Cycle 1: Rational design and expression of enzymes

Design：

To improve the stability of plastic-degrading enzymes, we hope to use machine learning to predict stable mutations of the enzymes. At the same time, in order to improve the acid-base tolerance of plastic-degrading enzymes, the predicted glycosylation sites are combined with the results of machine learning to find several optimal mutation sites, and then the mutated enzymes are used for the degradation of PET plastic. Through these operations, we expect to comprehensively improve the degradation rate and efficiency of plastic-degrading enzymes.

Figure 1. The DBTL cycle of the machine learning process.

We performed a comprehensive virtual mutation scan (https://mutcompute.com/view/8J17) for the modified enzyme using MutCompute to obtain a discrete probability distribution for the structural adaptation of all 20 standard amino acids at each position of IsPETase^PA (crystal structure, PDB: 8J17). The predicted distributions are rendered to the protein crystal structure to identify poorly adaptable positions of wild-type amino acid residues that may be better adapted by substitutes, and then ranked by the predicted probability (fold change in fit). Meanwhile, O-and N-glycosylation sites can be predicted by inputting the amino acid sequence on two sites, YinOYang 1.2-DTU Health Tech-Bioinformatic Services and NetNGlyc 1.0-DTU Health Tech-Bioinformatic Services, respectively. After finding the glycosylation site, the machine learning listed points and the protein on Snapgene sequence comparison, check the number of corresponding protein, if found consistent, and machine learning to detect the possibility of mutation site comparison again, if the site are consistent, and the site also belongs to the selected glycosylation site, so the site is determined as mutation site, determine all the mutation site, according to the highest mutation probability for follow-up experiments. Finally, four mutation sites (S29A, T59S, T122P, and N183A) were selected at IsPETase^PA.

The N,O-glycosylation sites predicted by DTU Health Tech

Stable mutation sites as predicted by machine learning.

Figure 2. Plot of the mutation sites for IsPETase^PA.

Build:

Four single-site mutant plasmids were obtained by designing mutant primers and using KOD amplification.

Figure 3. Mutation of the IsPETase^PA deglycosylation sites.

Test:

Wet lab validation

In wet experiments, we designed mutant primers, build single point mutant plasmids, and by KOD Neo PCR amplification, mutant DNA, and run glue verification, PCR purification, DPnI enzyme digestion background plasmid, PCR purification of linear DNA, by homologous recombination, and large intestine transformation, shock into the Pichia yeast, mutation of the enzyme degradation efficiency of PET plastic in fermentation experiments.

Figure 4 .KOD amplification, verified by glue running. 1：29 Mutation site；2：59 Mutation site；3：122 Mutation site；4: 183 Mutation site.

The plasmids of the four IsPETase^PA single mutation sites were 8507bp, verified by glue, and the bands were correct.

After introducing the constructed plasmids into E. coli and sequencing them successfully, we linearized the plasmid and introduced the mutant plasmid into Pichia yeast by electroshock.

Figure 5. KOD amplification, verified by glue running. 4：29 Mutation site；5：59 Mutation site；6：122 Mutation site；7: 183 Mutation site.

The plasmids of the four IsPETase^PA single mutation sites were 8507bp, verified by glue running, with correct bands and successful linearization. Therefore, we introduced it into Pichia pastoris, designed primers, and performed PCR verification of yeast colonies.

Figure 6. a. PCR of colonies at the deglycosylation site number 29. b. PCR of the deglycosylation site number 59. c. PCR of the deglycosylation site at position 122. d. PCR of the deglycosylation site at position 183.

All sequence lengths were 819bp with correct bands, and plasmids at four IsPETase^PA single mutation sites were successfully driven into Pichia pastoris.

Figure 7 .KOD amplification, verified by glue running. 1：92 Mutation site；2：169 Mutation site；3：190 Mutation site；4: 212 Mutation site；5：223 Mutation site.

The plasmids of the five FAST-PETase-212 / 277 single mutation sites were 8576bp, verified by glue with correct bands.

After introducing the successful constructed plasmids into E. coli and sequenced them successfully, we linearized the plasmid and introduced the mutant plasmid into Pichia yeast by electroshock.Therefore, we introduced it into Pichia pastoris, designed primers, and performed PCR verification of yeast colonies.

