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Draft genome sequence of a di-(2-ethylhexyl) phthalate-degrading bacterium, Sphingobium fluviale DEHP-117
Korean J. Microbiol. 2023;59(4):353-355
Published online December 31, 2023
© 2023 The Microbiological Society of Korea.

Hye Kyeong Kang, Sang Eun Jeong, Ye-Ji Hwang, Kook-Il Han, Mi-Hwa Lee, and Byung-Gon Ryu*

Environmental Microbiology Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea
Correspondence to: *E-mail: tesiakaist@gmail.com; Tel.: +82-54-530-0872; Fax: +82-54-530-0879
Received October 16, 2023; Revised November 29, 2023; Accepted December 2, 2023.
Abstract
In this study, draft genome of a di-(2-ethylhexyl) phthalate-degrading bacterium, Sphingobium fluviale DEHP-117 isolated from the sedimentary soil around the plastic-producing industry (Gumi, South Korea) in March, was analyzed. The final genome assembly consists of a total length of 3,590,970 bp and a total of 3,518 coding DNA sequences (CDSs), 44 transfer RNA (tRNA), and 3 ribosomal RNA (rRNA) were predicted.
Keywords : Sphingbium fluviale DEHP-117, biological degradation, di-(2-ethylhexyl) phthalate, draft genome
Body

Di-(2-ethylhexyl) phthalate (DEHP) is one of the most widely used phthalic acid esters (PAEs) to improve the flexibility and integrity of plastic and/or polymeric materials, accounting for 50% (w/w) of global phthalate production (Liu and Zhang, 2016). DEHP, which is not bound chemically to the polymer matrix, easily migrate to the environment (Latorre et al., 2012). In addition, the DEHP is also difficult to degrade because of its low water stability (0.285 mg/L at 24°C) and high octanol-water partitioning coefficient (log Kow=7.5) (Latorre et al., 2012). These characteristics and negative effects on health and the environment have made DEHP contamination a public concern (Wang et al., 2015). Several Gram-negative bacterial strains that are capable of degrading DEHP, especially members of the genera Gordonia, Rhodococcus, Sphingobium, and Burkholderia have been isolated from various environmental sources (Change and Zylstra, 1998; Han et al., 2020; Nahurira et al., 2017; Wang et al., 2018, 2019; Zhang et al., 2020; Zhao et al., 2018). We newly isolated Sphingobium fluviale DEHP-117 obtained from the plastic-producing industry and sequenced the genome to understand fundamental insights into the metabolic pathways for DEHP degradation.

A sedimentary soil sample was collected at the site of a plastic-producing industry (Gumi, South Kroea) in March. The enrichment cultures were obtained by incubating the sample at 25°C aerobically with shaking (150 rpm) for 2 weeks in a mineral salt medium (MSM) with 50 ppm DEHP as the sole carbon source. The enrichment-cultured samples were diluted in phosphate-buffered saline (PBS, pH = 7.4). The diluted samples were then spread on R2A agar plates (BD Difco) and incubated at 25°C and 150 rpm for 2 weeks to obtain a single colony, named DEHP-117. The 16S rRNA gene of the colony was PCR amplified using the universal 27f and 1492r primers (Lu et al., 2006), and the resulting sequence (1,419 bp) was compared with 16S rRNA gene sequences of all type strains using the Nucleotide Similarity Search tool.

Genomic DNA of stain DEHP-117 grown in R2A broth at 25°C was extracted using a DNA purification kits (MaxwellTM 16 Purification Kots; Promega). The genomic DNA was sequenced by the PacBio RSII system with an error correction by Illumina Hiseq 4000 sequencing. Briefly, the sequencing library (total 20 kb) was constructed using a PacBio DNA Template Prep Kit 1.0 and analyzed by single-molecule real-time (SMRTbellTM) sequencing at Macrogen, Inc. De novo assembly of the sequencing reads was performed through the hierarchical genome assembly process (HGAP 2, version 2.3.0), and paired-end reads (101 bp) obtained from the Illumina sequencing were mapped on the assembled contig for error corrections (pilon, v1.22). The genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016).

A total subreads (914,522,589 bp; mean subread length, 8,170 bp; N50, 10,749 bp) generated by PacBio Sequel system were used for de novo genome assembly. In order to validate accuracy of this assembly, total of 6,343,010 quality-filtered paired-end Illumina reads (mapped reads, 6,340,842; coverage, 99.87%; mapping depth, 253.8-fold) were mapped. The draft genome of strain DEHP-117 consisted of 5-contigs with a total length of 3,590,970 bp (max length, 1,414,423 bp; min length, 72,932 bp; mean length, 718,194; N50, 1,116,002 bp), and the GC content of the genomic DNA was 62.7%. The estimated genome length was 3,597,203 bp with 33.78 K-mer coverage and 0.03 heterozygosity. A total of 3,518 coding DNA sequences (CDSs), 44 transfer RNA (tRNA), and 3 ribosomal RNA (rRNA) were discovered. Functional annotation and KEGG pathway mapping were performed using BlastKOALA v3.0 (Kanehisa et al., 2016). Of the predicted genes, 1,658 genes were categorized into 243 functional pathways; 9 and 56 genes that may be involved, respectively, in aromatic compound degradation, xenobiotic degradation, and metabolism were detected. Among them, the gens of phthalate 4,5-dioxyganase (gene locus_tag: OOT33_16340) and 4,5-dihydroxyphthalate decarboxylase (gene locus_tag:OOT33_16350) involved in the phthalate degradation are observed in strain DEHP-117. The genome information for Sphingobium fluviale DEHP-117 will greatly contribute to understanding the DEHP degrading pathway.

Strain and nucleotide sequence accession numbers

The genome sequence and raw sequencing reads for strain DEHP-117 were deposited under GenBank accession number JAPFBJ000000000, BioProject accession number PRJNA897091, and BioSample accession number SAMN31577775.

적 요

본 연구에서는 플라스틱 생산 공장 인근 하천 퇴적토에서 분리한 프탈레이트[di-(2-ethylhexyl) phthalate, DEHP] 분해 미생물인 Sphingobium fluviale DEHP-117의 유전체를 분석하였다. 유전체의 크기는 3,590,970 bp이며 3,518개의 암호화 DNA 염기서열, 44개의 운반 RNA, 3개의 리보솜 RNA 염기서열 포함한다.

Acknowledgments

This work was carried out with support from a Nakdonggang National Institute of Biological Resources grant (project number NNIBR202303102) funded by the Ministry of Environment, South Korea.

Conflict of Interest

The authors declare that they have no conflict of interest.

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