A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against PET and polystyrene

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Taunt, Marcello Berto, Harry O. Jackson, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2668920/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jun, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for the low-cost synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and polystyrene (PS) was investigated via atomic force microscopy, revealing evidence of degradation of both plastics. Biological sciences/Biotechnology/Expression systems Biological sciences/Biotechnology/Molecular engineering Biological sciences/Biotechnology/Protein delivery Biological sciences/Biophysics/Nanoscale biophysics atomic force microscopy Chlamydomonas chloroplast expression PETase polyethylene terephthalate polystyrene Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Natural polymers such as lignin, starch, chitin, and cellulose are water-insoluble macromolecules present in the environment, and although such polymers are generally recalcitrant to physical and chemical degradation, nature has evolved enzymes for their breakdown 1 . Plastics are instead synthetic polymers designed specifically for their high resistance to degradation. Plastics are central to modern life and their production has expanded tremendously during the last few decades owing to their versatile properties and low cost. Although an increasing number of microorganisms capable of degrading plastic polymers have been isolated and the enzymes involved metabolically characterized, additional studies are needed to identify novel enzymes and associated degradation pathways for the wide range of different plastics 1 – 5 . The challenge of understanding and optimizing plastic enzymatic degradation closely emulates that of enzymatic depolymerization of polysaccharides 6 – 8 . Indeed, strategies that have been used to understand and improve glycoside hydrolases, including the development of quantitative assays for measuring enzyme (or enzyme cocktail) performance, can serve as inspiration for more quantitative metrics for comparing plastic-degrading enzymes and enzyme mixtures 9 , 10 . Owing to its robust mechanical properties and high post-consumer recycling costs, polyethylene terephthalate (PET) is one of the most abundant plastics in the world. PET accumulates in our environment without significant microbial conversion 2 – 5 . The constant flux of new PET into the global market has produced enormous amounts of waste with a long biodegradation timescale. This waste contributes to global pollution, especially for marine ecosystems, and poses a threat to human and animal health 11 – 14 . Similarly, polystyrene (PS) is a high demand plastic, ranking fourth in the world’s consumption of plastics and constituting 10% of total plastic waste 15 . PS has various applications and is widely used in households, industries, healthcare and more. Its disposal incurs incineration costs, depletion of valuable carbon resources and energy consumption. It can be converted into its monomer styrene and aromatic hydrocarbons such as toluene and ethyl benzene by thermal degradation 15 – 17 . In 2016, Yoshida et al. discovered and isolated two new enzymes from Ideonella sakaiensis , a bacterium which was able to grow using PET as the main carbon and energy source 18 . These enzymes are a polyethylene terephthalate hydrolase (PETase), which can convert PET into mono(2-hydroxyethyl) terephthalic acid (MHET) and mono(2-hydroxyethyl) terephthalate hydrolase, responsible for the conversion of MHET to terephthalic acid (TPA) and ethylene glycol (EG) 18 – 21 . Given that PET and PS waste is mainly disposed of via incineration or thermal degradation, the possibility of biological degradation of such waste to non-toxic monomers represents an attractive and greener solution to reduce pollution. Although PS-degrading organisms have been identified and characterized 22 , the enzymes responsible for the degradation of polystyrene are completely unknown. For production of enzymes for research and industrial purposes, the exploitation of the native plastic-degrading microorganism is not always possible and organisms that are easier to culture and engineer for heterologous expression are required 23 . To this end, microalgae represent an attractive biotechnology platform for the synthesis of recombinant proteins 24 – 26 . The advantages of using microalgae as opposed to traditional heterotrophic platforms of Escherichia coli , yeast, or CHO cells are: (i) the low-cost phototrophic cultivation of the alga in sterile, controlled photobioreactors using simple and inexpensive medium 27 ; (ii) the generally recognized as safe (GRAS) status of a number of algal species, including the chlorophyte Chlamydomonas reinhardtii 28 ; (iii) the availability of the chloroplast as a unique biosynthetic and storage compartment within the cell that contains its own minimal genetic system 29 – 31 ; and (iv) a growing interest and adoption of enabling synthetic biology principles for creating bespoke cell factories using microalgae 32 . Whilst several recent studies have reported the production of PETase in microalgal species through nuclear engineering 33 , 34 , the use of the chloroplast for expression of foreign genes confers several benefits. These including precise insertion into the chloroplast genome (= “plastome”) via homologous recombination, high-level expression that is not subject to any gene-silencing mechanisms, the possibility of expressing multiple transgenes as operons and the accumulation of the recombinant proteins in a benign compartment where the formation of di-sulfide bonds occurs readily 35 . Here we report the synthesis of PETase from I. sakaiensis in the chloroplast of C. reinhardtii and demonstrate that the purified enzyme is active against both PET and PS. Results Generation of transplastomic C. reinhardtii expressing PETase . A synthetic gene encoding the mature form of the I. sakaiensis PETase was codon optimized for expression in the chloroplast and cloned into the pSRSapI destination vector, to generate plasmid pSRSapI:PETase (Figure S1 ). The bacterial PETase enzyme contains two disulphide bonds that are formed following secretion into the I. sakaiensis periplasm 36 . We therefore decided to retain the sequence for the N -terminal Sec-type signal peptide within the transgene design so that the enzyme would be similarly targeted into the thylakoid lumen. Previous work has shown that bacterial signal peptides can direct recombinant proteins into the thylakoid lumen, and that disulphide bond formation occurs more readily in this chloroplast compartment 37 . This was used to transform C. reinhardtii strain TN72 with the PETase gene integrated into the plastome at a neutral locus between psbH and trnE2 . Integration of the transforming DNA into TN72 also restores a wild-type copy of psbH , which is an essential photosynthesis gene 38 . Selection is therefore based on restoration of photosynthesis allowing the generation of transformants lacking any antibiotic-based selectable marker, with the only foreign DNA introduced into the plastome being the PETase coding sequence (Fig. 1 A). As the C. reinhardtii plastome is polyploid with ~ 40 copies per cell under phototrophic conditions 40 , transformant lines were restreaked several times to single colonies under selective conditions to drive the lines to homoplasmy where all plastome copies have the transgenic DNA. Two such lines (TN72:PETase.1 and TN72:PETase.2, hereafter) were checked for homoplasmy using a 3-primer PCR screen (Fig. 1 A). This showed a single band at 1037 bp in the TN72:PETase.1 and TN72:PETase.2 PCRs and a single band at 878 bp in the parental TN72 control PCRs, confirming correct integration of the PETase cassette and homoplasmy (Fig. 1 B). Furthermore, it was found that selection based on restoration of phototrophy established homoplasmy much more readily than antibiotic-based selection. This is probably due to the strong selective pressure of restoring the photosynthetic phenotype and to the reduced copy number of cells grown on minimal medium rather than the acetate-containing medium normally used for antibiotic-based selection 38 . Finally, PCR amplification and sequencing of the PETase cassette from both transformant lines confirmed than neither had acquired any mutations during plastome integration. PETase is expressed in the chloroplast . The TN72:PETase.1 cell line was characterized further in order to assess the level of recombinant protein produced and to determine whether the protein is correctly folded and functional. An evaluation of the PETase level was conducted by SDS-PAGE analysis of cell extracts with proteins bands in the 27 kDa region cut from the gel, trypsin-digested and subjected to tandem mass (MS/MS) spectrometry. As shown in Supplementary Figure S2 protein sequence coverage for PETase of 27% was obtained, confirming unambiguously the presence of PETase in the strains. Other bands from constitutive proteins were