Figure 8 .1：IsPETase^PA; 2: Deglycosylation of IsPETase^PA；3：29 mutant IsPETase^PA4：59 mutant IsPETase^PA；5：122 mutant IsPETase^PA；6：183 mutant IsPETase^PA Correct deglycosylation was verified by protein glue.

Molecular docking validation:

We used AutoDock to dock IsPETase^PA with Polyethylene terephthalate (PET) and 1- (2-hydroxyethyl) 4-methyl terephthalate) (HEMT), analyzed and evaluated the results, and found that the mutations selected by machine learning and predicted glycosylation sites were indeed helpful to improve the heat resistance, acid-base tolerance and catalytic activity of the enzyme.

Figure 9. Molecular docking of IsPETase^PA to PET before and after the mutation. a. Molecular docking of unmutated IsPETase^PA with PET. b. Molecular docking of SER-29-ALA and PET. c. Molecular docking of THR-59-SER to PET. d. Molecular docking of THR-122-PRO to PET. e. Molecular docking of ASN-183-ALA to PET. The Score can be seen as a binding free energy.

Figure 10.Results of IsPETase^PA docking with PET.

Figure 11. Docking of the IsPETase^PA with HEMT before and after the mutation. a. Molecular docking of unmutated IsPETase^PAwith HEMT. b. Molecular docking of SER-29-ALA and HEMT. c. Molecular docking of THR-59-SER with HEMT. d. Molecular docking of THR-122-PRO with HEMT. e. Molecular docking of ASN-183-ALA to HEMT. The Score can be seen as the binding free energy.

Frigure 12. Results of IsPETase^PA docking with HEMT.

Analysis of docking results:

Based on the docking results, considering the binding free energy, root mean square deviation (RMSD), number and length of hydrogen bonds from Autodock, we found that the mutations at positions 122 and 183 are the best mutations for IsPETase^PA.

Learn:

Through experimental and molecular docking verification, we can show that machine learning of MutCompute and mutation of N, O-glycosylation sites can effectively improve the degradation efficiency of plastic-degrading enzymes. Therefore, in order to further improve the degradation efficiency of plastic-degrading enzymes, further massive machine learning prediction and mutation is necessary. Combined with the verification of experiments and molecular docking, the optimization iteration is constantly carried out, that is, better plastic degradative enzymes can be obtained.

Cycle 2: Optimization of promoters and signal peptides

Design:

The common promoters of Pichia pastoris are AOX1, GAP, etc., and the common signal peptides are α signal peptide and MF signal peptide.However, how to select a stronger promoter and signal peptide to improve the secretion of Pichia pastoris exogenous protein is a hot issue, and it is cumbersome to seek high-expression promoter and signal peptide through mutation, so we choose the way of matching the promoter and signal peptide to screen out a stronger promoter and signal peptide assembly mode.α

Build:

We constructed four recombinant plasmids，AOX1-MF-IsPETase^PA，AOXm-MF-IsPETase^PA,GAP-MF-IsPETase^PA，AOXm-α-IsPETase^PA.

Test:

Glue running verification and glue recovery

1: AOXm-IsPETase skeleton(8240bp)；2: MF-IsPETase skeleton(7628bp)

	ng/µL	A260/280	A260/230
AOXm-IsPETase skeleton	38.9	1.84	1.42
MF-IsPETase skeleton	167.9	1.81	1.31

1: AOX1

1: α-signal peptide 2: GAP

	ng/µL	A260/280	A260/230
α-signal peptide	128.8	1.89	0.21
GAP	207.3	1.86	0.64
AOX1	120	1.85	1.15

Colony PCR, verification of Recombinant Plasmid by Gel-running

Strain: α-signal peptide(267bp)

Strain: AOX1(944bp)

Strain: GAP(477bp）

The above four plasmids were successfully constructed and introduced into E. Coil, and the sequence was verified successfully.

Recombinant plasmid linearization

1: AOXm-α-signal peptide-IsPETase^PA，2: GAP-MF-IsPETase^PA；3: AOX1-MF-IsPETase^PA

PCR products were purified and electroporated to transfer the plasmid into yeast, and then we performed yeast colony PCR.

