Rapid Evolution of Plastic-Degrading Enzymes Prevalent in the Global Ocean
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bioRxiv preprint doi: https://doi.org/10.1101/2020.09.07.285692; this version posted September 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Title: Rapid Evolution of Plastic-degrading Enzymes Prevalent in the Global Ocean 2 3 Authors: Intikhab Alam1*, Nojood Aalismail2, Cecilia Martin2, Allan Kamau1, Francisco J. 4 Guzmán-Vega5, Tahira Jamil1, Afaque A. Momin5, Silvia G. Acinas3, Josep M. Gasol3, Stefan T. 5 Arold5,6, Takashi Gojobori1, Susana Agusti4 and Carlos M. Duarte1,2*. 6 Affiliations: 7 1Computational Bioscience Research Center (CBRC), King Abdullah University of Science and 8 Technology, Thuwal 23955, Saudi Arabia 9 2Red Sea Research Centre (RSRC) and Computational Bioscience Research Center (CBRC), 10 King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia 11 3Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar-CSIC, Pg. 12 Marítim de la Barceloneta 37-49, 08003, Barcelona, Spain 13 4Red Sea Research Centre (RSRC) and Computational Bioscience Research Center (CBRC), 14 King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia 15 5King Abdullah University of Science and Technology (KAUST), Computational Bioscience 16 Research Center (CBRC), Biological and Environmental Science and Engineering, Thuwal, 17 23955-6900, Saudi Arabia 18 6Centre de Biochimie Structurale, CNRS, INSERM, Université de Montpellier, 34090 19 Montpellier, France 20 . 21 *Correspondence to: [email protected], [email protected] 22 23 Short title: Plastic-degrading Enzymes Prevalent in the Global Ocean 24 25 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.07.285692; this version posted September 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 26 Abstract: 27 28 Estimates of marine plastic stocks, a major threat to marine life (1), are far lower than expected 29 from exponentially-increasing litter inputs, suggesting important loss factors (2, 3). These may 30 involve microbial degradation, as the plastic-degrading polyethylene terephthalate enzyme 31 (PETase) has been reported in marine microbial communities (4). An assessment of 416 32 metagenomes of planktonic communities across the global ocean identifies 68 oceanic PETase 33 variants (oPETase) that evolved from ancestral enzymes degrading polycyclic aromatic 34 hydrocarbons. Twenty oPETases show predicted efficiencies comparable to those of laboratory- 35 optimized PETases, suggesting strong selective pressures directing the evolution of these 36 enzymes. We found oPETases in 90.1% of samples across all oceans and depths, particularly 37 abundant at 1,000 m depth, with a strong dominance of Pseudomonadales containing putative 38 highly-efficient oPETase variants in the dark ocean. Enzymatic degradation may be removing 39 plastic from the marine environment while providing a carbon source for bathypelagic microbial 40 communities. 41 42 Exponential growth in plastic production along with poor waste management practices have led 43 to over 150 Tg plastic waste delivered to the ocean since 1950 (1), where it harms marine life, 44 from zooplankton to whales (5). Synthetic plastic polymers are derived from oil hydrocarbons 45 and designed to be durable in the environment (6, 7), being largely resistant to microbial 46 degradation. However, a newly-evolved plastic-degrading enzyme, polyethylene terephthalate 47 hydrolase (PETase), was recently discovered in a Japanese waste processing plant (8). This 48 PETase was inferred to have evolved since 1970, when the polyethylene terephthalate polymer 49 was introduced at industrial scale (9). The PETase catalyzes the hydrolysis of polyethylene 50 terephthalate (PET) plastic to monomeric mono-2-hydroxyethyl terephthalate (MHET), which is 51 ultimately broken down into non-hazardous monomers, terephthalate and ethylene glycol, by 52 MHETases. The efficiency of currently known PETases seems, however, to be low, and it has 53 been suggested that in the environment it only achieves a breakdown from micro- to 54 nanoplastics, rather than full degradation (10). 