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lastics with desirable properties such as degradation (2, 3). About 56 million tons of PET Department of Applied Biology, Faculty of Textile Science,
Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto
durability, plasticity, and/or transparency was produced worldwide in 2013 alone, prompt- 606-8585, Japan. 2Department of Biosciences and
have been industrially produced over the ing further industrial production of its mono- Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku,
past century and widely incorporated into mers, terephthalic acid (TPA) and ethylene glycol Yokohama, Kanagawa 223-8522, Japan. 3Life Science
consumer products (1). Many of these pro- (EG), both of which are derived from raw petro- Materials Laboratory, ADEKA, 7-2-34 Higashiogu, Arakawa-ku,
Tokyo 116-8553, Japan. 4Department of Polymer Science,
ducts are remarkably persistent in the environ- leum. Large quantities of PET have been intro- Faculty of Textile Science, Kyoto Institute of Technology,
ment because of the absence or low activity of duced into the environment through its production Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. 5Ecology-
catabolic enzymes that can break down their and disposal, resulting in the accumulation of PET Related Material Group Innovation Research Institute, Teijin,
plastic constituents. In particular, polyesters con- in ecosystems across the globe (4). Hinode-cho 2-1, Iwakuni, Yamaguchi 740-8511, Japan.
*Present address: Department of Polymer Chemistry, Graduate
taining a high ratio of aromatic components, such There are very few reports on the biological School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-
as poly(ethylene terephthalate) (PET), are chem- degradation of PET or its utilization to support 8530, Japan. †Corresponding author. E-mail: kmiyamoto@bio.
ically inert, resulting in resistance to microbial microbial growth. Rare examples include mem- keio.ac.jp (K.M.); bika@kit.ac.jp (K.O.)
0
Weight loss (mg)
10
20
30
40
50
PET 60
film 0 20 40 60 80
Cultivation time (days)
1 1 2 3
2 0
6 10
Weight loss (mg)
3
4 6 20
4
30
5
5 40
50
8 60
7 7 8
0 20 40 60
9 10 9 10
Cultivation time (days)
Fig. 1. Microbial growth on PET. The degradation of PET film (60 mg, 20 × vitamins) medium was changed weekly. (D to F) SEM images of I. sakaiensis
15 × 0.2 mm) by microbial consortium no. 46 at 30°C is shown in (A) to (C). cells grown on PET film for 60 hours. Scale bars, 1 mm. Arrow heads in the left
The MLE (modified lettuce and egg) medium (10 mL) was changed biweekly. panel of (D) indicate contact points of cell appendages and the PET film surface.
(A) Growth of no. 46 on PET film after 20 days. (B) SEM image of degraded PET Magnifications are shown in the right panel. Arrows in (F) indicate appendages
film after 70 days. The inset shows intact PET film. Scale bar, 0.5 mm. (C) Time between the cell and the PET film surface. (G) SEM image of a degraded PET
course of PET film degradation by no. 46. PET film degradation by I. sakaiensis film surface after washing out adherent cells. The inset shows intact PET film.
201-F6 at 30°C is shown in (D) to (H).The YSV (yeast extract–sodium carbonate– Scale bar, 1 mm. (H) Time course of PET film degradation by I. sakaiensis.
a mixture of bacteria, yeast-like cells, and proto- Biotechnology Information taxonomy database There are currently few known examples of
zoa, whereas the culture fluid was almost trans- under identifier 1547922). In addition to being esterases, lipases, or cutinases that are capable
parent (Fig. 1A). This consortium degraded the found in the culture fluid, cells were observed on of hydrolyzing PET (8, 9). To explore the genes
PET film surface (fig. S1) at a rate of 0.13 mg cm–2 the film (Fig. 1D) and appeared to be connected involved in PET hydrolysis in I. sakaiensis 201-
day–1 at 30°C (Fig. 1C), and 75% of the degraded to each other by appendages (Fig. 1E). Shorter F6, we assembled a draft sequence of its genome
PET film carbon was catabolized into CO2 at 28°C appendages were observed between the cells and (table S1). One identified open reading frame
(fig. S2). the film; these might assist in the delivery of se- (ORF), ISF6_4831, encodes a putative lipase that
