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PETase from Ideonella sakaiensis, a plastic-degrading bacterium

Noah Wallace, Dr. Todd D. Gruber

Christopher Newport University, Department of Molecular Biology and Chemistry

Abstract

PETase is an enzyme that is capable of degrading polyethylene terephthalate, the plastic commonly found in plastic water bottles We have developed assays to measure rates of plastic degradation by this enzyme and have cloned the PETase enzyme into an expression plasmid under an inducible promoter for use in E coli bacteria We have expressed the enzyme and can detect activity via a colorimetric assay using p-nitrophenyl acetate (PNPA), a small molecule substrate analog This assay is straightforward and useful We have additionally investigated the growth of Ideonella sakaiensis, the bacterium from which PETase was cloned, on different sources of PET Further, we have developed an assay in which plastic degradation can be monitored spectroscopically, opening the possibility of selecting for improved mutants of the parent organism Finally, we have determined that Ideonella likely forms a biofilm on the PET plastic in order to begin its degradation process

Other strains of bacteria have been found that can deteriorate other types of plastic and it is believed that there are many other forms of bacteria that have evolved the ability to metabolize plastic as a carbon source We believe an important next step in our research path is developing methodology to screen for other forms of plastic degrading bacteria Current methodologies are sparse and take on average several months, but we believe with our assays we can develop a methodology that cuts this screening time significantly Further environmental screening needs to be done to find these bacteria to better understand the enzymes that are used in this process Developing this methodology has three proposed steps

Objective 1: Separation of Ideonella from E coli Objective 2 Reisolating of Ideonella from an inoculated soil sample Objective 3: Isolation of a new plastic degrading bacteria

Background Information

Plastic waste is currently one of the worlds biggest problems.

In 2018 more than 400 million tons of plastic was produced, and 40 percent of it was single use plastic that was then thrown away1. Current recycling processes are lackluster and can not accommodate plastic contaminated by food or by the environment, such as ocean plastic. New methods of eliminating plastic waste need to be developed. Current research is already being conducted in our lab and others, using Ideonella sakaiensis a species of bacteria that can metabolize polyethylene terephthalate, a plastic common in plastic packaging. Ideonella is difficult to work with, proving challenging to genetically modify and grow in large quantities. Developing a procedure to better screen for plastic degrading bacteria needs to be performed to observe how other species of the bacteria break down plastic and potentially find a strain of bacteria that is easier to work with.

Previous Work

PET (polyethylene terephthalate) plastic has varying level of crystallinity depending on the plastics intended use and manufacturing process Highly crystalline plastic is impermeable to liquids and gasses and is well suited for containing liquids for applications in the plastic water bottle industry Amorphous plastic has some permeability to gasses and liquids and is common in clamshell containers for produce Ideonella can attach to amorphous plastic and degrade it but cannot attach to and degrade crystalline plastic Current literature has brushed around this topic but we have been able to quantify it We have also been able to show that plastic can be converted between different levels of crystallinity through melting at high temperatures By freezing the plastic quickly we can obtain amorphous plastic, letting the plastic cool slowly yields crystalline plastic This data can be seen in the graph below

Research Objective 1: Separation of Ideonella from E. coli

We have already begun research in ways separate our plastic degrading bacteria from other bacteria. The enzyme that breaks the polymer bonds is a strong esterase that also catalyzed reactions in p-nitrophenyl acetate, as well as fluorescein diacetate (FDA). Utilizing FDA in a top agar allows for the identification of bacteria that produce this esterase. Developing this technique further to better screen for our bacteria is the first step in being able to identify the bacteria and isolate it from other bacteria in a sample. By mixing the bacteria of interest with another bacteria that has different colony morphology allows for easier identification of the bacteria of interest. A further project of the lab has been screening for constituently active petase among Ideonella mutants as seen in figure 1. We believe screening between Ideonella and E. coli to be more straightforward. By isolating our bacteria that we know degrades plastic from E. coli, another bacteria with a different morphology that is easy to differentiate, we can formulate a procedure that can isolate plastic degrading bacteria from soil samples. We have used PNPA assays in our mutagenesis experiments and can utilize a similar procedure in this screening process.

Research Objective 3: Isolation of a new plastic degrading bacteria

Research steps one and two are meant to build competency and test a procedure to confirm that the procedure works, and it is possible to isolate a plastic degrading bacterium. Our procedure is based on past research by other labs who isolated plastic degrading bacteria. We will finalize our modified procedure and begin our attempt to isolate a bacteria species locally. This will be performed through environmental sampling of areas locally that are known to have plastic contamination, including recycling centers, land fills, and junk yards. The samples will then be put through a similar screening method in the hopes of finding a bacteria that can use plastic as a carbon source. Ideonella is currently the only known strain of bacteria to have the PETase enzyme to break the polymer links of PET plastic. The hope is to find another bacteria that can also deteriorate PET, but other forms of plastic will also be used in a larger scope of the project to identify any strain of bacteria that can break down previously thought non-biodegradable plastic.