AOXm-α-signal peptide-IsPETase^PA

GAP-MF-IsPETase^PA

AOX1-MF-IsPETase^PA

The bands were correct and the three recombinant plasmids were successfully introduced into Pichia pastoris. This was followed by a five-day fermentation to determine the total protein concentration.

At the same time, 25 microliters of samples were taken and the protein gel was run to verify its content.

1: AOXm-α-signal peptide-IsPETase^PA,2: GAP-MF-IsPETase^PA;3: AOX1-MF-IsPETase^PA;4: AOXm-MF-signal peptide-IsPETase^PA

Through experiments, it was found that the total protein concentration was the highest for five consecutive days with the combination of promoter and signal peptide of AOXm-MF, and the widest band after running protein gel. Therefore, among the combinations of AOX1-MF, AOXm-MF, GAP-MF and AOXm-α, the combination of AOXm-MF was optimized to achieve the highest amount of enzyme expressing IsPETase^PA.

Learn:

It can be seen that in the future, we can mutate the existing promoters and signal peptides, screen the promoters and signal peptides with higher protein expression after mutation, and then arrange and combine them to improve the expression of plastic degradation enzymes.

Cycle 3：Construction of the Dual-Enzyme System

Design:

The researchers in the article Enhancing the biodegradation of bis(2-hydroxyethyl) terephthalate by an IsPETase^PA and MHETase dual-enzyme system found that the synergistic effect of MHETase and IsPETase^PA can enhance the degradation capacity ¹. Inspired by this, our goal is to construct a dual-enzyme system to improve enzyme efficiency in plastic degradation.

We reviewed numerous studies and identified three highly efficient plastic-degrading enzymes: IsPETase^PA, FAST-PETase-212/277 and MHETase ¹².

IsPETase^PA:

At ambient temperature (30°C) and pH 7.0, the activity of IsPETase on PET film and BHET is 120.0, 5.5, and 88.0 times higher than that of previously identified enzymes TfH, LCC, and FsC, respectively. Its activity on highly crystalline commercial bottle-derived PET is at least 20 times higher than other PET hydrolases. Furthermore, IsPETase exclusively degrades PET and does not degrade other types of polyester plastics ³. Engineering efforts have been focused on improving the performance of the native enzyme. Some studies have rationalized the design of PETase to enhance degradation efficiency^{4 5}, and site-saturation mutagenesis was performed on key sites of IsPETase, resulting in the S92P/D157A (IsPETase^PA) variant, which exhibited a 24.75-fold increase in activity compared to the wild type at 40°C⁶. Moreover, the complex procedures and high costs of protein purification limit the industrial application of enzymes^{7 8}. Therefore, in practical applications, unpurified whole cells or crude enzyme extracts are preferred, and these crude fermentation products can be directly used for degradation.

FAST-PETase-212/277:

Researchers developed a potent PET hydrolase called FAST-PETase using industrial yeast Pichia pastoris. Through molecular engineering, they removed two N-chain glycosylation sites, further improving its performance. The strain FAST-PETase-212/277 was produced in a 30-L fermenter with antibiotic selection and co-expression of chaperones, yielding over 3 g/L. Notably, the crude fermentation product can be directly used to decompose PET without the need for purification. In a 10-L reactor system, within 24 hours, 0.5 mg enzyme g⁻¹ PET achieved over 95% degradation of post-consumer PET. These results demonstrate the economic feasibility of large-scale PET degradation using PET hydrolases produced in modern fermentation facilities ².

MHETase:

The addition of MHETase catalyzes the complete hydrolysis of MHET, an intermediate produced during PET degradation. When combined with IsPETase, MHETase significantly enhances PET degradation efficiency, similar to the improvements observed with IsMHETase, making it a viable candidate for constructing dual-enzyme PET degradation systems ⁹. We constructed two dual-enzyme systems: the MHETase-IsPETase^PA system and the MHETase-FAST-PETase-212/277 system. In these systems, IsPETase^PA/FAST-PETase-212/277 efficiently degrades PET, yielding intermediate products MHET and BHET. MHETase further converts these intermediates into TPA and EG. Additionally, IsPETase^PA/FAST-PETase-212/277 exhibits some degradation activity toward BHET. This synergistic effect not only ensures the complete conversion of intermediates to TPA, thereby preventing inhibition of subsequent reactions by intermediate products, but also improves the overall plastic degradation efficiency, enabling the sustainable utilization of the resulting products.