Using limiting dilutions of consortium no. 46 creted enzymes into the film (Fig. 1, D and F). shares 51% amino acid sequence identity and
that were cultured with PET film to enrich for The PET film was damaged extensively (Fig. 1G) catalytic residues with a hydrolase from Ther-
microorganisms that are nutritionally dependent and almost completely degraded after 6 weeks at mobifida fusca (TfH) (fig. S4 and table S2) that
on PET, we successfully isolated a bacterium capa- 30°C (Fig. 1H). In the course of subculturing no. exhibits PET-hydrolytic activity (10). We purified the
ble of degrading and assimilating PET. The strain 46, we found a subconsortium that lost its PET corresponding recombinant I. sakaiensis proteins
represents a novel species of the genus Ideonella, degradation capability. This subconsortium lacked (fig. S5) and incubated them with PET film at
for which we propose the name Ideonella sakaiensis I. sakaiensis (fig. S3), indicating that I. sakaiensis 30°C for 18 hours. Prominent pitting developed on
201-F6 (deposited in the National Center for is functionally involved in PET degradation. the film surface (Fig. 2A). Mono(2-hydroxyethyl)
Thermobifida group
4OYY
(Humicola insolens)
ADV92528
(T. fusca)
TfH
ADV92526
ADV92527
(T. cellulosilytica)
(T. cellulosilytica)
1000
BAO42836.1
668 (Saccharomonospora viridis)
1000 1000
FsC 986
1000
MHET O O
HO CH2 CH2 O C C OH
LCC
O O
HO C C OH
10 mV
TPA BHET O O
HO CH2 CH2 O C C O CH2 CH2 OH PETase
18h
0h 0.1
18 19 20 21 22 23 24 ADH43200.1
Retention time (min) (Bacillus subtilis)
pNP-acetate (C2)
PETase pNP-butyrate (C4)
pNP-caproate (C6) 100 LCC
pNP-caprylate (C8) PETase
PETase
TfH 10-1
TfH
TfH
LCC 10-2
TPA b/a(C2)
LCC
MHET b/a(C4)
TPA 10-3 FsC
BHET b/a(C6)
FsC b/a(C8) FsC MHET
10-4
0 40 80 120 0 0.1 0.2 0.3 -5 -4 -3 -2 -1 0 0 0.4 0.8 0 0.010 0.005 0.015 20 30 40 50 60 70 80
Apparent kcat (sec-1) Released compounds (mM) log10Ratio Apparent kcat (sec-1) Released compounds (mM) Temperature (°C)
Fig. 2. ISF6_4831 protein is a PETase. Effects of PETase on PET film are panel to those in the leftmost panel). All reactions were performed in pH
shown in (A) and (B). PET film (diameter, 6 mm) was incubated with 50 nM 7.0 buffer at 30°C. PET film was incubated with 50 nM enzyme for 18 hours.
PETase in pH 7.0 buffer for 18 hours at 30°C. (A) SEM image of the treated (E) Activity of the PET hydrolytic enzymes for highly crystallized PET
PET film surface. The inset shows intact PET film. Scale bar, 5 mm. (B) High- (hcPET). The hcPET (diameter, 6 mm) was incubated with 50 nM PETase
performance liquid chromatography spectrum of the products released from or 200 nM TfH, LCC, or FsC in pH 9.0 bicine-NaOH buffer for 18 hours at
the PET film. (C) Unrooted phylogenetic tree of known PET hydrolytic en- 30°C. (F) Effect of temperature on enzymatic PET film hydrolysis. PET film
zymes. The GenBank or Protein Data Bank accession numbers (with the or- (diameter, 6 mm) was incubated with 50 nM PETase or 200 nM TfH, LCC,
ganism source of protein in parentheses) are shown at the leaves. Bootstrap or FsC in pH 9.0 bicine-NaOH buffer for 1 hour. For better detection of the
values are shown at the branch points. Scale bar, 0.1 amino acid substitutions released products in (E) and (F), the pH and enzyme concentrations were
per single site. (D) Substrate specificity of four phylogenetically distinct PET determined based on the results shown in figs. S6 and S7, respectively.
hydrolytic enzymes (b/a indicates the ratio of the values in the middle-left Error bars in (D) to (F) indicate SE (n ≥ 3).
*
last four digits of the ORF number). Two-sided P 4831
**
PETase
**
values were derived from Baggerly’s test of the (4831)
**
differences between the means of two indepen- O O
0224
**
**
dent RNA sequencing experiments (*P < 0.05; HO CH2 CH2 O C C OH
* **
**P < 0.01). Colors correspond to the steps in
**
0076
(B). (B) Predicted I. sakaiensis PET degradation
pathway. The cellular localization of PETase and MHETase
* **
O O (0224)
**
0077
MHETase was predicted first (supplementary text,
TPA degradation
HO C C OH
section S1). Extracellular PETase hydrolyzes PET
****
0227
to produce MHET (the major product) and TPA.
