Progress 05/15/23 to 12/16/24

Outputs
Target Audience:This project focuses on reuse of polyethylene terephthalate (PET) monomers, or ethylene glycol and terephthalic acid (TPA). Other breakdown products of PET are monohydroxyethyl terephthalate (MHET) and bis-hydroxyethyl terephthalate (BHET).PET is a component of plastic bottles, clear plastic food containers, and polyester textiles. Our research group analyzed transient expression of a gene encoding a PET degrading enzyme, PHL7, in corn embryos. We demonstrated that the corn derived enzyme was active on amorphous PET containers (producing BHET), and on polyester textiles, producing BHET. The target audience for this information includes researchers interested in environmental issues, government agencies such as EPA that focus on environmental issues, companies involved in remediation and environmental issues. non-governmnet agencies focused on enviroonmental issues, textile recyclers and plastic manufacturers intersted in a circular economy. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant provided funds to hire a full time technician to assist with testing and analysis of the enzyme on sources of plastic. We hired a recent biotechnology major, Nao Mitsuguchi, who worked with us for 8 months, the duration of the phase I grant. He learned many skills in the laboratory as well as developed presentation skills for the lab meetings. He is Japanese by birth, he spent much time improving his English in presentations. In addition, two other employees, Enio Duque and Milena Aguirre,developed advanced skills with manipulating bacterial cells, growth and management of donor plants in the greenhouse, transient transformation processes, and tissue culture processes. How have the results been disseminated to communities of interest?Two sources of dissemination have been accomplished: 1. a poster presentation was made at the American Society of Plant Biologists in Honolulu in June 2024. 2. a provisional patent was filed on PETase activity from recombinant corn enzymes was filed in June 2024. What do you plan to do during the next reporting period to accomplish the goals?We did not receive a phase II grant so no further activity is anticipated.

Impacts
What was accomplished under these goals? Objective:GreenLab proposes to use the corn kernel biofactory to commercialize plastic degrading enzyme(s). An enzyme, PHL7, identified in and cloned from a bacterium discovered in a compost bin, has been shown to degrade PET into monomers at a rate that is commercially viable. One of the issues with industrial enzymes is always cost. Fermentation has limitations of cost of facilities for scaling up production. The corn system solves those issues by providing an easily scalable agricultural system--corn grain. Thus, corn produces enzymes cost-effectively--the objective of this work. Task 1: Build three vectors for PHL7 with a high-activity promoter and three subcellular targeting sites and move intoA. tumefaciens, EHA101. We were quite successful with this task. We built our 3 target vectors with the globulin-1 embryo promoter targeted to the ER, the cell wall and the vacuole. In addition we built a vector with cell wall targeting that had a C-terminal 6-histidine tag for facile purification. To ensure we had an active enzyme (although PHL7 was our prime target) we also built a vector with a cell wall targeted proteinfor an engineered enzyme called Fast PETase. All vectors were archived as both E. coli constructs and as EHA101 strains. Task 2: Transient expression of the three PHL7 vectors in immature embryos of High II maize. The five constructs described in Task 1 were co-cultivated with High II embryos that were harvested at about 13 days after pollination when the embryos were about 3 mm in length. Embryos were plated on non-selective plant medium and kept in the dark for 5 days. Havested embryos were stored at -80 degrees until ground and extracted. Task 3: Analysis of transiently expressed PHL7 for activity on PET and PEF. Although all vectors produced detectable PETase activity using the NitroPhenyl Butarate assay, only one extract had detectable activity on PET. We expected the PDD construct (embryo expressed, cell wall localized) to have the best activity because a histidine tag had been attached to the 5' end for purification and enrichment. However, it appears that the his-tagged protein was not stable and showed very little enzymatic activity on PET. In contrast, the same construct, embryo expressed with cell wall targeting (PDA) produced enzymatic activity on PET. PDB (embryo expressed with ER targeting) also showed enzymatic activity, but less than PDA. The PDA vector was used to begin to transform corn for stable regenerants, but none were recovered.

