Experiments

Content

Enzyme production Plastic depolymerization Biomass production References

On this page, you can read more about the experiments we carried out throughout our project. We have divided the project into three different parts: enzyme production, plastic depolymerization, and biomass production. To see more detailed information about all the lab notes from the experiments check out our lab book and protocols.

Introduction

Since 2016, when Ideonella sakaiensis 201-F6, was isolated from outside a bottle-recycling facility in Japan1, the research in plastic degrading enzymes has been a hot topic. Current plastic recycling methods are either physicochemical or thermal treatments2. Their downfall is that they are energy-intensive and often lead to the generation of secondary pollutants or toxic byproducts. This is why enzymatic biodegradation has become an attractive alternative. Among various types of plastics, polyethylene terephthalate (PET) is one of the most commonly used plastics worldwide and its accumulation in the environment is a global problem. PET is mainly used for packaging, textiles, and bottles, and it can take hundreds of years to degrade in the environment. Ideonella sakaiensis 201-F6 can use PET as its major energy and carbon source due to two native enzymes it produces, PETase and MHETase1. PETase hydrolyzes PET into mono(2-hydroxyethyl) terephthalate (MHET), while MHETase further cleaves MHET into environmentally benign monomers, terephthalic acid (TPA), and ethylene glycol (EG)1 as illustrated in Figure 1. This process is known as enzymatic depolymerization, and it has several advantages over conventional physical or chemical methods, such as lower energy consumption, reduced greenhouse gas emissions, and higher selectivity. However, enzymatic PET degradation still has many limitations, such as slow processing times and low degradation rates, which limit upscaling and achieving industrial levels2.

Figure 1. The PET depolymerization pathway.

Many naturally occurring microorganisms can depolymerize and biodegrade Polyethylene terephthalate polymers. However, in nature, the degradation rate is very slow. Enzymatic depolymerization can deconstruct plastics into smaller aromatic building blocks, which microbes can break down more rapidly. Thereby, the monomers obtained from the enzymatic depolymerization of PET can be used as substrates for growing microorganisms that can use them as carbon sources3. Several bacteria and fungi have been reported to be able to metabolize TPA and EG. When microbes metabolize waste products they break down the particles into useful metabolites and other necessary compounds for growth. When microbes grow they produce biomass with high protein content. In recent years many microbe-based food products have appeared into the market in an effort to have more sustainable alternatives to meat. Single-cell proteins (SCP) are microbial cells, which can be used as a source of nutrients for human or animal consumption. The protein content of microbial biomass can vary depending on the strain, the culture medium, and the growth conditions. The nutritional value and safety of microbial biomass can be assessed by analyzing its amino acid composition, essential fatty acids, vitamins, minerals, and potential toxins or allergens.

Enzyme production

Scientific background

Various enzymes exhibiting PET depolymerization activity have been identified and investigated, like cutinases, PETase, MHETase, lipases, and esterases4. The wild-type enzymes PETase and MHETase of Ideonella sakaiensis 201-F6 display higher enzymatic activity and specificity for PET than the other reported wild-type enzymes at comparatively low temperatures (30–40 °C)4. In efforts to make the enzymatic degradation of plastic more sustainable and industrially applicable, there has been much focus on improving the enzymatic activity and thermal stability of PETase. For this purpose, FAST-PETase (Functional, Active, Stable, and Tolerant PETase) was designed via machine learning-aided engineering5. It contains five mutations compared to the wild type and shows improved PET-hydrolytic activity in a wide range of temperatures and pH5.

Extracellular secretion of PETase is also essential for the application of this enzyme in PET degradation. Secretion greatly facilitates downstream processing and significantly reduces the production costs of recombinant proteins. There have been recent studies where extracellular expression of PETase has been reported4.

E. coli is a useful organism for recombinant protein production due to its availability, easy cultivation, established cloning techniques, and rapid protein expression. In bacteria, two major export pathways are capable of transporting proteins outside of the cell plasma membrane, the general secretion - Sec pathway - and the twin-arginine translocation or Tat pathway6. The activation of the protein export systems requires a specific signal peptide to the amino-terminal end of the desired target protein and the pathway6. In E. coli, the translocation of proteins usually happens via the Sec-dependent pathway, signal recognition particle (SRP) pathway, or twin-arginine translocation (TAT) pathway, mediated by N-terminal signal peptides4. Commonly used signal peptides for the production of extracellular recombinant proteins in E. coli include pelB, phoA, malE, or lamB. pelB has been used in the pET-vector expression system4. pelB in particular has been shown to be compatible with PETase4, while lamB is suitable for the WT-MHETase construct.