**
MHETase, a predicted lipoprotein, hydrolyzes MHET TPATP (0076/0077)
****
O2
0228
to TPA and EG. TPA is incorporated through the
**
NADPH + H+
TPA transporter (TPATP) (17) and catabolized by
**
0230 TPADO (0227/0228/0230)
****
TPA 1,2-dioxygenase (TPADO), followed by 1,2- NADP+
dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate de- COOH COOH COOH
**
0229
****
hydrogenase (DCDDH). The resultant PCA is
DCDDH Pca34
PCA degradation
****
**
TPA-Na HOO C
described in the supplementary text (section S2). OH HOO C
PET film OH
****
CO2 O2
0627 HO OC OH
BHET
**
NADP+ NADPH + H+ OH
ORF#
(ISF6_XXXX)
or PET film (fig. S9 and table S3). The catabolic RE FERENCES AND NOTES 16. P. Kersey et al., Nucleic Acids Res. 33, D297–D302
genes for TPA and the metabolite protocatechuic 1. V. Sinha, M. R. Patel, J. V. Patel, J. Polym. Environ. 18, 8–25 (2005).
(2010). 17. M. Hosaka et al., Appl. Environ. Microbiol. 79, 6148–6155
acid (PCA) were up-regulated dramatically when (2013).
2. R. J. Müller, I. Kleeberg, W. D. Deckwer, J. Biotechnol. 86,
cells were cultured on TPA-Na, BHET, or PET film. 87–95 (2001).
This contrasted with genes for the catabolism 3. D. Kint, S. Munoz-Guerra, Polym. Int. 48, 346–352 AC KNOWLED GME NTS
of maltose (Fig. 3A), which involves a pathway (1999). We are grateful to Y. Horiuchi, M. Uemura, T. Kawai, K. Sasage, and
distinct from the degradation of TPA and EG, 4. L. Neufeld, F. Stassen, R. Sheppard, T. Gilman, Eds., The New S. Hase for research assistance. We thank D. Dodd, H. Atomi,
Plastics Economy: Rethinking the Future of Plastics (World T. Nakayama, and A. Wlodawer for comments on this manuscript.
indicating efficient metabolism of TPA by I. Economic Forum, 2016); www3.weforum.org/docs/WEF_The_ This study was supported by grants-in-aid for scientific research
sakaiensis. The transcript level of the PETase- New_Plastics_Economy.pdf. (24780078 and 26850053 to S.Y.) and the Noda Institute for
encoding gene during growth on PET film was 5. T. Nimchua, H. Punnapayak, W. Zimmermann, Biotechnol. J. 2, Scientific Research (S.Y.). The reported nucleotide sequence data,
the highest among all analyzed coding sequen- 361–364 (2007). including assembly and annotation, have been deposited in the
6. T. Nimchua, D. E. Eveleigh, U. Sangwatanaroj, H. Punnapayak, DNA Data Bank of Japan, European Molecular Biology Laboratory,
ces (table S4), and it was 15, 31, and 41 times as J. Ind. Microbiol. Biotechnol. 35, 843–850 (2008). and GenBank databases under the accession numbers
high as when bacteria were grown on maltose, 7. Materials and methods are available as supplementary BBYR01000001 to BBYR01000227. All other data are reported in
TPA-Na, and BHET, respectively. This suggests that materials on Science Online. the supplementary materials. The reported strain Ideonella
the expression of PETase is induced by PET film 8. D. Ribitsch et al., Biocatalysis Biotransform. 30, 2–9 sakaiensis 201-F6T was deposited at the National Institute of
(2012). Technology and Evaluation Biological Resource Center as strain
itself and/or some degradation products other 9. W. Zimmermann, S. Billig, Adv. Biochem. Eng. Biotechnol. 125, NBRC 110686T and at Thailand Institute of Scientific and
than TPA, EG, MHET, and BHET. 97–120 (2010). Technological Research as strain TISTR 2288T.
The expression levels of the PETase gene in the 10. R. J. Müller, H. Schrader, J. Profe, K. Dresler, W. D. Deckwer,
four different media were similar to those of ano- Macromol. Rapid Commun. 26, 1400–1405 (2005).
11. S. Sulaiman et al., Appl. Environ. Microbiol. 78, 1556–1562 SUPPLEMENTARY MATERIALS
ther ORF, ISF6_0224 (fig. S10), indicating similar (2012).
regulation. ISF6_0224 is located adjacent to the www.sciencemag.org/content/351/6278/1196/suppl/DC1
12. C. M. Silva et al., J. Polym. Sci. A Polym. Chem. 43, 2448–2450
C
of a bacterium that might have obtained the nec-
essary set of genes through lateral gene transfer. hromosomal aberrations at 22q13 that de- SHANK3 are also associated with nonsyndromic
A limited number of mutations in a hydrolase, lete or inactivate one SH3 and multiple autism spectrum disorder (ASD) and intellectual
such as PET hydrolytic cutinase, that inherently ankyrin repeat domains 3 (SHANK3) allele disability (1–4). Genetic ablation of Shank3 in
targets the natural aliphatic polymer cutin may are genetic hallmarks of Phelan-McDermid mice yields ASD-like behavioral phenotypes and
have resulted in enhanced selectivity for PET. syndrome (PMDS). De novo mutations in synaptic dysfunction (5–10), the latter of which
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