Build:

Our design involves constructing the dual-enzyme system through the homologous recombination and restriction enzyme digestion method in genetic engineering. This method combines the specific cleavage ability of restriction enzymes with the high precision of homologous recombination, used for constructing gene knock-in or knock-out vectors. The approach involves designing a vector containing homologous arms of the target gene and using restriction enzymes to cut both the vector and the target genome, allowing the vector to integrate or replace the genome at homologous regions through homologous recombination. MHETase, IsPETase^PA, and FAST-PETase-212/277 were subjected to single and double enzyme digestions, with BamH I used to cut the fragment in the single digestion, and BamH I and BglII used to cut the vector in the double digestion, generating sequences that are neither BglII nor BamH I sites ¹⁰.

Figure 13 Flowchart of Homologous Recombination Steps

Figure 14 MHETase-IsPETase^PA plasmid

Figure 15 MHETase-FAST-PETase-212/277 plasmid

Simultaneously dephosphorylate to prevent plasmid self-ligation, and use T4 ligase to ligate the vector and fragments, resulting in two dual-enzyme systems: MHETase-IsPETase^PA and MHETase-FAST-PETase-212/277.

Test:

We successfully constructed the dual-enzyme systems using the gene engineering method of homologous recombination and restriction enzyme digestion (single enzyme digestion for fragments and double enzyme digestion for vectors). The recombinant plasmids of the MHETase-IsPETase^PA and MHETase-FAST-PETase-212/277 dual-enzyme systems were transformed into Escherichia coli by heat shock. Colony PCR was used to verify whether the plasmids were successfully constructed.

Figure 16 Colony PCR verification of MHET-IsPETase^PA a 5000 bp marker was used, and the correct bands for MHET-IsPETase^PA appeared in colonies 2 and 3, both around 3000 bp.

Figure 17 Colony PCR verification of MHETase-FAST-PETase-212/277: a 5000 bp marker was used, and the correct bands for MHETase-FAST-PETase-212/277 appeared in colonies 1, 4, and 5, all around 3000 bp.

Colonies containing the correct plasmid were selected and cultured for plasmid extraction. The plasmids were then linearized and electroporated into Pichia pastoris chassis cells. Yeast colony PCR was performed to further verify the successful transformation of the recombinant plasmid in Pichia pastoris.

Figure 18 Yeast colony PCR verification of MHETase-FAST-PETase-212/277 (left) and MHET-IsPETase^PA (right): a 5000 bp marker was used. The correct bands for MHETase-FAST-PETase-212/277 appeared in colonies 1, 5, 6, 8 and 10, all around 3000 bp. The correct bands for MHET-IsPETase^PA appeared in colonies 3, 4, 5, 6, 7, 8, and 10. These colonies can be inoculated into BMGY for fermentation.

Inoculate the selected colonies into BMGY medium for fermentation to measure the expression levels of recombinant products (determining protein concentration). At the same time, monitor the dual-enzyme degradation efficiency using HPLC analysis, observing improvements in enzyme expression levels and degradation efficiency.

Figure 19 Protein concentration changes over the 5-day fermentation period.

Learn:

Using homologous recombination and restriction enzyme digestion, we successfully constructed the dual-enzyme systems MHETase-IsPETase^PA and MHETase-FAST-PETase-212/277. Recombinant plasmid transformation and expression were verified in Escherichia coli and Pichia pastoris. Additionally, colonies were selected for activation, fermentation, and protein expression measurement, showing an overall upward trend in dual-enzyme protein concentration. The expression level of MHETase-IsPETase^PA peaked on the 4th day at approximately 4.5 mg/ml, while MHETase-FAST-PETase-212/277 reached its highest expression level on the 2nd day at 5.75 mg/ml, representing a 91.67% increase compared to the 3 mg/ml crude fermentation extract of FAST-PETase-212/277 mentioned in the literature. This demonstrates a significant enhancement in the protein expression levels of the dual-enzyme systems.

