Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation

Abstract

Background: The biological degradation of plastics is a promising method to counter the increasing pollution of our planet with artificial polymers and to develop eco-friendly recycling strategies. Polyethylene terephthalate (PET) is a thermoplast industrially produced from fossil feedstocks since the 1940s, nowadays prevalently used in bottle packaging and textiles. Although established industrial processes for PET recycling exist, large amounts of PET still end up in the environment-a significant portion thereof in the world's oceans. In 2016, Ideonella sakaiensis, a bacterium possessing the ability to degrade PET and use the degradation products as a sole carbon source for growth, was isolated. I. sakaiensis expresses a key enzyme responsible for the breakdown of PET into monomers: PETase. This hydrolase might possess huge potential for the development of biological PET degradation and recycling processes as well as bioremediation approaches of environmental plastic waste.

Results: Using the photosynthetic microalga Phaeodactylum tricornutum as a chassis we generated a microbial cell factory capable of producing and secreting an engineered version of PETase into the surrounding culture medium. Initial degradation experiments using culture supernatant at 30 °C showed that PETase possessed activity against PET and the copolymer polyethylene terephthalate glycol (PETG) with an approximately 80-fold higher turnover of low crystallinity PETG compared to bottle PET. Moreover, we show that diatom produced PETase was active against industrially shredded PET in a saltwater-based environment even at mesophilic temperatures (21 °C). The products resulting from the degradation of the PET substrate were mainly terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) estimated to be formed in the micromolar range under the selected reaction conditions.

Conclusion: We provide a promising and eco-friendly solution for biological decomposition of PET waste in a saltwater-based environment by using a eukaryotic microalga instead of a bacterium as a model system. Our results show that via synthetic biology the diatom P. tricornutum indeed could be converted into a valuable chassis for biological PET degradation. Overall, this proof of principle study demonstrates the potential of the diatom system for future biotechnological applications in biological PET degradation especially for bioremediation approaches of PET polluted seawater.

Keywords: Diatoms; PETase; Plastic degradation; Plastic pollution; Polyethylene terephthalate.

Fig. 1

Expression and localization of PETase-GFP in the diatom P. tricornutum. PETase^R280A-GFP (see schematic of fusion protein) was expressed successfully in the diatom. Confocal laser scanning microscopy showed that the recombinant protein localized in the ER and most likely other compartments of the secretory pathway. Secretion of the fusion protein could not be analyzed via this method. The lower part of the figure shows a wild type control in which no recombinant protein is expressed. Only plastid autofluorescence but no GFP signal was detectable. SP signal peptide, GFP green fluorescent protein, TL transmitted light, PAF plastid autofluorescence, Merge overlay of GFP and PAF

Fig. 2

Secretion analysis of PETase-FLAG. a Schematic of the expressed recombinant protein AP_SP-PETase^R280A-FLAG. b Western Blot after SDS-gel separation of the cell pellet (10 µg of total protein) and medium fractions (total precipitated protein fraction) of 50 ml cultures (induced at OD₆₀₀ = 0.4) expressing AP_SP-PETase^R280A-FLAG. Detection of recombinant proteins was conducted using an antibody against the FLAG-tag (α-FLAG). As control for intracellular proteins, an alpha-tubulin antibody (α-Tubulin) was used. Wild type medium and cell pellet fractions as well as a FLAG positive control lysate (Rockland, FLAG+) served as control protein fractions. AP_SP-PETase^R280A-FLAG clone 2 showed the highest expression and secretion efficiency (middle), whereas complete secretion of the recombinant protein was only achieved by clone 1 (left). As shown by the control via alpha-tubulin detection (right), presence of PETase^R280A-FLAG in the medium fraction was not due to cell lysis. A signal in the range of the calculated molecular mass of AP_SP-PETase^R280A-FLAG (30.4 kDa) could only be observed for clone 2 (left and middle). The dominant signals detected by the FLAG-tag antibody appeared at molecular masses of approximately 40 and 50–55 kDa in AP_SP-PETase^R280A-FLAG clone 1, 2 and 3. Calculated molecular masses: AP_SP-PETase^R280A-FLAG: 30.4 kDa; FLAG-tag, 1 kDa; PETase^R280A-FLAG: 28.5 kDa; FLAG+, 60 kDa. AP alkaline phosphatase, SP signal peptide, WT wild type, AP_# AP_SP-PETase^R280A-GFP clone #. Numbers beside/on the Western Blots indicate molecular masses of the marker (PageRuler™ Prestained 10–180 kDa Protein Ladder) bands in kDa

Fig. 3

Scanning electron microscopic analysis of PET bottle film degradation by PETase-FLAG secreted from P. tricornutum. As depicted in the upper left part, untreated PET and PET incubated with wild type cells on an f/2 agar plate overflowed with 2 ml f/2 liquid medium showed, besides a usually smooth surface, occasional stress marks. In contrast, PET incubated for 5 weeks with cells expressing AP_SP-PETase^R280A-FLAG (clone 1), was lanced by holes, dents, furrows and cavities when inspected via SEM (see also Additional file 1: Figure S5). In a specific area of the PET disk a structure (imprint) similar to the form of a P. tricornutum cell (fusiform morphotype) from which several holes and furrows originated was detected (see also Additional file 1: Figure S6). AP alkaline phosphatase, SP signal peptide, WT wild type, AP_1 AP_SP-PETase^R280A-GFP clone 1

Fig. 4

SEM and UHPLC analysis of PETG film degradation by PETase-FLAG secreted from P. tricornutum. A small piece of PETG film was incubated with 1 ml of supernatant (medium fraction) of a 50 ml culture expressing AP_SP-PETase^R280A-FLAG (clone 1, induced for 4 days) and wild type and analyzed via SEM. As shown in the upper part, similar but more area-wide changes in the surface of the PETG film as observed in the solid approach (PET bottle film degradation by AP_SP-PETase^R280A-FLAG clone 1 on solid medium, Fig. 3) were detected. The wild type control (lower left) did not show any significant aberrations in the surface structure of the PETG film. UHPLC analysis (lower right) of the medium fractions after 1 week of incubation with the PETG film at 30 °C revealed production of TPA and MHET in sample AP_1 (AP_SP-PETase^R280A-FLAG clone 1), which were absent from the wild type control. PETG polyethylene terephthalate glycol, SN supernatant, AP alkaline phosphatase, SP signal peptide, WT wild type, AP1/AP_1 AP_SP-PETase^R280A-FLAG clone 1

Fig. 5

UHPLC analysis of the medium fraction of shredded PET incubated with PETase-FLAG producing clone 2. The experiment was performed in 50 ml f/2-medium containing AP_SP-PETase^R280A-FLAG expressing clone 2 (AP2) and approximately 5 g of shredded PET. At T₀ the cultures were adjusted to an OD₆₀₀ of 0.4 and expression of the recombinant protein (AP_SP-PETase^R280A-FLAG) was induced with nitrate. Samples of 1 ml were taken at individual time points (T, 1 day = 24 h) and fresh nitrate was supplemented to the cultures at T₃, T₆ and T₁₀. The standards used for UHPLC analyses are shown on the lower right. Note that the concentrations of standard compounds are not equal. BHET bis(2-hydroxyethyl) terephthalic acid, MHET mono(2-hydroxyethyl) terephthalic acid, TPA terephthalic acid, WT wild type, AP2 AP_SP-PETase^R280A-GFP clone 2