bamh I restriction endonuclease, linearized the circular plasmid, and then digested the IsPETase^PA plasmid with Bamh I and Bgl II restriction endonuclease to obtain the IsPETase^PA fragment and its skeleton, and linked it with T4 ligase. Finally, the multicopy plasmid was linearized with BamH I or Bgl II restriction enzymes and verified by running glue.

We also transferred the plasmids with correct bands to E. coli TOP 10, then obtained a single colony by thinning the coated plate, and performed colony pcr to take the bacteria with correct bands, expand culture, extract the plasmids, and linearize them. The linearized plasmids were introduced into Pichia Pastoris GS115 by electrotransfer for expression.

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