Our future work can focus on further improving the efficiency of enzyme-based plastic degradation and expanding the construction of dual-enzyme systems. By mutating the enzymes MHETase and Is-PETase ^PA and then constructing the dual-enzyme system, we aim to increase enzyme activity and substrate-binding ability, ultimately achieving more efficient plastic degradation.

We have already used machine learning to mutate the two plastic-degrading enzymes and tested their substrate-binding capabilities through molecular docking, selecting the optimal mutant enzymes. In the future, machine learning could be used to modify plastic-degrading enzymes and construct dual-enzyme systems, addressing issues such as low degradation efficiency and providing an effective solution.

Cycle 4：Multi-Copy Molecular Experiment

Design:

Increasing the copy number of the target gene can often effectively enhance the expression level of the desired protein. However, an excessively high copy number may impose endoplasmic reticulum stress on the engineered strain, leading to reduced expression levels. Therefore, an appropriate gene copy number is beneficial for the expression of exogenous proteins. Currently, constructing multi-copy expression vectors in vitro and transforming yeast to obtain multi-copy strains is one of the commonly used techniques ¹.

We aim to increase the copy number to enhance protease expression, as higher enzyme expression contributes to improved degradation efficiency. In terms of multi-copy numbers, we constructed a multi-copy gene expression cassette based on the existing patent A Method for Constructing Yeast Multi-Copy Expression Vectors. This method involves first selecting a first vector and a second vector containing the target gene, then using a pair of isocaudamer restriction enzymes and a reference enzyme to digest the first and second vectors to obtain the sub-sequences and parent sequences of the target gene. The target vector containing a reference fragment is obtained, and rapid screening is performed using the reference fragment to acquire the multi-copy expression vector. This method allows for the quick and accurate construction of a multi-copy vector containing a specific gene and copy number, reducing the probability of false positives caused by random ligation direction of expression cassettes, direct ligation of vectors, or incomplete digestion in conventional multi-copy expression vector construction methods. The process is simple and highly efficient ¹.

Figure 20 Schematic Diagram of the Construction of Yeast Multi-Copy Expression Vectors

We increased the copy number of IsPETase ^PAand FAST-PETase-212/277 by constructing in vitro multi-copy expression vectors using the pAO815 series vectors developed by Invitrogen. This method utilizes the isocaudamer restriction sites BglII and BamHI at both ends of the expression cassette. After digesting the expression cassette with these enzymes, the cassette is inserted into the BamHI site to produce tandem repeat expression cassettes ¹.

Multi-copy plasmids were constructed using the isocaudamer method (single and double enzyme digestion) with BglII and BamHI. The cut sites of BglII and BamHI were ligated with T4 ligase, forming sequences that are neither BglII nor BamHI, resulting in two-copy IsPETase PA-2C and FAST-PETase-212/277-2C.

Build:

Figure 21 Flowchart for Constructing Dual-Copy IsPETase PA-2C and FAST-PETase-212/277-2C Expression Vectors

Figure 22 IsPETase PA-2C plasmid

Test:

We successfully constructed the dual-copy plasmids IsPETase PA-2C and FAST-PETase-212/277-2C, and transformed them into Escherichia coli for colony PCR identification to select the correct colonies.

Figure 23 After transforming FAST-PETase-212/277-2C into Escherichia coli and performing colony PCR, gel electrophoresis was used for identification. A 5000 bp marker was selected, and lanes 3 and 4 showed double bands, with the larger band above 3000 bp and the smaller band around 1000 bp.

Plasmids were extracted from E. coli containing the successfully constructed plasmids and were transformed into Pichia pastoris using linearized electroporation. IsPETase PA-2C was successfully identified, and yeast colony PCR further confirmed the successful transformation and expression of the recombinant product. The purified strain was then inoculated into BMGY medium for activation.

Figure 24 Yeast colony PCR for IsPETase PA-2C showed double bands, with a 15,000 bp marker used. Lanes 1, 2, 3, 4, 5, and 6 displayed correct bands, all below 1000 bp, around 800 bp. These colonies can be inoculated into BMGY for fermentation.

