Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae

Abstract

Background: For decades, plastic has been a valuable global product due to its convenience and low price. For example, polyethylene terephthalate (PET) was one of the most popular materials for disposable bottles due to its beneficial properties, namely impact resistance, high clarity, and light weight. Increasing demand of plastic resulted in indiscriminate disposal by consumers, causing severe accumulation of plastic wastes. Because of this, scientists have made great efforts to find a way to biologically treat plastic wastes. As a result, a novel plastic degradation enzyme, PETase, which can hydrolyze PET, was discovered in Ideonella sakaiensis 201-F6 in 2016.

Results: A green algae, Chlamydomonas reinhardtii, which produces PETase, was developed for this study. Two representative strains (C. reinhardtii CC-124 and CC-503) were examined, and we found that CC-124 could express PETase well. To verify the catalytic activity of PETase produced by C. reinhardtii, cell lysate of the transformant and PET samples were co-incubated at 30 °C for up to 4 weeks. After incubation, terephthalic acid (TPA), i.e. the fully-degraded form of PET, was detected by high performance liquid chromatography analysis. Additionally, morphological changes, such as holes and dents on the surface of PET film, were observed using scanning electron microscopy.

Conclusions: A PET hydrolyzing enzyme, PETase, was successfully expressed in C. reinhardtii, and its catalytic activity was demonstrated. To the best of our knowledge, this is the first case of PETase expression in green algae.

Keywords: Bioremediation; Chlamydomonas reinhardtii; Microalgae; PET hydrolase (PETase); Plastic degradation; Polyethylene terephthalate (PET).

Fig. 1

Structure of backbone plasmid and plasmid construction strategy. Bla and Sh-ble represent Ampicillin and Zeocin resistance gene, respectively. Ori represents origin of replication for E. coli

Fig. 2

Selection of PETase transformants. a, b Final culture solution of CC-124 (a) and CC-503 (b) transformants. c, d PCR results of CC-124 (c) and CC-503 (d) transformants for confirming the gene integration. Numbers represent clone #. WT1 and WT2 represent CC-124 and CC-503 wild type, respectively

Fig. 3

Expression of PETase in C. reinhardtii. a, b Western blot results of CC-124 (a) and CC-503 (b) transformants. Numbers represent clone #. Coomassie brilliant blue staining was performed for each western blot as a standard

Fig. 4

Catalytic activity assay of PETase produced by C. reinhardtii. a Schematic diagrams of the activity assay experiment strategies. b–d HPLC profiles of PETase powder incubation experiments: 2 weeks (b), 3 weeks (c) and 4 weeks (d) after incubation. Green and red lines indicate CC-124 wild type and CC-124_PETase #11 lysates, respectively

Fig. 5

The results of scanning electron microscopy. a, c PET surface after 4 weeks incubation with CC-124 wild type (a) and CC-124_PETase #11 (c) lysate. b PET surface after 2 weeks incubation with CC-124_PETase #11. The magnitudes were ×20,000 for all samples. Small and large red boxes represent original and zoomed pictures, respectively