55 56 The discovery, and subsequent biotechnological improvement (11) of this spontaneously-evolved 57 plastic-degrading enzyme offers hope that plastic waste can be degraded in waste facilities, 58 thereby preventing it from entering the ocean (12). However, this still leaves the fate of the > 150 59 Tg plastic waste already delivered to the ocean unresolved. Recent estimates show that 99% of 60 the plastic that entered the oceans cannot be accounted for, since inventories of plastic floating 61 on the surface of the ocean account to ~ 1% of the expected load (2), pointing at the operation of 62 processes responsible for major losses of plastic at sea (3). One such process may involve the 63 microbial degradation of plastics. Microplastics, the numerically dominant marine debris, have 64 developed a characteristic microbial community, referred to as the marine plastisphere (10). If 65 the PETase enzyme evolved in waste treatment plants from ancestral Ideonella sakaiensis (I. 66 sakaiensis) genes (8), and provided the current prevalence of polyethylene terephthalate as debris 67 in the marine environment, it is possible that other PETases may have evolved independently in 68 plastisphere ocean microbes as well. The huge population of prokaryotes in the global ocean, 69 estimated to contribute 1029 cells distributed among 1010 different OTUs (13), provides enough bioRxiv preprint doi: https://doi.org/10.1101/2020.09.07.285692; this version posted September 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 70 population size, genetic diversity and rate of growth for mutations(14) to independently evolve 71 PETase activity, which would enable microorganisms to access a novel, widely available organic 72 substrate. Indeed, a recent assessment of PETase abundance in marine and terrestrial 73 environments, confirmed the presence of the PETase gene in 31 of the 108 environmental marine 74 samples examined, all from surface waters (4). However, a global assessment of the presence, 75 abundance and diversity of PETases in the whole ocean, extending to the deep ocean, the largest 76 microbial habitat in the biosphere (15), is yet to be conducted. 77 78 Results 79 Here we report the prevalence of PETases in the global ocean microbiome and provide a 80 phylogeny suggesting Oceanospirillales to be the parental clade leading to 68 identified variants 81 of marine PETase. We do so on the basis of a thorough exploration of PETase through gene 82 catalogs from two global-ocean expeditions, Tara Oceans (16) and Malaspina (17), delivering 83 416 metagenomes collected from 204 individual ocean locations across the Indian, Pacific and 84 Atlantic Oceans from surface to the deep sea (Fig. 1). 85 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.07.285692; this version posted September 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. PETase abundance (FPKM) ) log10(dat$Abundance + 0.1) h t −11.00-−1 0.510.0000.51.01011.5−1.0-−1 0.50.000.51.011.5−11.00-−1 0.510.0000.511.0011.5−11.00-−1 0.510.0000.511.0011.5−11.00-1−0.510.000 0.511.001 1.5−101.0-1−0.510.000 0.511.001 1.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5 p 10 10 10 e ) 100 D m ( 1 $ t 2 h 102 t a p 3 d 4 e 1( 04 Depth (m) 0 D 5 1 1 2 3 4 5 6 7 8 9 10 11 g 33 [] o 33 [] l 0 1000 2000 3000 4000 0 1000 2000 3000 4000 90˚S 90° N 9090° ˚NN Ocean Data View 0 m A B 60˚S 6060°˚NN 6060° ˚NN 1000 m 30˚S 3030° ˚NN 3030° N˚N 2000 m EQ e d u EQ t EQ 3000 m 0° i 0° t 30˚N a L 4000 m 3030° ˚SS 3030° ˚SS 60˚N w e i V 5000 m w 0 e i 60˚S a 90˚N 60° S 60˚S V t 60° S a a D t a D 32 [] n 33 a n 6000 m e a c e O c 90° S O 180˚W 90˚W0˚ 90˚E 180˚E 9090° ˚SS 180° W 90° W 0° 90° E 180° E 0 1000 2000 3000 4000 33 [] ) ) h h 60˚N t t 1 p p 1 2 3 4 5 6 71 8 2 9 3 10 4 11 5 6 7 8 9 10 11 ) s e e 100 0 0 e m 0.8 D l e D D ( s 1 1 p $ $ h 2 t t 30˚N a t 10 2 2 m a a 0.6 T p 3 3 a d d s E e 4 ( ( 4 140 D P n 0 0 5 5 0.4 o 1 1 i h t t EQ-1 0 1 -1 0 1 i g -1 0 1 -1 0 1 -1 0 1 g −1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−−11.00 −0.510.00.00 0.50.511.001.01.5−11.51.00−1.0−0.5−0.510.00 0.00.50.511.00 1.01.5−11.001.5−−0.51.010.0−00.50.50.011.000.51.5−11.01.00 −1.50.5−11.00.00−0.510.01.00 1.50.5−101.01.0−0.51.510.0−01.00.5−0.511.000.01.50.51.01.5c −1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5−1.0−0.50.00.51.01.5 w o o 0.2 a l l log10(dat$Abundance + 0.1) log10(dat$Abr undance + 0.1) F PETase abundance (FPKM) 0 30˚S 8 w e 1 2 i M E V 6 K 60˚S a 3 4 t P 0 a 0 F D 5 6 n E 4 a e 7 8 S c ± O C 180˚W 90˚W0˚ 90˚E n 180˚2E a e 20200000 M 0 8 ) m ( AM3 M F h t 6 K BM4 p P e 40400000 F CM5 D E 4 S ± n w 2 a e i e V a M 60600000 t a 0 D n 0 0 0 0 0 0 a 0 0 0 Seafloor depth (m) e 0 0 0 c 1 5 0 0 0 0 O - - 1 2 3 3 - - - 180˚W 90˚W0˚ 90˚E 180˚E 0 0 > 0° 0 0 0 0 180° W 90° W 90° E 180° E 1 0 0 0 5 0 0 1 2 33 [] Longitude Depth (m) 86 87 88 Fig.