The goal of the experiments

We wanted to design an iGEM-compatible recombinant system of FAST-PETase and MHETase for E. coli that secretes the enzymes. To build the expression strains, we did restriction cloning, using the Xhol and Ndel restriction enzymes to cut both the gene inserts and the pRSFDuet-1 vector. The ligated expression vector was transferred first into cloning host E. coli TOP10 to confirm the successful cloning, and later to expression host E. coli T7. We had plans to further optimize the enzymes by adding SpyCatcher/SpyTag domains into our products, thus binding the secreted enzymes into a scaffold or to each other, further optimizing enzyme activity. We wanted to optimize the expression conditions and purify the enzymes by affinity chromatography using His-tags and analyze the activity by measuring the release of TPA from PET using HPLC.

The success of the experiments

We successfully designed the constructs for FAST-PETase and MHETase based on recent literature. Both constructs were optimized for translation and transcription by calculating the optimal sites for the inserts and by removing rare codons. By adding the signal peptides lamB and pelB respectively into our MHETase and FAST PETase constructs as shown in Figure 2, we facilitated secretion into the culture media. We successfully cloned FAST-PETase into both TOP10 and T7 expression hosts. We then induced the expression of FAST-PETase by adding IPTG and studied the expression of the enzyme by SDS-PAGE.

Figure 2. FAST-PETase and MHETase inserts. More detailed illustrations are available on our design page.

For the MHETase, we never got the correct size bands in the restriction analysis of the clones. We suspect that this may be due to a mix-up with inserts. Unfortunately, we did not have the time or funds to purify our enzymes or do activity analysis, due to many difficulties in the cloning process. Due to the same problems, we also did not have time to compare the effectiveness of SpyCather/SpyTag parts, even though they were designed. Our engineering page outlines the most central phases of our wet lab work.

Plastic depolymerization

Scientific background

The depolymerization of PET by enzymes is a complex process that depends on several factors, such as the type and form of the PET plastic and its crystallinity, the enzyme concentration and ratio, the reaction temperature and pH, and the presence of additives or co-solvents7. PET polymers are large molecules that have a crystalline structure. Often plastics do not have a homologous structure, but a mix of regular crystals (crystalline region) and irregular groups (amorphous region)7. This is what makes them durable and flexible, but also hard to degrade or recycle. PET-based plastics possess a high degree of crystallinity (30–50%), which is one reason for the low rate of microbial degradation in nature (~>50 years)7.

The enzymatic degradation of plastics occurs in two stages: 1) adsorption of enzymes on the polymer surface, and 2) followed by hydrolysis of the bonds7. The enzymes responsible for breaking down PET are α/β-hydrolases7. They possess a Nucleophil-His-Acid catalytic triad, in this case, Serine-His-Aspartame, which efficiently catalyzes a variety of different substrates7. The process of breaking down PET is initiated by serine oxygen attacking the carbonyl carbon within the ester bonds of PET7. The reaction is facilitated by the negatively charged aspartate, which stabilizes the positively charged histidine residue7. The charge transfer results in ester bond hydrolysis by enabling serine to perform a nucleophilic attack, leading to the breakdown of the ester bond7. PETase, one of these enzymes, can specifically hydrolyze ester bonds found in aromatic polyesters. It plays a crucial role in converting PET into mono-2-hydroxyethyl terephthalate (MHET). On the other hand, MHETase is responsible for breaking down MHET into terephthalic acid (TPA) and ethylene glycol (EG).

The goal of the experiments

For this experiment, we wanted to use the purified enzymes to depolymerize PET into TPA and EG. We wanted to test different types and forms of PET (e.g., films, commercial packages, and untreated and pretreated plastics) with different reaction parameters (e.g., temperature, pH, enzyme concentration) to determine the optimal conditions for efficient depolymerization. To analyze the kinetics and products of the enzymatic reaction we wanted to use HPLC to quantify the produced monomers and demonstrate the degradation by taking pictures using scanning electron microscopy (SEM).

The success of the experiments

As we did not have time to purify the enzyme, we attempted the depolymerization without purification by adding different plastics into the media to which our expression host had secreted the enzymes. After a period of time, we measured the loss of mass. Unfortunately, no reduction in mass was observed.