We can begin to test our procedure by trying to isolate bacteria that have potential plastic degrading properties.

Steps

1. Environmental sampling at local land fills, junk yards, and recycling centers

2. Procedure to isolate bacteria

3. Cataloging bacteria and testing for plastic degradation effectiveness.

Significance

The figure above illustrates the two enzymes Ideonella utilizes to first cleave the polymer chain of plastic and then break the monomer down into two products. The terephthalic acid can be recovered to make new plastic, and the ethelene glycol is used by the bacteria as a carbon source.

Some literature has shown that purified PETase the active enzyme in Ideonella can degrade highly crystalline plastic Our research has shown that when another carbon source is available the bacteria stops producing the enzyme The next step in the research method would be to mutate the bacteria to try and force the bacteria to always produce the enzyme – producing constituent PETase regardless of conditions This has not been very succeful as the bacteria is challenging to work with an we have not been able to force a plasmid into the bacteria As such, we tried random mutations trying to keep the bacteria producing constituent PETase through random mutation with UV light This methodology required a screening procedure, and this is how we developed the PNPA Top Agar assay that we will later use in high throughput screening of new species of bacteria Random mutations is a long, often unfruitful endeavor, we have had some success, but it has been limited

Through our different experiments we have developed new methodologies that we believe can be leveraged in an effort to cut down the screening time of isolating new species of plastic degrading bacteria Building a library of different species could be very beneficial to the scientific community Finding a bacteria with a constituently active PETase enzyme, a bacteria that more readily accepts foreign plasmids, or a bacteria that can adhere to and degrade highly crystalline plastic would all push the field significantly forward Current screening methodology takes between four and six months We believe that with our new assays we can cut the screening time down considerably Faster screening time for environmental samples will allow for more screening It is believed there ae many forms of plastic degrading bacteria that have evolved independently from one another, we just need to find them

Research Objective 2: Isolation of Ideonella from an inoculated soil sample

A sample of our known plastic degrading bacteria will be added to a sample of soil and we will attempt to reisolate. This will be done by following a few known procedures used for environmental samplings - Serial dilutions and limiting dilutions procedures. Rationale: This will allow us to test our procedure to determine if it works to find plastic degrading bacteria. By adding our bacteria that we know can deteriorate plastic we have a positive control for the experiment. Our preliminary work making minimal media plates that only Ideonella can grow on have been successful and we can use these plates to isolate the bacteria from unwanted species.

Plastic is a leading environmental concern and there need to be new and innovative ways to get rid of plastic. Current recycling processes are lacking and unable to fully remove plastic from the environment. There have been several forms of bacteria that can break down a number of different plastics but other strains need to be researched. Ideonella was the first bacteria discovered to degrade PET plastic but is still ineffective at degrading highly crystalline plastic. Much of our single use plastic is highly crystalline so further work needs to be done to combat this plastic. Understanding new ways to degrade plastic with the final goal of plastic removal from the environment is of interest. Having a library of different bacteria that can degrade plastic can help show new ways to accomplish this goal. Building a procedure to effectively sample for such specific bacteria needs to be developed.

References

1.https://www.bbc.co.uk/newsround/42810179J.

2.https://www.sciencealert.com/new-plastic-munching-bacteriacould-fuel-a-recycling-revolution

Yang, Y., Yang, J., & Jiang, L. (2016). Comment on "A bacterium that degrades and assimilates poly(ethylene terephthalate)". Science, 353(6301), 759–759. doi 10.1126/science.aaf8305

Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji H., Maeda, Y., … Oda, K. (2016). A bacterium that degrades and assimilates poly(ethyleneterephthalate). Science, 351(6278), 1196–1199. doi:10.1126/science.aad6359 ability to fully digest plastic

Plating our bacteria on minimal media M9 Agar plates

Our cultured bacteria (white) as well as a contaminant (Yellow)

Acknowledgements

This work was supported by a 2018 Small Project Research Grant from VAS and by funds from CNU to TDG. Additional preliminary experiments on PETase assay development, Ideonella culturing procedures, and alternative ways to measure degradation were performed by Mary Adams, Andrew Chafin, Diamonte Jones, Carson Pittman (HRA), Caroline Tsui, and Sam Tyler.