analysed revealing the presence of malate dehydrogenase of 30 kDa (Figure S3) and cytochrome c at 10 kDa (Figure S4). Purification of recombinant PETase from transgenic C. reinhardtii . PETase has the classic α/β hydrolases fold, but despite the sequence similarity to cutinases and lipases, the highly polarized surface charge of PETase creates a dipole that gives the enzyme an isoelectric point of 9.6 20 that allows a first step of purification by cationic exchange HiPrep ™ SP HP 16/10. The protein was successfully recovered using an optimized two-step chromatography approach as shown in Fig. 2 A and 2 B. Elution of PETase from the HiPrep ™ SP HP 16/10 column was conducted by linear gradient of NaCl, with the target protein eluting at 20–30% concentration. Several other peaks were observed on the chromatogram during this phase indicating the presence of several endogenous protein contaminants. Fractions highlighted in red in Fig. 2 A were loaded on the size exclusion chromatography (SEC) column, a HiLoad ™ Superdex ™ 75 column. During SEC a group of low-concentration proteins eluted first (peak 6 in Fig. 2 B); followed by the majority of the proteins as three separate peaks labelled 8–10 (due to the presence of two small shoulders), 11 and 12 (Fig. 2 B). All fractions were analyzed by SDS-PAGE (Fig. 2 C). The gel shows that fraction 8–10 contained a major protein with a molecular weight greater than 35 kDa, that was analysed by MS/MS revealing the presence of malate dehydrogenase (Supplementary Figure S3). Sample 12 revealed a band with a molecular weight of approximately 27 kDa, consistent with that of mature PETase. The band was analysed via MS/MS spectrometry confirming its identity as PETase with a 51% sequence coverage (Fig. 2 D). Sample 12 also contained a mitochondrial cytochrome c that was identified by MS/MS spectrometry and UV-visible spectroscopy (Supplementary Figure S4 and Fig. 2 E). Since this cytochrome has a pI = 9.39, it eluted with PETase during the cationic exchange chromatography. Moreover the difference in molecular weights between PETase (27 kDa) and cytochrome c (12 kDa) was not sufficiently different for the two proteins to be efficiently separated by size exclusion chromatography. The recombinant PETase is active. To investigate the activity of the algal-expressed PETase, PET and PS substrates were incubated with the enzyme and control solutions then assessed by semi-contact atomic force microscopy (AFM) imaging in air (Fig. 3). It is clear that the recombinant PETase can modify the morphology of both PET and PS samples. Specifically, in samples incubated with the enzyme, the formation of holes can be observed by the presence of darker spots, while no holes are visible in the control samples (i.e. the same experimental procedures without the enzyme). To quantitatively evaluate the morphological changes on the surfaces due to PETase activity, the average surface roughness of the samples σ rms and the lateral correlation length ξ were calculated. These variables correspond to a measure of the texture of the surface, and the distance range over which points in one region of space are correlated with those in another region, respectively 41 (Fig. 3). PETase activity resulted in a decrease of ξ in both materials: PET samples (Fig. 3A and 3B) displayed a decrease by an order of magnitude, from 2.2 ± 0.6 µm (control sample) to 0.22 ± 0.08 µm (when incubated with enzyme), while, for PS samples (Fig. 3C and 3D), a reduction of 60% was observed, from 0.53 ± 0.1 µm (control sample) to 0.21 ± 0.03 µm (when incubated with enzyme). The average values of ξ were obtained from three images of each sample and interestingly, after incubation with the enzyme, they were similar (≅0.2 µm) for both materials (Fig. 4 B). Since there were no other sources of degradation in sample reactions we could ascribe those values exclusively to the PETase activity, and, in particular, the presence, the dimension and the density of the holes on the surfaces confirmed the functionality of the enzyme. Conversely, the surface roughness variation (Δ σ rms ) significantly increased for PS samples (from 4.5 ± 0.6 nm to 22 ± 3 nm with Δ σ rmsPS = + 17.5 nm) , while it does not change for PET samples (from 4.8 ± 0.5 nm to 4.4 ± 0.4 nm with Δ σ rmsPET =-0.4 nm ) (Fig. 4 A and 4 B). This may suggest that the chemical and physical stress suffered by the plastic samples during the incubation could affect more one material than the other. A possible explanation is that the technical-grade PET foil is more resilient to stress than postconsumer PS foil (in fact Δ σ rmsPS < Δ σ rmsPET ), but, in any case both materials were affected by the enzyme activity. Discussion PETase is a recently discovered hydrolase enzyme acting on PET 18 . With a view to its potential significance in an industrial context, in this work we describe a platform for producing such enzymes in the chloroplast of C. reinhardtii where we obtained ∼ 5 mg of pure product from 40 g of wet biomass (yield of ≅ 0.012%). While the recombinant protein yield is lower than that of bacterial systems 42 , microalgae offer several potential advantages, such as reduced scale-up costs and use of the algal biomass for extraction of useful byproducts; carotenoids, pigments, proteins, and vitamins that can be used for the production of nutraceuticals, animal feed additives, cosmetics, or for energy production 26 , 43 – 46 . Expression in the chloroplasts has the advantage of precision targeting of transgenic DNA into a selected site in the plastome via homologous recombination, stable and high-level expression of transgenes in the absence of selection, and the ability to compartmentalize and target potentially toxic recombinant proteins within the chloroplast. Moreover, the use of photosynthetic restoration in the transformation of strain TN72 avoids the use of selection markers based on antibiotic-resistance genes and circumvents concerns over escape of such genes via lateral gene transfer. Finally, the pilot scale PETase production carried out here (10 L culture medium), allowed for the first time the expression of a sufficient amount of recombinant protein for purification, and this will facilitate further characterization of the enzyme kinetics for different PETase variants. Interestingly, the purification of the recombinant PETase by means of cationic exchange chromatography resulted in the presence of a cytochrome c with a pI similar to that of the PETase (9.39 and 9.65, respectively) in the final fraction, implying that the two proteins stayed together during all the purification steps. Considering this result, one potential explanation for the unprecedented hydrolytic activity on post-consumer material containing mainly polystyrene could be due to the presence of cytochrome c in the reaction mixture. Cytochrome c is a well characterized and ubiquitous protein that can interact with various molecules (i.e. phospholipids through electrostatic and hydrophobic bonds) 47 – 50 , and in some cases (e.g. M80A mutant) it can exert a catalytic activity 51 – 53 , but primarily cytochrome c is an electron transfer protein working only in the presence of electron donors that, in this specific case, were not added to the reaction mixture. Therefore, we can exclude that within the reaction conditions tested in this paper cytochrome c contributed to the enzymatic reaction between PETase and PS. An explanation for the PS activity could be found in the structure of PS, that, unlike PET, has the aromatic rings perpendicular to the polymer chain, thus providing Van der Waals interactions between the aromatic ring of Trp185 present in the active site of PETase and the surface exposed aromatic rings of the plastics. This could be a hypothesis but the scope of this work was the production of an active form of PETase in the microalgal chloroplast and we have successfully obtained a transgenic strain of C. reinhardtii expressing an active heterologous PETase. The unexpected novelty was the discovery of its unprecedented activity on PS, paving the way to new perspectives of investigation on the mechanism of this reaction. Methods Materials. Chlamydomonas reinhardtii strain TN72 (CC-5168, Chlamydomonas Resource Center: www.chlamycollection.org ) was used to generate the transgenic lines in this study as described previously 38 , 54 . All lines were maintained on tris-acetate-phosphate (TAP) medium supplemented with 1.5% agar unless otherwise stated. Liquid cultures were cultivated in TAP medium at 25°C, with agitation at 125 rpm. All lines were illuminated with continuous white light at 50 µE/m 2 /s, with the light sensitive TN72 parental line protected with a white paper cover to reduce light intensity by approximately an order of magnitude. Cloning procedures. DNA manipulations were performed following standard protocols 55 , including transformation of chemically competent E. coli DH5α. Oligonucleotide primers were purchased from Eurofins Genomics (Ebersberg, Germany) while restriction enzymes and T4 DNA ligase for cloning procedures are purchased from NEB (Ipswich, MA, USA) and Thermo Scientific (Waltham, MA,USA).. The coding sequence for I. sakaiensis PETase (PETase Uniprot: A0A0K8P6T7; Gene: ISF6_4831; EC:3.1.1.101), together with a Strep-tag at the N terminus, was codon-optimized for the C. reinhardtii chloroplast using the Codon Usage Database ( www.kazusa.or.jp/codon ) with SapI and SphI sites added for cloning into the pSRSapI transformation vector (Figure S1 ) 56 . The DNA was synthesized by IDT (Integrated DNA Technologies, Inc., Coralville, Iowa-USA), and was cloned into the transformation vector by standard molecular techniques 55 . Transformation of the C. reinhardtii chloroplast. C. reinhardtii was transformed by the glass bead method as described previously 38 , 57 . 