Biomass production

Scientific background

Many naturally occurring microorganisms can depolymerize and biodegrade polyethylene terephthalate polymers. From these, the most notable regarding our project were Pseudomonas putida, Rhodococcus opacus, Comamonas testosteroni, and Bacillus subtilis. They are all common soil microbes, that have been documented to be able to use PET degradation monomers as carbon sources8. Ethylene glycol has been shown to be metabolized by various microbes through the sequential oxidation of EG to glyoxylate (KEGG, R00476) via glycolaldehyde (KEGG, R01781) and glycolate (KEGG, R01333) as intermediates8. TPA, in turn, requires specific enzymes to turn it into protocatechuate, which is then further changed to central carbon metabolism intermediates8. Efficient metabolization of EG and TPA has been achieved by laboratory evolution and by metabolic engineering since natural uptake is quite conservative8.

The goal of the experiments

The goal of this section is to use the monomers obtained from enzymatic depolymerization as substrates for growing microorganisms that can produce protein-rich biomass. First, we wanted to know if the strains selected actually could grow on minimal media with PET monomers as a carbon source, and conduct laboratory evolution on the microbes to get better growth. We wanted to evaluate the growth rate and quality of the microbial biomass under different culture conditions making pure cultures as well as growing the bacteria in co-cultures to see if they have synergistic actions or whether they would have competitive relations and inhibit the growth. We also wanted to analyze the nutritional value and safety of the biomass for potential applications as food or feed. We were also modeling possible end products of metabolizing TPA and EG and considering genetical engineering of E. coli and P. putida for better TPA and EG uptake, respectively.

The success of the experiments

We selected P. putida, R. opacus, and C. testosteroni as the strains of bacteria for metabolizing TPA and EG. We found that P. putida KT2440 was the best candidate for our system, as they have high growth rates, yields, and protein contents when cultured in M9 minimal medium supplemented with TPA and EG. We analyzed the uptake of monomers by performing HPLC. We also performed co-cultures of these strains to enhance their synergistic interactions. We analyzed the protein content by Bradford analysis, and we found that protein was generated from biomass of R. opacus cultivated on the minimal media containing TPA which accounted for 3.4 µg/ml.

However, we did not have enough guidance or resources to analyze other compounds of the biomass, such as the amino acid composition, essential fatty acids, vitamins, minerals, and potential toxins or allergens of our biomass.

References

  1. Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016;351(6278):1196–1199. doi:10.1126/science.aad6359
  2. Kushwaha A, Goswami L, Singhvi M, Kim BS. Biodegradation of poly(ethylene terephthalate): Mechanistic insights, advances, and future innovative strategies. Chemical Engineering Journal. 2023;457:141230. doi:10.1016/j.cej.2022.141230
  3. Schaerer LG, Wu R, Putman LI, Pearce JM, Lu T, Shonnard DR, Ong RG, Techtmann SM. Killing two birds with one stone: chemical and biological upcycling of polyethylene terephthalate plastics into food. Trends in Biotechnology. 2023;41(2):184–196. doi:10.1016/j.tibtech.2022.06.012
  4. Shi L, Liu H, Gao S, Weng Y, Zhu L. Enhanced Extracellular Production of IsPETase in Escherichia coli via Engineering of the pelB Signal Peptide. Journal of Agricultural and Food Chemistry. 2021;69(7):2245–2252. doi:10.1021/acs.jafc.0c07469
  5. Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, Acosta DJ, Alexander BR, Cole HO, Zhang Y, et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature. 2022;604(7907):662–667. doi:10.1038/s41586-022-04599-z
  6. Freudl R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microbial Cell Factories. 2018;17(1):52. doi:10.1186/s12934-018-0901-3
  7. Urbanek AK, Kosiorowska KE, Mirończuk AM. Current Knowledge on Polyethylene Terephthalate Degradation by Genetically Modified Microorganisms. Frontiers in Bioengineering and Biotechnology. 2021;9:771133. doi:10.3389/fbioe.2021.771133
  8. Tiso T, Winter B, Wei R, Hee J, de Witt J, Wierckx N, Quicker P, Bornscheuer UT, Bardow A, Nogales J, et al. The metabolic potential of plastics as biotechnological carbon sources – Review and targets for the future. Metabolic Engineering. 2022;71:77–98. (Substrates for Metabolic Engineering). doi:10.1016/j.ymben.2021.12.006