Korean Journal of Microbiology (2020) Vol. 56, No. 1, pp. 80-82 pISSN 0440-2413 DOI https://doi.org/10.7845/kjm.2020.9129 eISSN 2383-9902 Copyright ⓒ 2020, The Microbiological Society of Korea

Complete genome sequence of ginsenosidivorax KHI-28T, a ginsenoside-converting bacterium, isolated from sediment

1 2 2 1 1,3 Young Woo Lee , Byoung Hee Lee , Ki-Eun Lee , Soon Youl Lee , and Wan-Taek Im * 1Department of Biotechnology, Hankyong National University, Anseong 17579, Republic of Korea 2Microorganism Resources Division, National Institute of Biological Resources, Incheon 22689, Republic of Korea 3AceEMzyme Co., Ltd., Academic Industry Cooperation, Anseong 17579, Republic of Korea

퇴적층으로부터 분리한 진세노사이드 전환 능력이 있는 T Mucilaginibacter ginsenosidivorax KHI-28 의 유전체 서열 분석

이영우1 ･ 이병희2 ･ 이기은2 ･ 이순열1 ･ 임완택1,3* 1국립한경대학교 농업생명과학대학 생명공학과, 2국립생물자원관, 3(주)에이스엠자임

(Received October 28, 2019; Revised February 14, 2020; Accepted February 14, 2020)

Mucilaginibacter ginsenosidivorax KHI-28T (KACC 14955T = bacteriaceae, order , class Sphingobacteria, T LMG 25804 ) is Gram-stain-negative, strictly aerobic, non- and phylum (Ludwig et al., 2011). Currently, the motile, non-spore-forming, and rod-shaped. M. ginsenosidivorax genus is comprised of more than 51 [https://www. was isolated from sediment in a river in Gapcheon, Daejeon, bacterio.net], including M. dorajii (Kim et al., 2010), M. South Korea. Strain KHI-28T had β-glucosidase activity, polysacchareus (Han et al., 2012), M. lappiensis (Männistö et responsible for transforming ginsenosides Rb1 and Re into Rg2 and C-K, respectively. The complete genome of M. ginseno- al., 2010), M. composti (Cui et al., 2011), and M. rigui (Baik et sidivorax KHI-28T revealed a single circular chromosome al., 2010). Strains of this genus are Gram-stain-negative, non- comprising 7,811,413 bp, with a G + C content of 43.1%. Several motile, non-spore-forming, and rod-shaped. The G + C content glycoside hydrolase-encoding genes were found, which might of the genomic DNA of this genus ranges from 39.1 to 49.8%. contribute in converting major ginsenosides to minor ginseno- During the screening of ginsenoside-converting strains, a sides and they were expected strong pharmacological effects. Gram-stain-negative, rod-shaped, pale-pink pigmented bacterium In addition, genes encoding nucleotide excision repair enzymes Mucilaginibacter ginsenosidivorax KHI-28T, which could and virulence factors were found. convert major ginsenosides into minor ginsenosides, was Keywords: Mucilaginibacter ginsenosidivorax, complete genome, isolated from the sediment in a river in Gapcheon, Daejeon, glycoside hydrolase, PacBio RS II, sediment South Korea (Kim et al., 2013). The ginsenoside conversion ability test of the M. ginsenosidivorax KHI-28T was determined using the method described by Kim et al. (2013). Thus, The genus Mucilaginibacter was first described by Pankratov complete genome sequencing of strain KHI-28T was carried et al. (2007) and subsequently amended by Urai et al. (2008) out. This strain is available from the host institute and from two and Baik et al. (2010); this genus belongs to the family Sphingo- culture collections (= KACC 14955T = LMG 25804T). T *For correspondence. E-mail: [email protected]; Genomic DNA of M. ginsenosidivorax KHI-28 was extracted Tel.: +82-31-670-5335; Fax: +82-31-670-5339 T Complete genome sequence of M. ginsenosidivorax KHI-28 ∙ 81