400 mL transformation cultures were grown to early log phase (1–2 x 10 6 cells/ mL) and harvested by centrifugation at 3000 x g . Cells were resuspended in sterile high salt minimal (HSM) medium to a density of 2 x 10 8 cells/ml, and 300 µL added to sterilized 5-ml tests tube containing 300 mg sterile 400–625 µm diameter glass beads and 10 µg plasmid DNA. Tubes were agitated by vortex at maximum speed for 15 seconds, then mixed with 4 ml molten HSM supplemented with 0.5% agar at 42°C and immediately spread on HSM plates supplemented with 1.5% agar and 100 µg/mL ampicillin. Plates were sealed with parafilm and incubated at 25°C in dim light (~ 2 µE/m 2 /s) overnight then transferred to moderate light (~ 50 µE/m 2 /s) 29,38 . Transformant colonies were picked after 3–4 weeks and restreaked to single colonies on selective media until homoplasmy was achieved, typically after 2–3 restreaks. Genotyping of transformant lines . Lines were assessed by PCR analysis using a three-primer strategy as described previously 38 In each case a forward flanking primer was designed outside of the left homology arm used for transformation, with reverse primers designed within the parental and transformed cassettes respectively. Details of primers used are given in the supplementary data (Table S5). Insertion of the target PETase CDS was further confirmed by PCR amplification of the expression cassette followed by Sanger sequencing of the PCR product. Total genomic DNA is extracted from a small amount of cells using the Chelex 100 method 58 and PCR amplification is carried out with Phusion DNA polymerase (Thermo Scientific) according to the manufacturer’s instructions. In this strategy (see Fig. 1 A), a plastome specific primer (P1) is used in conjunction with two other primers in a three primer reaction. A second primer (P2) binds to a terminator element ( T rbcL ) in the parental TN72 strain, in which the endogenous psbH gene (essential for photosynthesis) is disrupted with an aminoglycoside 3″-adenylyltransferase (aadA) expression cassette, to generate a band of 878 bp. The third primer (P3) binds to a promoter element ( P psaA ) in the transformed genotype, containing the PETase expression cassette and the restored of psbH to give a band of 1037 bp. Dilution control PCR (1:100 and 1:200) were performed with TN72 DNA, to ensure that the PCR reaction was sufficiently sensitive to detect the parental DNA in the TN72:PETase PCR if the parental DNA were present at low/single copy numbers. Scale up cultivation, cell breakage, and preparation of soluble protein extract. Chlamydomonas reinhardtii expressing PETase cells line was inoculated in 20 ml of liquid TAP medium and grown at, 25°C, 100 rpm and intensity light of 50 µE m − 2 s − 1 for 4 days. Cell growth was monitored over time using a Bürker chamber and the rate of increase was obtained. Cells were let grown until a concentration of 4 x 10 6 cells/ml was reached and then an inoculum of 2 x1 0 5 cells/ml was scaled twice, each with 1:10 dilutions. A further scale up was conducted using a 10 L homemade bioreactor. Cells were harvested by centrifugation at 3000 g, 4°C for 15 min and disrupted by 20 sonication cycles of 1 min each. In order to remove cell debris and hydrophobic proteins, a precipitation step in presence of (NH4) 2 SO 4 1M was then followed by dialysis vs 25 mM phosphate buffer pH 7 and finally loaded into the column. Protein Purification. PETase was purified exploiting the calculated pI of 9.6 and the molecular weight of 27559 Da, using a two-step reliable purification protocol with a cationic exchange HiPrep ™ SP HP 16/10 (Cytiva) chromatography. The eluted fractions containing PETase were collected and concentrated by Amicon® membrane ultrafiltration spin columns with an 5000 Da cutoff to a < 2 ml volume, and loaded on a size exclusion SEC separation using a HiLoad ™ Superdex ™ 75 (GE Healthcare) column. PETase was eluted from the HiPrep ™ SP HP 16/10 column across a 25mM Phosphate pH 7 / Phosphate 25 mM pH 7, 0.5 M NaCl gradient. Fractions containing the protein were pooled, concentrated, and loaded onto the SEC column and eluted isocratically in the presence of 200 mM phosphate buffer pH 7; 0.15 M NaCl. The eluted fractions from the two chromatographic steps were assessed by SDS-PAGE and western blotting. Tandem mass spectrometry For UHPLC–HRMS analysis, dry extracted peptides were resuspended in 50 µL of a mixture of water:acetonitrile : formic acid 95:3:2, sonicated for 10 minutes at room temperature and centrifuged at 12100 g for 10 minutes. A Thermo Scientific Dionex Ultimate 3000 195 UHPLC coupled to a Thermo high-resolution Q Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) was used for the analyses. Centroided MS and MS2 spectra were recorded from 200 to 2000 m/z in Full MS/dd-MS² (TOP2) mode. Precursor dynamic exclusion (6 seconds) and apex triggering (1–5 seconds) were set; peptide-like isotope pattern ions were preferred. The mass spectrometer was calibrated before the start of the analyses; an initial segment (0.1–0.7 minutes) with a lock mass (391.28429) was included in the MS 204 method. For protein identification, raw data, converted into mascot generic format using MsConvert (v. 3.0.10730, ProteoWizard tools; 25), were searched against Swiss-Prot for peptide sequences and an in-house database. Trypsin was selected as the proteolytic enzyme; oxidized methionine (M) was set as variable modifications while carbamidomethylation of cysteine (C) was set as fixed modifications in the search parameters. One missed cleavage was allowed. Mass tolerances were set at 10 ppm for the precursor ions (peak detection mismatch #13C = 1) and 0.5 Da for all the samples. An automatic decoy database search was used to estimate the false discovery rate; probability threshold was trimmed to get a FDR < 1%. Activity assays. Purified recombinant PETase was tested against 0.25 mm polyethylene terephthalate (PET) film and postconsumer packaging mainly composed of polystyrene (PS). 200 µL digestion reactions were set up with 6 x 4 mm pieces of clean substrate, 10 µL of purified protein fraction or a control solution in Phosphate buffer 25 mM pH 7. Reaction tubes were incubated at 30°C shaking (250 rpm) for 96 hours. Solid substrate was then removed and rinsed in subsequent steps with 1% SDS. Protein activity was assessed by analyzing the plastic pieces after incubation with the enzyme by atomic force microscopy (AFM). Morphological characterization of PET and PS samples was performed using an NT-MDT SMENA Solver platform (Moscow, Russia); the analysis was performed in semi-contact mode and the images analyzed using Gwyddion 2.61 freeware ( http://gwyddion.net ) Declarations Acknowledgements Giulia Di Rocco acknowledges Life Science Department “FAR2021.”and “RICHIESTA DI FINANZIAMENTO DI AZIONI DI MOBILITÁ NELL’AMBITO DEL PROGRAMMA DI COLLABORAZIONE SCIENTIFICA DELL’UNIVERSITÁ DI MODENA E REGGIO EMILIA CON UNIVERSITÁ E ISTITUZIONI STRANIERE (2018). Marcello Berto acknowledges Life Science Department “FAR2021”. Availability of Data and Materials The datasets used or analysed are available on https://doi.org/10.5281/zenodo.7740875 . References Chen, C. C., Dai, L., Ma, L. & Guo, R. T. Enzymatic degradation of plant biomass and synthetic polymers. Nat. Rev. Chem. 4, 114–126 (2020). Shah, A. A., Hasan, F., Hameed, A. & Ahmed, S. Biological degradation of plastics: A comprehensive review. Biotechnol. 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The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism. Front. Plant Sci. 12, 1–15 (2021). Moog, D. et al. Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation. Microb. Cell Fact. 18, 1–15 (2019). Kim, J. W. et al. Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae. Microb. Cell Fact. 19, 1–9 (2020). Tran, M., Zhou, B., Pettersson, P. L., Gonzalez, M. J. & Mayfield, S. P. Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts. Biotechnol. Bioeng. 104, 663–673 (2009). Seo, H. et al. Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli. Biochem. Biophys. Res. Commun. 508, 250–255 (2019). Bally, J. et al. Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins. Plant Biotechnol. J. 6, 46–61 (2008). Wannathong, T., Waterhouse, J. C., Young, R. E. B., Economou, C. K. & Purton, S. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Appl. Microbiol. Biotechnol. 