Efficient secretion of a plastic degrading enzyme from the green algae Chlamydomonas reinhardtii

Abstract

Plastic pollution has become a global crisis, with microplastics contaminating every environment on the planet, including our food, water, and even our bodies. In response, there is a growing interest in developing plastics that biodegrade naturally, thus avoiding the creation of persistent microplastics. As a mechanism to increase the rate of polyester plastic degradation, we examined the potential of using the green microalga Chlamydomonas reinhardtii for the expression and secretion of PHL7, an enzyme that breaks down post-consumer polyethylene terephthalate (PET) plastics. We engineered C. reinhardtii to secrete active PHL7 enzyme and selected strains showing robust expression, by using agar plates containing a polyester polyurethane (PU) dispersion as an efficient screening tool. This method demonstrated the enzyme's efficacy in degrading ester bond-containing plastics, such as PET and bio-based polyurethanes, and highlights the potential for microalgae to be implemented in environmental biotechnology. The effectiveness of algal-expressed PHL7 in degrading plastics was shown by incubating PET with the supernatant from engineered strains, resulting in substantial plastic degradation, confirmed by mass spectrometry analysis of terephthalic acid formation from PET. Our findings demonstrate the feasibility of polyester plastic recycling using microalgae to produce plastic-degrading enzymes. This eco-friendly approach can support global efforts toward eliminating plastic in our environment, and aligns with the pursuit of low-carbon materials, as these engineered algae can also produce plastic monomer precursors. Finally, this data demonstrates C. reinhardtii capabilities for recombinant enzyme production and secretion, offering a "green" alternative to traditional industrial enzyme production methods.

Keywords: Chlamydomonas reinhardtii; Climate-neutral economy; Environmental biotechnology; Microalgae; PETase; Secretion; Sustainable plastic recycling.

Fig. 1

Overview of the vector design, the transformation workflow, and an experimental result. (A) Schematic representation of the vector used, including the chimeric Par1 promoter, bleomycin resistance gene, F2A auto-cleavable peptide, SP7 signal peptide, rbcs2 introns, and the rbcs2 terminator region. (B) Workflow for generating transformants with halos. (C) A typical result of the transformed cells with halos, indicating successful expression and secretion of the target protein as designed in the vector. Selection on TAP media plates containing zeocin 15 µg/mL and Impranil DLN at 0.5% (v/v).

Fig. 2

Enzymatic activity of PHL7 produced in C. reinhardtii. (A) Cleavage of ester bond activity in the supernatant by Fluorescein DiAcetate (FDA) assay. (B) Relative absorption reduction per day of Impranil DLN. Wild-type cells are the parental CC1690 strains (green). pJP32PHL7 0.5% Impranil DLN are the transformants picked from the selection plates containing zeocin 15 ug/mL and 0.5% Impranil DLN (purple). pJP32PHL7 0.75% Impranil DLN are the transformants picked from the selection plates containing zeocin 15 ug/mL and 0.75% Impranil DLN (magenta). A violin plot and a box plot superimpose the bin dot plot to summarize statistics.

Fig. 3

PHL7 glycosilation on the secretory pathway. (A) Schematic representation of pJP32PHL7 vector corresponding to sample loaded in lane “PHL7” on zymogram. (B) Schematic representation of pJP32PHL7dg (non-glycosylated PHL7) that corresponds to sample loaded in lane “PHL7dg” on zymogram. C) SDS zymogram gel with 1% v/v Impranil DLN containing Precision Plus Protein Unstained Protein Standards, Strep-tagged recombinant (Bio-Rad Laboratories #1610363) and 10X concentrated supernatant samples from pJP32PHL7 (PHL7), pJP32PHL7dg (PHL7dg), and wild-type parental cc1690 strain (wt) (from left to right). The gel displayed 2 halos (i.e., transparent bands) in the PHL7 lane and 1 halo in the PHL7dg lane after 1 day of incubation in a 100 mM potassium phosphate buffer solution, pH 8.0 at 37 °C.

Fig. 4

Growth curves and enzyme activity profiles of the parental line (wt) and the recombinant pJP32PHL7 (PHL7) strains over time. (A) The growth curves of the wild-type (wt, green circles) and the mutant (PHL7, magenta triangles) strains, measured as optical density at 750 nm (OD750) over time. Each point represents the mean OD750 at a given time point, with vertical black error bars indicating the standard deviation across biological replicates (n = 3). (B) Depicts enzyme activity, expressed as the percentage change in optical density per day (% Δ OD/day) of Impranil DLN, for the same strains over time. Mean enzyme activity is shown with black error bars representing the standard deviation across biological replicates (n = 3). The same symbol patterns were used, the wild-type (wt, green circles) and the mutant (PHL7, magenta triangles).

Fig. 5

Terephthalic Acid (TPA) release during PET degradation experiment. (A) The plot shows the TPA concentration (mg equivalent of TPA per liter, calculated by absorbance at 240 nm) over time for wild-type and PHL7 strains, measured during the enzymatic degradation of PET. The absorbance values were normalized to the initial value at time point t0, and the TPA concentration was calculated using the standard curve. Each data point represents biological replicates’ mean TPA concentration (± SD). The TPA concentration trends after day two were statistically analyzed using linear models. Strain-specific differences in TPA production were observed, with the wild type shown in green and PHL7 in purple. n = 2 biological replica, and n = 21 technical replica (B) Mass spectrometry plot with intensity (y-axis) versus mass-to-charge ratio (m/z) for the range of 110 to 170 m/z, with two peaks highlighted corresponding to TPA (Terephthalic acid) in wild-type (wt) and PHL7 samples. The TPA standard signal was scaled down by a factor of 10 for visual clarity, and its signals are displayed in the negative range to differentiate them from sample spectra.