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Noda Institute for Scientific Research GRANT2012 Young Investigator Research Grant

Metabolic and enzymatic analyses of a poly(ethylene terephtha­late) assimilation bacteriumShosuke YoshidaDepartment of Biosciences and Informatics, Keio University

Research aims

nitorophenyl (pNP)-butyrate.

Poly(ethylene terephthalate) (PET) is one of the mostcommonly used plastic materials because it is chemicallyinert and non-biodegradable. The Oda group (Kyoto Institute of Technology) screened for PET-degrading bacteriafrom the natural environment and successfully isolated aPET-assimilating bacterium Ideonella sp. No. 201-F6strain. This discovery allowed us to identify novel enzymesfrom the bacterium that degrade PET, an unnatural material. The aim of this study was to use genomic informationof the No. 201-F6 strain, which we ascertained with nextgeneration sequencing, to understand the unique enzymaticscheme that allows PET to be used as a sole carbon source.

Results

Methods(1) Whole genome analysis of Ideonella sp. No. 201-F6strainFifty-one million reads with an average length of 74.9 ntwere obtained by sequencing. Denovo assembly analysisconstructed 2892 contigs, ranging from 93,997 nt to 187 nt,from the single reads. The RAST server annotated 3960 putative genes from the contigs.
(2) Discovery of PET-hydrolyzing enzymeThe genes of 42 putative esterases and 8 putative lipaseswere found in the annotated No. 201-F6 genes. One of theputative lipases, ORF2645 protein, showed 51% identitywith a cutinase from Thermobifida fusca and 82% identitywith PBSA (Poly{(tetramethylene succinate)-co-adipate})depolymerase from Acidovorax delafieldii.4)(1) Whole genome analysis of Ideonella sp. No. 201-F6strainAfter culturing No. 201-F6 strain cells in a nutrient-richmedium, genomic DNA was extracted, fragmented with anAcoustic Solubilizer (Covaris Inc.), and converted to a library for next-generation sequencing by using a Paired-EndSample Preparation Kit (Illumina Inc.). Sequencing with aread length of 80 nt was performed using the Genome Analyzer IIx (Illumina Inc.). The reads were assembled intocontigs by using CLC Genomics Workbench (CLC bioInc.). Genes were annotated on the Rapid Annotation usingSubsystems Technology (RAST) server.

Conclusion(2) Discovery of PET-hydrolyzing enzymeThe annotated putative genes of the No. 201-F6 strainwere searched in silico for hydrolases such as esterases andlipases. Similarly, the sequence data were searched forhomologs of cutinases, which have been reported as PEThydrolyzing enzymes.1,2,3)

In this study, we successfully identified a novel PET hydrolase, ORF2645 protein, from a PET-assimilating bacterium Ideonella sp. No. 201-F6 strain. Detailed biochemicalcharacterization of this protein is ongoing. By using RNASeq analysis, we are attempting to identify additional No.
201-F6 proteins that may be involved in PET degradation.

(3) Functional analysis of the ORF2645 proteinThe codons of ORF2645 gene were optimized forexpression in Escherichia coli, and the whole gene wassynthesized. The optimized gene was ligated into thepET-21b(+) vector. Proteins were expressed in BL21CodonPlus(DE3)-RIPL (Agilent Inc.). Proteins werepurified by nickel-affinity chromatography. The activity ofpurified proteins was evaluated using a PET film,bis(2-hydroxyethyl)terephthalic acid (BHET), and para-Fig. 1. Enzymatic degradation of PET film surface by ORF2645 protien(SEM images)(3) Functional analysis of ORF2645 proteinCrater-shaped pits were formed on the surface of PETfilm by the activity of purified ORF2645 protein (Fig. 1).
This protein also is enzymatically active on BHET, andproduced mono(2-hydroxyethyl) terephthalic acid with aturnover rate of 6.3 s−1. The turnover rate for hydrolysis ofpNP-butyrate by this enzyme was 30 s−1.

52ReferencesSulaiman, S., Yamato, S., Kanaya, E., Kim, J.-J., Koga, Y., Takano,K., and Kanaya, S. (2012) Isolation of a novel cutinase homologwith polyethylene terephthalate-degrading activity from leaf-branchcompost by using a metagenomic approach. Appl. Environ. Microbiol. 78: 1556–62.
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