100, 5467–5477 (2016). McLaughlin, J. A. et al. The Synthetic Biology Open Language (SBOL) Version 3: Simplified Data Exchange for Bioengineering. Front. Bioeng. Biotechnol. 8, 1–15 (2020). Lau KW, Ren J, W. M. (2000). Redox modulation of chloroplast DNA replication in Chlamydomonas reinhardtii. Antioxid Redox Signal 529–535 (2000). doi: 10.1089/15230860050192305 Di Lauro, M. et al. Liquid-Gated Organic Electronic Devices Based on High-Performance Solution-Processed Molecular Semiconductor. Adv. Electron. Mater. 3, 1700159 (2017). Rosano, G. L., Morales, E. S. & Ceccarelli, E. A. New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 28, 1412–1422 (2019). Chisti, Y. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306 (2007). Spolaore, P., Joannis-Cassan, C., Duran, E. & Isambert, A. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87–96 (2006). Deng, Y. et al. Microalgae for nutrient recycling from food waste to aquaculture as feed substitute: a promising pathway to eco-friendly development. J. Chem. Technol. Biotechnol. 96, 2496–2508 (2021). Fabris, M. et al. Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy. Frontiers in Plant Science 11, (2020). Parray, Z. A. et al. Interaction of polyethylene glycol with cytochrome c investigated via in vitro and in silico approaches. Sci. Rep. 11, 1–16 (2021). Krasnikov, B. F. et al. Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ. Am. J. Physiol. - Cell Physiol. 300, 1193–1203 (2011). Ranieri, A. et al. Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen. J. Biol. Inorg. Chem. 20, (2015). Di Rocco, G. et al. The enthalpic and entropic terms of the reduction potential of metalloproteins: Determinants and interplay. Coord. Chem. Rev. 445, 214071 (2021). Ranieri, A. et al. Electrocatalytic Properties of Immobilized Heme Proteins: Basic Principles and Applications. ChemElectroChem 6, (2019). Lancellotti, L. et al. Adsorbing surface strongly influences the pseudoperoxidase and nitrite reductase activity of electrode-bound yeast cytochrome c. The effect of hydrophobic immobilization. Bioelectrochemistry 136, 107628 (2020). Lancellotti, L. et al. Urea-induced denaturation of immobilized yeast iso-1 cytochrome c: Role of Met80 and Tyr67 in the thermodynamics of unfolding and promotion of pseudoperoxidase and nitrite reductase activities. Electrochim. Acta 363, (2020). Davies, D. R. & Plaskitt, A. Genetical and structural analyses of cell-wall formation in Chlamydomonas reinhardi. Genet. Res. 17, 33–43 (1971). Green MR, S. J. Molecular cloning: a laboratory manualk . (2013). Young, R. E. B. & Purton, S. Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: A strategy for the isolation of nuclear mutations that affect chloroplast gene expression. Plant J. 80, 915–925 (2014). Kindle, K. L., Richards, K. L. & Stern, D. B. Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U. S. A. 88, 1721–1725 (1991). Werner, R. & Mergenhagen, D. Mating Type Determination of Chlamydomonas reinhardtii by PCR. Plant Mol. Biol. Report. 16, 295–299 (1998). Additional Declarations No competing interests reported. Supplementary Files DiRoccoSupplementarymaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 20 Jun, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 08 May, 2023 Reviews received at journal 14 Apr, 2023 Reviewers agreed at journal 30 Mar, 2023 Reviewers invited by journal 30 Mar, 2023 Editor assigned by journal 26 Mar, 2023 Editor invited by journal 17 Mar, 2023 Submission checks completed at journal 17 Mar, 2023 First submitted to journal 08 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2668920","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":184301650,"identity":"17ce38a1-5e78-4289-b970-af6894d0e4f4","order_by":0,"name":"Giulia Di Rocco","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDACZgbGAwkMDIxtEK4NSAQMePBoYUDWkobQglsPUAsQMzZA2IcRwri0yLvzGBx4UMMg2yd9+OCHHxXnE7ezcyc+rqhgkLHHocXwMFBLwjEG4za+tGTJnjO3E3c28242PHMGt8MMm0Fa2BgS23h4zJgZ224nbjjMu02ysY2Qln9wLeegWv7h1iLPDNSS2AbXcgCqpQG3FgNmtoIDiX0Sxm08bCC/JBsDtWw2bDgmwcNzAIct/Yc3PvzxzUZ2fg8zKMTsZDecP7vxYUONjT17Aw5bIEZJYEhgisBtwWHUKBgFo2AUjAIEAAB3vFPOmTcvOAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Modena and Reggio Emilia","correspondingAuthor":true,"prefix":"","firstName":"Giulia","middleName":"Di","lastName":"Rocco","suffix":""},{"id":184301651,"identity":"98243cc4-86bd-4f8b-aa07-1a3c7a022d92","order_by":1,"name":"Henry N. Taunt","email":"","orcid":"","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Henry","middleName":"N.","lastName":"Taunt","suffix":""},{"id":184301652,"identity":"d8a7ad92-91be-4790-bda2-e758145a467a","order_by":2,"name":"Marcello Berto","email":"","orcid":"","institution":"University of Modena and Reggio Emilia","correspondingAuthor":false,"prefix":"","firstName":"Marcello","middleName":"","lastName":"Berto","suffix":""},{"id":184301653,"identity":"598d790f-8c10-4eee-a6df-808567303ab3","order_by":3,"name":"Harry O. Jackson","email":"","orcid":"","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Harry","middleName":"O.","lastName":"Jackson","suffix":""},{"id":184301654,"identity":"b25b06f6-5437-49c1-bdfb-7bd8a0f1cbc3","order_by":4,"name":"Daniele Piccinini","email":"","orcid":"","institution":"University of Modena and Reggio Emilia","correspondingAuthor":false,"prefix":"","firstName":"Daniele","middleName":"","lastName":"Piccinini","suffix":""},{"id":184301655,"identity":"41639e0b-620e-4cd9-b98e-e6f81867522a","order_by":5,"name":"Alan Carletti","email":"","orcid":"","institution":"University of Modena and Reggio Emilia","correspondingAuthor":false,"prefix":"","firstName":"Alan","middleName":"","lastName":"Carletti","suffix":""},{"id":184301656,"identity":"c1dd7a64-e9f2-4bf4-a100-02b36fdd43a6","order_by":6,"name":"Saul Purton","email":"","orcid":"","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Saul","middleName":"","lastName":"Purton","suffix":""}],"badges":[],"createdAt":"2023-03-08 09:59:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2668920/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2668920/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-37227-5","type":"published","date":"2023-06-20T21:18:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":34596391,"identity":"3e9dfdf1-36ac-44c9-8cfd-7cc54863fe2e","added_by":"auto","created_at":"2023-03-21 15:39:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80479,"visible":true,"origin":"","legend":"\u003cp\u003eA) Transformation of TN72 with the pSRSapI:PETase plasmid. Phototrophic selection selection simultaneously restores \u003cem\u003epsbH\u003c/em\u003e and replaces the \u003cem\u003eaadA\u003c/em\u003e cassette. with a\u003cem\u003e \u003c/em\u003ePETase expression cassette. The coding sequences, promoters and UTR elements of each cassette are illustrated using standard SBOL glyphs\u003csup\u003e39\u003c/sup\u003e. PCR primers used for genotype diagnostics are indicated: primer P1 binds upstream from the insertion site in both genotypes and gives either an 878 bp band in combination with primer P2 (binds to the TN72 plastome) or a 1037 bp band with primer P3 (binds to the transformed plastome). B) PCR results confirming the homoplasmic state of transformants TN72:PETase.1 and TN72:PETase.2. A single band of 878 bp is observed in the TN72 control, whereas a 1037 bp band is detected for the transformant lines. 1:100 and 1:200 dilutions of the TN72 DNA validate the sensitivity of the assay for detecting even a single copy of the parental DNA remaining in the transformant chloroplast.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-2668920/v1/0e64f489fabe5257fe846370.png"},{"id":34596395,"identity":"7fde5f3b-0f02-4368-83e9-ccafd244d026","added_by":"auto","created_at":"2023-03-21 15:39:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":810979,"visible":true,"origin":"","legend":"\u003cp\u003ePETase purification pipeline. A) HiPrep\u003csup\u003eTM\u003c/sup\u003e SP HP 16/10 chromatography B) Size exclusion chromatography (SEC-Superdex 75) was used to produce the final PETase purified product. Recombinant PETase is eluted in fraction 12 (highlighted in red), with significant enrichment of malate dehydrogenase (MDH) seen in fraction 8 – 10. C) SDS-PAGE for the fractions eluted during SEC chromatography; the bands of MDH (malate dehydrogenase), PETase and cytochrome \u003cem\u003ec\u003c/em\u003e were cut from the gel and the digested peptides extracted for MS analysis D) MS/MS result for the PETase protein sequence coverage. E) UV-vis spectrum with the peaks typical of a cytochrome \u003cem\u003ec\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-2668920/v1/61a043fad71d9a69192e5e4b.png"},{"id":34596393,"identity":"99e5a641-2944-43c9-b1b5-76b630cd1ee3","added_by":"auto","created_at":"2023-03-21 15:39:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1578443,"visible":true,"origin":"","legend":"\u003cp\u003eAFM topographical images of PET and PS samples following incubation with either recombinant PETase or a control solution. A) PET treated with a control solution, and B) treated with recombinant PETase. C) PS treated with a control solution, and D) treated with recombinant PETase. All images are 10 µm x 10 µm.