Publications

Progress 05/15/23 to 05/14/24

Outputs
Target Audience:Removing plastics and polyester from the environment, particularly landfills, is relevant to many sectors of society. Thus, society at large, municipalities, recyclers, and manufacturers of plastics and polyester will be the target audience. Only 9% of bottleslabelled number one (polyethelene terephthalate, PET)are recycled out of the 240 million tons discarded. Recycled plastics go into carpets, clothing, and lower quality thermoform plastics. Textiles, on the other hand, comprise 7.7% of all landfill waste. Polyester comprises more than half of textiles, creating more plastic (PET) waste. This project seeks to enzymatically degrade the PET waste from clothing and waste plastic in order to regenerated the material from the components generated by the enzymes. We seek to make the recycling and reuse industry cost effective through our enzymes. Changes/Problems:For our Phase II we switched from plastic recycling to polyester recycling. The Phase II was not funded. What opportunities for training and professional development has the project provided?Several entry level personnel were trained in this project. A first-time technician was trained in company operating procedures and was responsible for most of the incubations. A more senior person worked on the transient transformation procedures and protein extractions. Our most senior scientist was trained on the new HPLC that was leased from ThermoFisher and was able to detect breakdown products of plastic and polyester. How have the results been disseminated to communities of interest?A poster presentation was made at the American Society for Plant Biology in 2024. A provisional patent application was filed. We have collaborated with a fabric recycling company to commercializa the technology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Task 1: Build three vectors for PHL7 with a high-activity promoter and three subcellular targeting sites and move into A. tumefaciens EHA101. Task 1 is complete. Our collaborators at Applied Biotechnology Institute built 5 vectors based on advice from the panel. The panel suggested we try a second gene, so we included FAST PETase (Lu et al., 2022). We also included one plant vector with a his-tag on the protein. The vectors are named for the project (PD = PET degradation) with the third letter as the designation for the plasmid structure including the promoter and targeting sequence. PDA, cell wall targeted; PDB, endoplasmic reticulum targeted; PDC, vacuole targeted; PDD, cell wall targeted with an N-terminal histidine tag; PDF, cell wall targeted TX fast PETase. PDE comprised the E. coli control version.Each of the plasmids was transformed into E. coli to maintain the construct. All vectors have been moved into EHA101 for transformation of maize. Task 2: Transient expression of the three PHL7 vectors in immature embryos of High II maize. Task 2 is complete. The total number of embryos treated with each vector is: PDA, 2591; PDB, 1697; PDC, 249; PDD, 2919; PDF, 244.A transformation-susceptible line of corn, Hi-II (Armstrong, 1991) is produced in-house. Hi-II seed is produced by fertilizing an ear from the female (B) with pollen from the male (A) plant. Hi-II seed is then planted 2 months before transformation. When ears emerge, they are self-pollinated. Embryos are harvested at approximately 14 days post pollination when they are 3-4 mm long. Embryos are co-cultivated with the A. tumefaciens strains, sonicated for 30 seconds, and plated on media for 5 days. Protein was extracted from all embryo sets. Task 3: Analysis of transiently expressed PHL7 for activity on PET. Task 3 was the most challenging. We have a control culture of the PHL7 gene in E. coli, PDE, which was utilized to establish analytical procedures--degrading plastics and textiles as well as colorimetric activity assays. PDE is high-expressing, has a his-tag and thus is easy to purify on a cobalt column. It is easily visible on stained gels, and on western blots with anti-his antibodies. We used it to establish a standard curve to measure TPA release and to quantify our colorimetric assay using para-Nitrophenyl Butyrate (NPB). It was also used to generate anti-PETase antibodies in a guinea pig. We expected the transient plant expression to be low and thus knew detection would be difficult. Two buffers were compared for enzyme extraction from the embryos. Fifty mM sodium acetate, pH 5.0, our standard extraction buffer was compared to 50 mM sodium phosphate pH 7.4, directly compatible with the enzyme assay. On a western blot, it was apparent that pH 5.0 sodium acetate extracted more PETase than the reaction buffer of pH 7.4 sodium phosphate. This was also reflected on the colorimetric standard curve using PDE. Calculated concentrations of the plant extracted PETase are PDA, 0.033 mg/mL; PDB, 0.033 mg/mL; PDC, 0.019 mg/mL; PDD partially purified, 0.024 mg/mL. No activity was shown in control extracts.Although activity was seen with the FAST PETase from TX, it was not further pursued since it was a back-up for PHL7, and PHL7 was active. The reaction with PET substrates required the use of different types of materials to identify one that was amenable to detection of breakdown products. HPLC was used to identify TPA, MHET, and BHET. These products were detected from PDA treated plastic sheets, showing success for potential products. Negative control reactions consisted of embryos treated with empty vector A. tumefaciens which were extracted in a similar fashion to the enzyme-containing vectors. Subsequent to container degradation, polyester textile degradation was incorporated into the Phase I because our Phase II commercialization plan focusedon recycling polyester with a partner rather than recycling PET containers. Polyester comprises PET in its crystalline form. We pretreated the polyester fabrics with 10 M sodium hydroxide for 24 hours to soften the crystalline form (Giraldo?Narcizo et al., 2023). The plant-based enzyme, PDA, released more TPA and BHET than control fabric pretreated with NaOH, demonstrating that the plant-made enzyme was active in polyester degradation.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Nathan Hood, Nao Mitsugutchi, Enio Duque, Milena Aguerra, Erin Egelkrout, Elizabeth Hood; 2024 Generation and Reuse of Microplastic Monomers Using Plant-Produced Recombinant Enzymes. American Society of Plant Biology.