using a MagAttract HMW DNA kit (Qiagen) and was purified pathway enzymes and excinuclease UvrABC complex, which using the chloroform wash method (shared protocol; Pacific notice and repair the structural changes caused by ionizing Biosciences). Genome sequencing was performed using the radiation (Truglio et al., 2006). Additionally, various glycometa- Pacific Biosciences RSII sequencing platform with a 20 kb bolism-related genes were identified, including those encoding SMRTbellTM template library at DNA Link Inc. Sequences α and β galactosidases, α-amylases, β-mannosidase, gluco- were assembled using the HGAP3 protocol (Pacific Biosciences) sylceramidase, β-N-acetylhexosaminidase, endo-1,4-β-xylanase, and the sequencing depth was 128.87. The genome sequence glycosyltransferase, malto-oligosyltrehalose synthase, pimeloyl- was annotated using the NCBI Prokaryotic Genome Automatic ACP methyl ester carboxylesterase or carboxylesterase type B, Annotation Pipeline (http://www.ncbi.nlm.nih.gov/books/NBK and S-formylglutathione hydrolase FrmB. 174280/). rRNAs and tRNAs were predicted using rRNAmmer The availability of the complete genome sequence of M. and tRNAscan-SE, respectively (Rhoads and Au, 2015). ginsenosidivorax KHI-28T will allow further functional and The complete genome of M. ginsenosidivorax KHI-28T comparative genome analyses to better understand the genomic consists of one circular chromosome of 7,811,413 bp with a traits involved in the conversion of plant secondary meta- G + C content of 43.1%. Of the 6,323 predicted genes found in bolites, as described by Siddiqi et al. (2017). the genome, 6,249 protein-coding genes, 9 rRNA genes (5S, 16S, and 23S), 55 tRNA genes, and 74 pseudogenes were Nucleotide sequence accession number identified. The majority of the protein-coding genes (98.82%) The complete genome sequence of Mucilaginibacter ginseno- were assigned a putative function, while the remaining sidivorax KHI-28T has been deposited in DDBJ/EMBL/NCBI predicted genes were annotated as hypothetical or conserved GenBank under accession number CP042437. hypothetical proteins. The genome statistics are described in Table 1. Analysis of the complete genome of M. ginsenosidivorax 적 요 KHI-28T showed that it encoded many glycosides and hydro- lases, including 17 β-glucosidases, 17 α-glucosidases, 13 α-L- 대한민국 대전 갑천의 퇴적층으로부터 분리한 Mucilagini- rhamnosidases, 10 α-L-arabinofuranosidases, and 39 β-xylosidases, bacter ginsenosidivorax KHI-28T 균주의 유전체 서열을 분석 which may be responsible for its ability to convert ginseng 하였다. 균주 KHI-28T는 진세노사이드 Rb1과 Re를 Rg2와 saponins (Kim et al., 2013). In addition, the genome annotation C-K로 각각 변환하는 역할을 하는 베타-글루코시데이즈 활성 also revealed other genes, for example, those encoding virulence 을 가지고 있었다. 균주 KHI-28T의 전체 유전체를 분석한 결 factors such as hemolysinA, zinc metalloprotease, ATP-dependent 과 단일 원형 염색체로 구성되어 있으며 그 크기는 7,811,413 metalloprotease FtsH, metalloprotease ybeY, protease HtpX bp였고 G + C 비율이 43.1%이었다. 균주 Gsoil 3017T는 메이 and CRISPR/Cas system-associated endonuclease Cas1 and 2. 져 진세노사이드를 마이너 진세노사이드로 전환하는 즉, 인삼 We also identified genes for the nucleotide excision repair 사포닌의 당 분해에 관여하는 여러 타입의 글라이코사이드 분 해에 관여하는 유전자를 가지고 있었다. 이러한 지놈 분석은 주요 진세노사이드가 우수한 약리학적 활성의 미량 진세노사 T Table 1. General features of M. ginsenosidivorax KHI-28 이드로 전환하는데 관여하는 유전자 특징을 이해하는데 큰 기 Features Value 여가 되었다. 또한 뉴클레오티드 제거 보수 효소와 독성 유발 Genome size (bp) 7,811,413 인자를 인코딩한 유전자가 발견되었다. G + C content (%) 43.1 Total genes 6,390 Pseudo genes 74 Protein-coding genes 6,249 Acknowledgments Number of rRNA genes (5S, 16S, 23S) 9 (3, 3, 3) This work was supported by grants from the National Number of tRNA genes 55

Korean Journal of Microbiology, Vol. 56, No. 1 82 ∙ Lee et al.

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