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-2668920/v1/3a6c2f14e532892ba6d171ee.png"},{"id":34597574,"identity":"c38edbc9-4619-4926-b37b-f7689ae2c6b6","added_by":"auto","created_at":"2023-03-21 15:47:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":113014,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological parameters extracted from AFM images. A) Roughness \u003cem\u003es\u003c/em\u003e\u003csub\u003e\u003cem\u003erms\u003c/em\u003e\u003c/sub\u003e and B) correlation length \u003cem\u003ex\u003c/em\u003e variation between PET and PS sample after immersion in control and PETase-containing solutions. Standard deviations from 3\u003cem\u003en\u003c/em\u003e technical replicate images of one substrate sample are reported as errors.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-2668920/v1/2b6769da1da1662cf7d00d50.png"},{"id":44731562,"identity":"8302fac3-f936-44df-882b-6f201d9184ff","added_by":"auto","created_at":"2023-10-16 21:45:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2449925,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2668920/v1/118dbc7c-af93-4bf6-b6f9-718283bca778.pdf"},{"id":34596394,"identity":"55a8f5dc-b609-4368-a898-ab4dcb32a73a","added_by":"auto","created_at":"2023-03-21 15:39:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":511747,"visible":true,"origin":"","legend":"","description":"","filename":"DiRoccoSupplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2668920/v1/36d1475aa20bc0e9a061dabc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against PET and polystyrene","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNatural polymers such as lignin, starch, chitin, and cellulose are water-insoluble macromolecules present in the environment, and although such polymers are generally recalcitrant to physical and chemical degradation, nature has evolved enzymes for their breakdown\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Plastics are instead synthetic polymers designed specifically for their high resistance to degradation. Plastics are central to modern life and their production has expanded tremendously during the last few decades owing to their versatile properties and low cost. Although an increasing number of microorganisms capable of degrading plastic polymers have been isolated and the enzymes involved metabolically characterized, additional studies are needed to identify novel enzymes and associated degradation pathways for the wide range of different plastics\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The challenge of understanding and optimizing plastic enzymatic degradation closely emulates that of enzymatic depolymerization of polysaccharides\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Indeed, strategies that have been used to understand and improve glycoside hydrolases, including the development of quantitative assays for measuring enzyme (or enzyme cocktail) performance, can serve as inspiration for more quantitative metrics for comparing plastic-degrading enzymes and enzyme mixtures\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Owing to its robust mechanical properties and high post-consumer recycling costs, polyethylene terephthalate (PET) is one of the most abundant plastics in the world. PET accumulates in our environment without significant microbial conversion\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The constant flux of new PET into the global market has produced enormous amounts of waste with a long biodegradation timescale. This waste contributes to global pollution, especially for marine ecosystems, and poses a threat to human and animal health\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Similarly, polystyrene (PS) is a high demand plastic, ranking fourth in the world\u0026rsquo;s consumption of plastics and constituting 10% of total plastic waste\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. PS has various applications and is widely used in households, industries, healthcare and more. Its disposal incurs incineration costs, depletion of valuable carbon resources and energy consumption. It can be converted into its monomer styrene and aromatic hydrocarbons such as toluene and ethyl benzene by thermal degradation\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn 2016, Yoshida et al. discovered and isolated two new enzymes from \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e, a bacterium which was able to grow using PET as the main carbon and energy source\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These enzymes are a polyethylene terephthalate hydrolase (PETase), which can convert PET into mono(2-hydroxyethyl) terephthalic acid (MHET) and mono(2-hydroxyethyl) terephthalate hydrolase, responsible for the conversion of MHET to terephthalic acid (TPA) and ethylene glycol (EG)\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Given that PET and PS waste is mainly disposed of via incineration or thermal degradation, the possibility of biological degradation of such waste to non-toxic monomers represents an attractive and greener solution to reduce pollution. Although PS-degrading organisms have been identified and characterized\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, the enzymes responsible for the degradation of polystyrene are completely unknown.\u003c/p\u003e \u003cp\u003eFor production of enzymes for research and industrial purposes, the exploitation of the native plastic-degrading microorganism is not always possible and organisms that are easier to culture and engineer for heterologous expression are required\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To this end, microalgae represent an attractive biotechnology platform for the synthesis of recombinant proteins\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The advantages of using microalgae as opposed to traditional heterotrophic platforms of \u003cem\u003eEscherichia coli\u003c/em\u003e, yeast, or CHO cells are: (i) the low-cost phototrophic cultivation of the alga in sterile, controlled photobioreactors using simple and inexpensive medium\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e; (ii) the generally recognized as safe (GRAS) status of a number of algal species, including the chlorophyte \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e; (iii) the availability of the chloroplast as a unique biosynthetic and storage compartment within the cell that contains its own minimal genetic system\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e; and (iv) a growing interest and adoption of enabling synthetic biology principles for creating bespoke cell factories using microalgae\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Whilst several recent studies have reported the production of PETase in microalgal species through nuclear engineering\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, the use of the chloroplast for expression of foreign genes confers several benefits. These including precise insertion into the chloroplast genome (= \u0026ldquo;plastome\u0026rdquo;) via homologous recombination, high-level expression that is not subject to any gene-silencing mechanisms, the possibility of expressing multiple transgenes as operons and the accumulation of the recombinant proteins in a benign compartment where the formation of di-sulfide bonds occurs readily\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Here we report the synthesis of PETase from \u003cem\u003eI. sakaiensis\u003c/em\u003e in the chloroplast of \u003cem\u003eC. reinhardtii\u003c/em\u003e and demonstrate that the purified enzyme is active against both PET and PS.