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BhrPETase N205G

Plastic pollution poses a serious threat to the global environment. One of the potential solutions, enzyme degradation, would be a suitable approach of dealing with plastic wastes. Among all plastic pollutions, more than 10% of them are Polyethylene terephthalate (PET). Thus, our team has been searching for possible PET hydrolases to break down PET. However, according to Nature's publishment on April 27, 2022, traditional PET hydrolases' enzymatic ability of degrading PET are easily affected by the fluctuation of temperature and pH value. Therefore, we decided to artificially mutate wild-type BhrPETase to increase the enzyme’s range of tolerance so that it can efficiently degrade PET under a wider range of environmental conditions, thereby enhance its potential application. BhrPETase was identified by the Shingo group in a metagenomic study on uncultured thermophiles and was deposited into the NCBI database by the group in 2018 and annotated as a PET hydrolase. As one of the most-confident mutants created in our lab, this basic part encodes mutated BhrPETase N205G.

Usage and Biology

To generate mutated variants, we have trained a Transformer AI model. This model predicts the top 10 potential mutation sites, which are likely to have significant impacts on the enzyme's structure and function. Next, we analyzed the top 10 potential sites via Meta's ESM-1b model to eliminate the silent mutations, which involve changes in nucleotides that do not altering the corresponding amino acids. This ensures that the mutations result in changes in the enzyme's structure and thereby its function. For further information, please check the model page on our wiki. https://2024.igem.wiki/basis-china/model

The BhrPETase N205G sequence is expressed in E.coli BL21(DE3) using the pET28a vector. The pET-28a is a classical plasmid vector used for protein expression in E.coli. This vector contains the T7 promoter, the lac operator, a ribosome binding site, the 6xHis sequence, and the T7 terminator. The T7 promoter is a strong promoter recognizable by T7 RNA polymerase, used to regulate gene expression of recombinant proteins. The lac operator can be activated by IPTG and used to control gene expression. The 6xHis sequence encodes for a tag that facilitates protein purification. Asides from the features included in the plasmid backbone, we added a signal peptide sequence — pELB — before the BhrPETase N205G sequence, which is inserted between the promoter and terminator.

We tested for successful plasmid construction and transformation into E.coli through colony PCR and gel electrophoresis. The following gel result demonstrates that the plasmid transformed into E.coli are correct. The plasmid should have a total of 891 base pairs and the results match.

Sequencing also demonstrated successful plasmid construction.

After confirming that the BhrPETase N205G is present in our chassis, we tested whether the bacteria can survive as usual with our genes. Thus, we coated the bacteria on petri dishes to observe their growth. After a night of incubation, E. coli grew over the plate, justifying that E. coli can survive with the gene encoding BhrPETase N205G.

Next, we tested the synthesis of BhrPETase N205G in E.coli through SDS PAGE. The results show that the enzymes synthesized in E.coli an secreted are in fact BhrPETase N205G.

To test the potential PET degradation efficiency of the BhrPETase N205G synthesized in E.coli we applied the p-nitrophenyl butryte degradation assay from the iGEM19_Toronto team (for more details, please see protocols). The following graph shows the enzyme activities of BhrPETase WT and IsPETase WT compared to the mutations N191S, M57L, W229F, N205G. The mutation BhrPETase N205G has a higher enzyme activity than BhrPETase WT at 30 min, and has certain potential to surpass the efficiency of BhrPETase if given more time.

Following the protocol, we ran the assay for 30 minutes. From the results above, we can observe that N205G only has a slightly higher efficiency compared to wild type BhrPETase. We hypothesize that N205G will significantly surpass BhrPETase if given more time. Hence, we created a math model to predict the pathway of the curves beyond 30 minutes. The math model supports our hypothesis, indicating that N205G will exhibit higher efficiency than WT BhrPETase if the assay is extended beyond 30 minutes.

To further measure the BhrPETase N205G efficiency we used a scanning electron microscope to directly observe changes in the plastic after degradation. Different plastic samples were placed in the culture medium of the engineered E. coli. After two weeks, the samples were observed under an SEM for any alterations in the surface of the plastic by technicians at Shenzhen University. The results demonstrated that plastic with low level of crystallinity were degraded under the exposure to PETase synthesized by our engineered E. coli. Further, the fact that plastic with high crystallinity did not show any significant changes addresses our hypothesis in Cycle 1: PET degradation is affected by the crystallinity of the plastic, which varies depending on its manufacturing process.The SEM allows us to see the changes of plastic pieces with our bare eyes.

SEM procedure:

1 Cultivate the bacteria with PET for 14 days.

2 Remove and clean with water

3 Soak in 72% ethanol for 10 minutes (sterilization)

4 Soak in 100% ethanol for 10 minutes

5 Replace the ethanol and soak again in 100% ethanol for half an hour.

6 Dry well in an ultra-clean bench

7 Hand over to engineer

After determining that our enzyme BhrPETase N205G expressed in E.coli is actually more efficient than wild type BhrPETase N205G, we moved on to expressed the gene in Synechococcus elongates PCC 7942. To achieve this we inserted the signal peptide and BhrPETase N205G sequence between the PpsbA2 and terminator Bba_B0015 of a transfer vector.

The reconstructed plasmids were transformed into cyanobacteria and coated on BG11 plates containing the antibiotic spectacularionomycin. Single colonies containing the BhrPETase N205G gene appeared after two weeks of incubation.

For more direct indication of PET degradation by BhrPETase N205G, we conducted another test using high-performance liquid chromatography. HPLC is a method to directly detect the presence of specific compound in a chemical mixture. During the degradation of PET by PETase, TPA is created as a byproduct; hence, the presence of TPA in the final product indicates degradation occurred. The results show that TPA is present after a 2 week incubation of PET with the engineered bacteria.

Sequence and Features

Reference

Lu, Hongyuan, et al. “Machine Learning-Aided Engineering of Hydrolases for Pet Depolymerization.” Nature News, Nature Publishing Group, 27 Apr. 2022, www.nature.com/articles/s41586-022-04599-z. Kato, Shingo, et al. “Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle.” Microbes and Environments, vol. 33, no. 1, Jan. 2018, pp. 107–10. https://doi.org/10.1264/jsme2.me17165.