Quantitative analysis of the protein products obtained from fermentation (measuring protein concentration) was conducted to verify that the multi-copy expression resulted in higher enzyme production, which helps improve degradation efficiency.

Figure 25 Comparison of Protein Concentration Changes of IsPETase^PA and IsPETase PA-2C at pH=6 and pH=7

Learn:

Using the isocaudamer method, we successfully constructed the dual-copy IsPETase PA-2C vector expression cassette and successfully transformed it into Escherichia coli and Pichia pastoris for identification. After activating the selected colonies and measuring protein expression levels during fermentation, we found that the expression of IsPETase PA-2C exhibited a more stable upward trend compared to IsPETase PA, which showed a declining trend and a significant reduction in enzyme activity. Specifically, IsPETase PA-2C reached its highest expression level of approximately 8.0 mg/ml on the fifth day, with higher protein expression under pH=6 conditions.

Unfortunately, the constructed FAST-PETase-212/277-2C was difficult to enrich on plates after identification in Pichia pastoris, and due to time constraints, we have not yet conducted colony activation for fermentation.

In the future, we will continue to improve the construction of the FAST-PETase-212/277-2C dual-copy expression cassette and perform verification experiments in fermentation. Additionally, we will further analyze and explore the effects of multi-copy numbers on plastic degradation, aiming to expand from the existing two-copy to four-copy, eight-copy, and other high-copy systems. However, an excessively high copy number may impose endoplasmic reticulum stress on the engineered strain, leading to reduced expression levels. Ongoing experiments will help us identify the optimal copy number and its impact on protein expression levels.

As mentioned earlier, we have already obtained high-efficiency mutant enzymes, so in the future, we will also focus on increasing the copy number based on these mutations. This will enhance enzyme activity while increasing the expression of highly efficient enzymes, providing valuable insights for the future prospects of enzyme-mediated plastic degradation.

Reference

Footnotes

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2. CHEN C-C, LI X, MIN J, et al. Complete decomposition of poly(ethylene terephthalate) by crude PET hydrolytic enzyme produced in Pichia pastoris [J]. Chemical Engineering Journal, 2024, 481: 148418. ↩ ↩²

3. Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL et al. , Characterization and engineering of a plastic-degrading aro-matic polyesterase. Proc Natl Acad Sci U S A 115:E4350‒ E4357 (2018). ↩

4. Son HF, Cho IJ, Joo S, Seo H, Sagong HY, Choi SY et al., Rational protein engineering of thermo-stable PETase from Ideonella sakaiensis for highly effi cient PET degradation. ACS Catal 9:3519‒ 3526 (2019). ↩

5. Wei Yi, Xiao Yunjie, Yang Haitao, Wang Zefang. Polyethylene terephthalate hydrolase IsPETase and its application prospect. Acta Microbiologica Sinica, 2023, 63(1): 15-29. ↩

6. Yin QD, Zhang JX, Ma S, Gu T, Wang MF, You SP et al., Effi cient polyethylene terephthalate biodegradation by an engineered Ideonella sakaiensis PETase with a fi xed substrate-binding W156 residue. Green Chem 26:2560‒ 2570 (2023). ↩

7. Fang YX, Chao KX, He J, Wang ZG and Chen ZM, High-effi ciency depolymerization/degradation of polyethylene terephthalate plas-tic by a whole-cell biocatalyst. 3 Biotech 13:138 (2023). ↩

8. Gercke D, Furtmann C, Tozakidis IEP and Jose J, Highly crystalline postconsumer PET waste hydrolysis by surface displayed PETase using a bacterial whole-cellbiocatalyst. ChemCatChem 13:3479‒ 3489 (2021). ↩

9. LIU Xinyue, GENG Wenchao, SUN Jinyuan, CHEN Zehua, CUI Yinglu, WU Bian. Structure motif guided mining of MHET hydrolase and development of a two-enzyme cascade for plastics depolymerization at mild temperature[J]. Chinese Journal of Biotechnology, 2024, 40(3): 773-785. ↩

10. Griswold KE, Kawarasaki Y, Ghoneim N, Benkovic SJ, Iverson BL, Georgiou G. Evolution of highly active enzymes by homology-independent recombination. Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10082-7. ↩