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGeneration of transplastomic\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eC. reinhardtii\u003c/span\u003e \u003cb\u003eexpressing PETase\u003c/b\u003e. A synthetic gene encoding the mature form of the \u003cem\u003eI. sakaiensis\u003c/em\u003e PETase was codon optimized for expression in the chloroplast and cloned into the pSRSapI destination vector, to generate plasmid pSRSapI:PETase (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The bacterial PETase enzyme contains two disulphide bonds that are formed following secretion into the \u003cem\u003eI. sakaiensis\u003c/em\u003e periplasm\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We therefore decided to retain the sequence for the \u003cem\u003eN\u003c/em\u003e-terminal Sec-type signal peptide within the transgene design so that the enzyme would be similarly targeted into the thylakoid lumen. Previous work has shown that bacterial signal peptides can direct recombinant proteins into the thylakoid lumen, and that disulphide bond formation occurs more readily in this chloroplast compartment\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This was used to transform \u003cem\u003eC. reinhardtii\u003c/em\u003e strain TN72 with the \u003cem\u003ePETase\u003c/em\u003e gene integrated into the plastome at a neutral locus between \u003cem\u003epsbH\u003c/em\u003e and \u003cem\u003etrnE2\u003c/em\u003e. Integration of the transforming DNA into TN72 also restores a wild-type copy of \u003cem\u003epsbH\u003c/em\u003e, which is an essential photosynthesis gene\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Selection is therefore based on restoration of photosynthesis allowing the generation of transformants lacking any antibiotic-based selectable marker, with the only foreign DNA introduced into the plastome being the PETase coding sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs the C. \u003cem\u003ereinhardtii\u003c/em\u003e plastome is polyploid with ~\u0026thinsp;40 copies per cell under phototrophic conditions\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, transformant lines were restreaked several times to single colonies under selective conditions to drive the lines to homoplasmy where all plastome copies have the transgenic DNA. Two such lines (TN72:PETase.1 and TN72:PETase.2, hereafter) were checked for homoplasmy using a 3-primer PCR screen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This showed a single band at 1037 bp in the TN72:PETase.1 and TN72:PETase.2 PCRs and a single band at 878 bp in the parental TN72 control PCRs, confirming correct integration of the PETase cassette and homoplasmy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, it was found that selection based on restoration of phototrophy established homoplasmy much more readily than antibiotic-based selection. This is probably due to the strong selective pressure of restoring the photosynthetic phenotype and to the reduced copy number of cells grown on minimal medium rather than the acetate-containing medium normally used for antibiotic-based selection\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Finally, PCR amplification and sequencing of the PETase cassette from both transformant lines confirmed than neither had acquired any mutations during plastome integration.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePETase\u003c/span\u003e \u003cb\u003eis expressed in the chloroplast\u003c/b\u003e. The TN72:PETase.1 cell line was characterized further in order to assess the level of recombinant protein produced and to determine whether the protein is correctly folded and functional. An evaluation of the PETase level was conducted by SDS-PAGE analysis of cell extracts with proteins bands in the 27 kDa region cut from the gel, trypsin-digested and subjected to tandem mass (MS/MS) spectrometry. As shown in Supplementary Figure S2 protein sequence coverage for PETase of 27% was obtained, confirming unambiguously the presence of PETase in the strains. Other bands from constitutive proteins were analysed revealing the presence of malate dehydrogenase of 30 kDa (Figure S3) and cytochrome \u003cem\u003ec\u003c/em\u003e at 10 kDa (Figure S4).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePurification of recombinant PETase from transgenic\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eC. reinhardtii\u003c/span\u003e. PETase has the classic α/β hydrolases fold, but despite the sequence similarity to cutinases and lipases, the highly polarized surface charge of PETase creates a dipole that gives the enzyme an isoelectric point of 9.6\u003csup\u003e20\u003c/sup\u003e that allows a first step of purification by cationic exchange HiPrep\u003csup\u003e\u0026trade;\u003c/sup\u003e SP HP 16/10. The protein was successfully recovered using an optimized two-step chromatography approach as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Elution of PETase from the HiPrep\u003csup\u003e\u0026trade;\u003c/sup\u003e SP HP 16/10 column was conducted by linear gradient of NaCl, with the target protein eluting at 20\u0026ndash;30% concentration. Several other peaks were observed on the chromatogram during this phase indicating the presence of several endogenous protein contaminants. Fractions highlighted in red in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA were loaded on the size exclusion chromatography (SEC) column, a HiLoad\u003csup\u003e\u0026trade;\u003c/sup\u003e Superdex\u003csup\u003e\u0026trade;\u003c/sup\u003e 75 column. During SEC a group of low-concentration proteins eluted first (peak 6 in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB); followed by the majority of the proteins as three separate peaks labelled 8\u0026ndash;10 (due to the presence of two small shoulders), 11 and 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). All fractions were analyzed by SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The gel shows that fraction 8\u0026ndash;10 contained a major protein with a molecular weight greater than 35 kDa, that was analysed by MS/MS revealing the presence of malate dehydrogenase (Supplementary Figure S3). Sample 12 revealed a band with a molecular weight of approximately 27 kDa, consistent with that of mature PETase. The band was analysed via MS/MS spectrometry confirming its identity as PETase with a 51% sequence coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Sample 12 also contained a mitochondrial cytochrome \u003cem\u003ec\u003c/em\u003e that was identified by MS/MS spectrometry and UV-visible spectroscopy (Supplementary Figure S4 and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Since this cytochrome has a pI\u0026thinsp;=\u0026thinsp;9.39, it eluted with PETase during the cationic exchange chromatography. Moreover the difference in molecular weights between PETase (27 kDa) and cytochrome \u003cem\u003ec\u003c/em\u003e (12 kDa) was not sufficiently different for the two proteins to be efficiently separated by size exclusion chromatography.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe recombinant PETase is active.\u003c/b\u003e To investigate the activity of the algal-expressed PETase, PET and PS substrates were incubated with the enzyme and control solutions then assessed by semi-contact atomic force microscopy (AFM) imaging in air (Fig.\u0026nbsp;3). It is clear that the recombinant PETase can modify the morphology of both PET and PS samples. Specifically, in samples incubated with the enzyme, the formation of holes can be observed by the presence of darker spots, while no holes are visible in the control samples (i.e. the same experimental procedures without the enzyme). To quantitatively evaluate the morphological changes on the surfaces due to PETase activity, the average surface roughness of the samples \u003cem\u003eσ\u003c/em\u003e\u003csub\u003e\u003cem\u003erms\u003c/em\u003e\u003c/sub\u003e and the lateral correlation length \u003cem\u003eξ\u003c/em\u003e were calculated. These variables correspond to a measure of the texture of the surface, and the distance range over which points in one region of space are correlated with those in another region, respectively\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;3). PETase activity resulted in a decrease of \u003cem\u003eξ\u003c/em\u003e in both materials: PET samples (Fig.\u0026nbsp;3A and 3B) displayed a decrease by an order of magnitude, from 2.2 \u0026plusmn; 0.6 \u0026micro;m (control sample) to 0.22 \u0026plusmn; 0.08 \u0026micro;m (when incubated with enzyme), while, for PS samples (Fig.\u0026nbsp;3C and 3D), a reduction of 60% was observed, from 0.53 \u0026plusmn; 0.1 \u0026micro;m (control sample) to 0.21 \u0026plusmn; 0.03 \u0026micro;m (when incubated with enzyme). The average values of \u003cem\u003eξ\u003c/em\u003e were obtained from three images of each sample and interestingly, after incubation with the enzyme, they were similar (\u0026cong;0.2 \u0026micro;m) for both materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Since there were no other sources of degradation in sample reactions we could ascribe those values exclusively to the PETase activity, and, in particular, the presence, the dimension and the density of the holes on the surfaces confirmed the functionality of the enzyme. Conversely, the surface roughness variation (Δ\u003cem\u003eσ\u003c/em\u003e\u003csub\u003e\u003cem\u003erms\u003c/em\u003e\u003c/sub\u003e) significantly increased for PS samples (from 4.5 \u0026plusmn; 0.6 nm to 22 \u0026plusmn; 3 nm with Δ\u003cem\u003eσ\u003c/em\u003e\u003csub\u003e\u003cem\u003ermsPS\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e=\u0026thinsp;+\u0026thinsp;17.5 nm)\u003c/em\u003e, while it does not change for PET samples (from 4.8 \u0026plusmn; 0.5 nm to 4.4 \u0026plusmn; 0.4 nm with Δ\u003cem\u003eσ\u003c/em\u003e\u003csub\u003e\u003cem\u003ermsPET\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e=-0.4 nm\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This may suggest that the chemical and physical stress suffered by the plastic samples during the incubation could affect more one material than the other. A possible explanation is that the technical-grade PET foil is more resilient to stress than postconsumer PS foil (in fact Δ\u003cem\u003eσ\u003c/em\u003e\u003csub\u003e\u003cem\u003ermsPS\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e\u0026lt;\u003c/em\u003e\u0026thinsp;Δ\u003cem\u003eσ\u003c/em\u003e\u003csub\u003e\u003cem\u003ermsPET\u003c/em\u003e\u003c/sub\u003e ), but, in any case both materials were affected by the enzyme activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePETase is a recently discovered hydrolase enzyme acting on PET\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. With a view to its potential significance in an industrial context, in this work we describe a platform for producing such enzymes in the chloroplast of C. \u003cem\u003ereinhardtii\u003c/em\u003e where we obtained \u0026sim; 5 mg of pure product from 40 g of wet biomass (yield of \u0026cong; 0.012%). While the recombinant protein yield is lower than that of bacterial systems\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, microalgae offer several potential advantages, such as reduced scale-up costs and use of the algal biomass for extraction of useful byproducts; carotenoids, pigments, proteins, and vitamins that can be used for the production of nutraceuticals, animal feed additives, cosmetics, or for energy production\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Expression in the chloroplasts has the advantage of precision targeting of transgenic DNA into a selected site in the plastome via homologous recombination, stable and high-level expression of transgenes in the absence of selection, and the ability to compartmentalize and target potentially toxic recombinant proteins within the chloroplast. Moreover, the use of photosynthetic restoration in the transformation of strain TN72 avoids the use of selection markers based on antibiotic-resistance genes and circumvents concerns over escape of such genes via lateral gene transfer. Finally, the pilot scale PETase production carried out here (10 L culture medium), allowed for the first time the expression of a sufficient amount of recombinant protein for purification, and this will facilitate further characterization of the enzyme kinetics for different PETase variants. Interestingly, the purification of the recombinant PETase by means of cationic exchange chromatography resulted in the presence of a cytochrome \u003cem\u003ec\u003c/em\u003e with a pI similar to that of the PETase (9.39 and 9.65, respectively) in the final fraction, implying that the two proteins stayed together during all the purification steps. Considering this result, one potential explanation for the unprecedented hydrolytic activity on post-consumer material containing mainly polystyrene could be due to the presence of cytochrome \u003cem\u003ec\u003c/em\u003e in the reaction mixture. Cytochrome \u003cem\u003ec\u003c/em\u003e is a well characterized and ubiquitous protein that can interact with various molecules (i.e. phospholipids through electrostatic and hydrophobic bonds)\u003csup\u003e\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and in some cases (e.g. M80A mutant) it can exert a catalytic activity\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, but primarily cytochrome \u003cem\u003ec\u003c/em\u003e is an electron transfer protein working only in the presence of electron donors that, in this specific case, were not added to the reaction mixture. Therefore, we can exclude that within the reaction conditions tested in this paper cytochrome \u003cem\u003ec\u003c/em\u003e contributed to the enzymatic reaction between PETase and PS.\u003c/p\u003e \u003cp\u003eAn explanation for the PS activity could be found in the structure of PS, that, unlike PET, has the aromatic rings perpendicular to the polymer chain, thus providing Van der Waals interactions between the aromatic ring of Trp185 present in the active site of PETase and the surface exposed aromatic rings of the plastics. This could be a hypothesis but the scope of this work was the production of an active form of PETase in the microalgal chloroplast and we have successfully obtained a transgenic strain of C. \u003cem\u003ereinhardtii\u003c/em\u003e expressing an active heterologous PETase. The unexpected novelty was the discovery of its unprecedented activity on PS, paving the way to new perspectives of investigation on the mechanism of this reaction.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials.\u003c/strong\u003e \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e strain TN72 (CC-5168, Chlamydomonas Resource Center: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.chlamycollection.org\u003c/span\u003e\u003c/span\u003e) was used to generate the transgenic lines in this study as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. All lines were maintained on tris-acetate-phosphate (TAP) medium supplemented with 1.5% agar unless otherwise stated. Liquid cultures were cultivated in TAP medium at 25\u0026deg;C, with agitation at 125 rpm. All lines were illuminated with continuous white light at 50 \u0026micro;E/m\u003csup\u003e2\u003c/sup\u003e/s, with the light sensitive TN72 parental line protected with a white paper cover to reduce light intensity by approximately an order of magnitude.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning procedures.\u003c/strong\u003e DNA manipulations were performed following standard protocols\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, including transformation of chemically competent \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha;. Oligonucleotide primers were purchased from Eurofins Genomics (Ebersberg, Germany) while restriction enzymes and T4 DNA ligase for cloning procedures are purchased from NEB (Ipswich, MA, USA) and Thermo Scientific (Waltham, MA,USA).. The coding sequence for \u003cem\u003eI. sakaiensis\u003c/em\u003e PETase (PETase Uniprot: A0A0K8P6T7; Gene: ISF6_4831; EC:3.1.1.101), together with a Strep-tag at the N terminus, was codon-optimized for the \u003cem\u003eC. reinhardtii\u003c/em\u003e chloroplast using the Codon Usage Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.kazusa.or.jp/codon\u003c/span\u003e\u003c/span\u003e) with SapI and SphI sites added for cloning into the pSRSapI transformation vector (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The DNA was synthesized by IDT (Integrated DNA Technologies, Inc., Coralville, Iowa-USA), and was cloned into the transformation vector by standard molecular techniques\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransformation of the\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eC. reinhardtii\u003c/span\u003e \u003cstrong\u003echloroplast.\u003c/strong\u003e \u003cem\u003eC. reinhardtii\u003c/em\u003e was transformed by the glass bead method as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. 400 mL transformation cultures were grown to early log phase (1\u0026ndash;2 x 10\u003csup\u003e6\u003c/sup\u003e cells/ mL) and harvested by centrifugation at 3000 x \u003cem\u003eg\u003c/em\u003e. Cells were resuspended in sterile high salt minimal (HSM) medium to a density of 2 x 10\u003csup\u003e8\u003c/sup\u003e cells/ml, and 300 \u0026micro;L added to sterilized 5-ml tests tube containing 300 mg sterile 400\u0026ndash;625 \u0026micro;m diameter glass beads and 10 \u0026micro;g plasmid DNA. Tubes were agitated by vortex at maximum speed for 15 seconds, then mixed with 4 ml molten HSM supplemented with 0.5% agar at 42\u0026deg;C and immediately spread on HSM plates supplemented with 1.5% agar and 100 \u0026micro;g/mL ampicillin. Plates were sealed with parafilm and incubated at 25\u0026deg;C in dim light (~\u0026thinsp;2 \u0026micro;E/m\u003csup\u003e2\u003c/sup\u003e/s) overnight then transferred to moderate light (~\u0026thinsp;50 \u0026micro;E/m\u003csup\u003e2\u003c/sup\u003e/s)\u003csup\u003e29,38\u003c/sup\u003e. Transformant colonies were picked after 3\u0026ndash;4 weeks and restreaked to single colonies on selective media until homoplasmy was achieved, typically after 2\u0026ndash;3 restreaks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping of transformant lines\u003c/strong\u003e. Lines were assessed by PCR analysis using a three-primer strategy as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e In each case a forward flanking primer was designed outside of the left homology arm used for transformation, with reverse primers designed within the parental and transformed cassettes respectively. Details of primers used are given in the supplementary data (Table S5). Insertion of the target PETase CDS was further confirmed by PCR amplification of the expression cassette followed by Sanger sequencing of the PCR product. Total genomic DNA is extracted from a small amount of cells using the Chelex 100 method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and PCR amplification is carried out with Phusion DNA polymerase (Thermo Scientific) according to the manufacturer\u0026rsquo;s instructions. In this strategy (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), a plastome specific primer (P1) is used in conjunction with two other primers in a three primer reaction. A second primer (P2) binds to a terminator element (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/sub\u003e) in the parental TN72 strain, in which the endogenous \u003cem\u003epsbH\u003c/em\u003e gene (essential for photosynthesis) is disrupted with an aminoglycoside 3\u0026Prime;-adenylyltransferase (aadA) expression cassette, to generate a band of 878 bp. The third primer (P3) binds to a promoter element (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003epsaA\u003c/em\u003e\u003c/sub\u003e) in the transformed genotype, containing the PETase expression cassette and the restored of \u003cem\u003epsbH\u003c/em\u003e to give a band of 1037 bp. Dilution control PCR (1:100 and 1:200) were performed with TN72 DNA, to ensure that the PCR reaction was sufficiently sensitive to detect the parental DNA in the TN72:PETase PCR if the parental DNA were present at low/single copy numbers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScale up cultivation, cell breakage, and preparation of soluble protein extract.\u003c/strong\u003e \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e expressing PETase cells line was inoculated in 20 ml of liquid TAP medium and grown at, 25\u0026deg;C, 100 rpm and intensity light of 50 \u0026micro;E m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 4 days. Cell growth was monitored over time using a B\u0026uuml;rker chamber and the rate of increase was obtained. Cells were let grown until a concentration of 4 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml was reached and then an inoculum of 2 x1 0\u003csup\u003e5\u003c/sup\u003e cells/ml was scaled twice, each with 1:10 dilutions. A further scale up was conducted using a 10 L homemade bioreactor. Cells were harvested by centrifugation at 3000 g, 4\u0026deg;C for 15 min and disrupted by 20 sonication cycles of 1 min each. In order to remove cell debris and hydrophobic proteins, a precipitation step in presence of (NH4)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 1M was then followed by dialysis \u003cem\u003evs\u003c/em\u003e 25 mM phosphate buffer pH 7 and finally loaded into the column.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein Purification.\u003c/strong\u003e PETase was purified exploiting the calculated pI of 9.6 and the molecular weight of 27559 Da, using a two-step reliable purification protocol with a cationic exchange HiPrep\u003csup\u003e\u0026trade;\u003c/sup\u003e SP HP 16/10 (Cytiva) chromatography. The eluted fractions containing PETase were collected and concentrated by Amicon\u0026reg; membrane ultrafiltration spin columns with an 5000 Da cutoff to a\u0026thinsp;\u0026lt;\u0026thinsp;2 ml volume, and loaded on a size exclusion SEC separation using a HiLoad\u003csup\u003e\u0026trade;\u003c/sup\u003e Superdex\u003csup\u003e\u0026trade;\u003c/sup\u003e 75 (GE Healthcare) column. PETase was eluted from the HiPrep\u003csup\u003e\u0026trade;\u003c/sup\u003e SP HP 16/10 column across a 25mM Phosphate pH 7 / Phosphate 25 mM pH 7, 0.5 M NaCl gradient. Fractions containing the protein were pooled, concentrated, and loaded onto the SEC column and eluted isocratically in the presence of 200 mM phosphate buffer pH 7; 0.15 M NaCl. The eluted fractions from the two chromatographic steps were assessed by SDS-PAGE and western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTandem mass spectrometry\u003c/strong\u003e For UHPLC\u0026ndash;HRMS analysis, dry extracted peptides were resuspended in 50 \u0026micro;L of a mixture of water:acetonitrile : formic acid 95:3:2, sonicated for 10 minutes at room temperature and centrifuged at 12100 g for 10 minutes. A Thermo Scientific Dionex Ultimate 3000 195 UHPLC coupled to a Thermo high-resolution Q Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) was used for the analyses. Centroided MS and MS2 spectra were recorded from 200 to 2000 m/z in Full MS/dd-MS\u0026sup2; (TOP2) mode. Precursor dynamic exclusion (6 seconds) and apex triggering (1\u0026ndash;5 seconds) were set; peptide-like isotope pattern ions were preferred. The mass spectrometer was calibrated before the start of the analyses; an initial segment (0.1\u0026ndash;0.7 minutes) with a lock mass (391.28429) was included in the MS 204 method. For protein identification, raw data, converted into mascot generic format using MsConvert (v. 3.0.10730, ProteoWizard tools; 25), were searched against Swiss-Prot for peptide sequences and an in-house database. Trypsin was selected as the proteolytic enzyme; oxidized methionine (M) was set as variable modifications while carbamidomethylation of cysteine (C) was set as fixed modifications in the search parameters. One missed cleavage was allowed. Mass tolerances were set at 10 ppm for the precursor ions (peak detection mismatch #13C\u0026thinsp;=\u0026thinsp;1) and 0.5 Da for all the samples. An automatic decoy database search was used to estimate the false discovery rate; probability threshold was trimmed to get a FDR\u0026thinsp;\u0026lt;\u0026thinsp;1%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivity assays.\u003c/strong\u003e Purified recombinant PETase was tested against 0.25 mm polyethylene terephthalate (PET) film and postconsumer packaging mainly composed of polystyrene (PS). 200 \u0026micro;L digestion reactions were set up with 6 x 4 mm pieces of clean substrate, 10 \u0026micro;L of purified protein fraction or a control solution in Phosphate buffer 25 mM pH 7. Reaction tubes were incubated at 30\u0026deg;C shaking (250 rpm) for 96 hours. Solid substrate was then removed and rinsed in subsequent steps with 1% SDS. Protein activity was assessed by analyzing the plastic pieces after incubation with the enzyme by atomic force microscopy (AFM). Morphological characterization of PET and PS samples was performed using an NT-MDT SMENA Solver platform (Moscow, Russia); the analysis was performed in semi-contact mode and the images analyzed using Gwyddion 2.61 freeware (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gwyddion.net\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiulia Di Rocco acknowledges Life Science Department \u0026ldquo;FAR2021.\u0026rdquo;and \u0026ldquo;RICHIESTA DI FINANZIAMENTO DI AZIONI DI MOBILIT\u0026Aacute; NELL\u0026rsquo;AMBITO DEL PROGRAMMA DI COLLABORAZIONE SCIENTIFICA DELL\u0026rsquo;UNIVERSIT\u0026Aacute; DI MODENA E REGGIO EMILIA CON UNIVERSIT\u0026Aacute; E ISTITUZIONI STRANIERE (2018).\u0026nbsp;Marcello Berto acknowledges Life Science Department \u0026ldquo;FAR2021\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used or analysed are available on\u0026nbsp;\u003cstrong\u003ehttps://doi.org/10.5281/zenodo.7740875\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen, C. 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Report. 16, 295\u0026ndash;299 (1998).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"atomic force microscopy, Chlamydomonas, chloroplast expression, PETase, polyethylene terephthalate, polystyrene","lastPublishedDoi":"10.21203/rs.3.rs-2668920/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2668920/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for the low-cost synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e in which the PETase from \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and polystyrene (PS) was investigated via atomic force microscopy, revealing evidence of degradation of both plastics.\u003c/p\u003e","manuscriptTitle":"A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against PET and polystyrene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-21 15:39:02","doi":"10.21203/rs.3.rs-2668920/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-05-08T19:49:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-04-14T08:50:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"cd8cb3e6-5d4c-4dc7-bf46-ac254961dca8","date":"2023-03-30T23:55:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-03-30T16:05:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-26T18:45:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-03-17T14:11:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-03-17T10:19:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-03-08T09:52:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa44e263-b8ad-45ad-9e79-53f9095cc41e","owner":[],"postedDate":"March 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":19998332,"name":"Biological sciences/Biotechnology/Expression systems"},{"id":19998333,"name":"Biological sciences/Biotechnology/Molecular engineering"},{"id":19998334,"name":"Biological sciences/Biotechnology/Protein delivery"},{"id":19998335,"name":"Biological sciences/Biophysics/Nanoscale biophysics"}],"tags":[],"updatedAt":"2023-10-16T21:29:29+00:00","versionOfRecord":{"articleIdentity":"rs-2668920","link":"https://doi.org/10.1038/s41598-023-37227-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-06-20 21:18:42","publishedOnDateReadable":"June 20th, 2023"},"versionCreatedAt":"2023-03-21 15:39:02","video":"","vorDoi":"10.1038/s41598-023-37227-5","vorDoiUrl":"https://doi.org/10.1038/s41598-023-37227-5","workflowStages":[]},"version":"v1","identity":"rs-2668920","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2668920","identity":"rs-2668920","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}