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Draft genome sequence of oligosaccharide-synthesizing Leuconostoc citreum SG255 isolated from radish kimchi in Korea

Sungyoon Kim1, Sulhee Lee2, and Young-Seo Park1* 1Department of Food Science and Biotechnology, Gachon University, Seongnam 13120, Republic of Korea 2Research Group of Healthcare, Korea Food Research Institute, Wanju 55365, Republic of Korea

올리고당을 합성하는 Leuconostoc citreum SG255 균주의 유전체 염기 서열

김성윤1 ･ 이설희2 ･ 박영서1* 1가천대학교 식품생물공학과, 2한국식품연구원 헬스케어연구단

(Received December 28, 2020; Revised March 14, 2021; Accepted March 15, 2021)

Leuconostoc citreum SG255, which was isolated from radish was reported that alternansucrase from L. mesenteroides NRRL kimchi, synthesized oligosaccharides using sucrose as a donor B-21297 catalyzes the synthesis of oligosaccharides using and maltose or lactose as an acceptor. In this study, the draft raffinose as an acceptor (Côté et al., 2009). Dextransucrases genome sequence of L. citreum SG255 was determined using from L. mesenteroides B-512FM catalyze the synthesis of Illumina Miseq platform. The draft genome consisted of 28 maltodextrins using maltose as an acceptor (Fu and Robyt, contigs and the genome size was 1,869,057 bp. The G + C content was 37.78% with 1,829 coding sequences, 3 rRNA 1990). In preliminary experiments, L. citreum SG255 isolated genes, and 51 tRNA genes. Leuconostoc citreum SG255 has from radish kimchi catalyzed the synthesis of oligosaccharides alternansucrase gene, which encodes for the enzyme synthesizing using sucrose as a donor and maltose or lactose as an acceptor oligosaccharides. (Park et al., 2019). Consequently, this strain was assumed to Keywords: Leuconostoc citreum, alternansucrase, draft genome have glycosyltransferase. sequence, Illumina Miseq, lactic acid bacteria The strain was incubated in MRS medium at 37°C for 16 h. Genomic DNA isolation was carried out using AccuPrep genomic DNA Extraction kit (Bioneer). Whole-genome Leuconostoc citreum was first reported in 1989 as L. sequencing of L. citreum SG255 was performed by Sanigen amelibiosum and L. citreum and was classified as same species, Co., Ltd. using the Illumina Miseq platform. Library construction retaining the nomenclature L. citreum in 1992 (Takahashi et al., was performed using Illumina TruSeq DNA library prep kit 1992). It is a Gram-positive, catalase-negative, non-motile, and and sequencing was performed using Illumina Miseq. Illumina facultative anaeorobe. It is known to synthesize exopolysac- Miseq platform provided 482.8× coverage of the genome, charides, such as dextransucrase and alternansucrase, through which was assembled de novo into 28 contigs. Quality control glycosyltransferase. These enzymes hydrolyze sucrose and was performed using FastQC v0.11.8 and trimming process link one glucose molecule to another through different types of was performed using Trimmomatic v0.38. Contamination was glycosidic linkages (van Hijum et al., 2006). Additionally, it is checked using BWA v0.7.17 for alignment followed by known that these enzymes can use other acceptor molecules. It Samtools v1.9. De novo assembly was performed using Spades v3.13.0. Genome annotation was conducted through NCBI *For correspondence. E-mail: [email protected]; Tel.: +82-31-750-5378; Fax: +82-31-750-5389 Prokaryotic Genome Annotation Pipeline (PGAP) and coding 70 ∙ Kim et al.

sequences were detected using GenMarkS+. To calculate alternansucrase [EC 2.4.1.140]. It was annotated as SH3-like genomic distance with other L. citreum strains, orthologous domain-containing protein in NCBI (NCBI GenBank locus tag average nucleotide identity (OrthoANI) was compared with 25 number: H2O16_RS08760). The SH3-like motif is a structural strains through OrthoANI tool. The formula, distance = 1 – distinction of alternansucrase (Wangpaiboon et al., 2019), and (OrthoANI/100), was used to calculate the the orthoANI values synteny analysis with other L. citreum strains showed that the (Lee et al., 2016). Each of the protein coding genes was gene encoding this enzyme from L. citreum SG255 was classified according to its function through the assignment of a identified as an alternansucrase gene (Oberto, 2013). The alter- Clusters of Orthologous Group (COG) code and was sorted nansucrase from L. citreum SG255 was expected to catalyze into COG categories (Tatusov et al., 1997). Carbohydrate the synthesis of various types of oligosaccharides using active enzymes were analyzed through dbCAN2 meta server different acceptor and receptor sugar molecules. (Jhang et al., 2018). Leuconostoc citreum SG255 was deposited in Korean Culture The draft genome of L. citreum SG255 consisted of 28 Center of Microorganisms under the deposit number KCCM contigs and 1,869,057 bp with G + C content of 38.8%. The 43402. number of protein coding sequences (CDSs) was 1,829 with 3 ribosomal RNA genes (5S, 16S, and 23S) and 51 transfer RNA Nucleotide sequence accession number genes (Table 1). Leuconostoc citreum SG255 was most similar The draft genome sequence of Leuconostoc citreum SG255 to L. citreum NRRL B-742 at OrthoANI value 99.49%. has been deposited in NCBI GenBank under the accession Classification into COG categories revealed that 421 genes number JACGMK010000001-JACGMK010000028. belonged to information storage and processing, 324 genes belonged to cellular processing, and 606 genes belonged to metabolism. Additionally, 28 genes belonged to code X 적 요 (Mobilome: prophages, transposons). However, 444 genes were either not assigned or poorly identified. Compared with 깍두기에서 분리된 Leuconostoc citreum SG255는 sucrose other strains with high OrthoANI value, the maximum 를 공여체로 하고 maltose 혹은 lactose를 수용체로 하여 올리 difference was only 14 (code L) except that of not assigned, 고당을 생성하였다. Leuconostoc citreum SG255의 유전체는 which was 74. When three search tools for carbohydrate active 1,869,057 bp의 28개의 contig로 구성된 염색체로 조합되었으 enzymes (CAZymes) annotation in dbCAN2 meta server, 며 G + C의 비율은 37.78%로 나타났다. 1,829개의 코딩 유전 namely, HMMER, DIAMOND, and Hotpep, 36 carbohydrate 자, 3개의 rRNA 유전자와 51개의 tRNA 유전자가 염색체 DNA active enzymes were commonly identified by all search tools. 에서 확인되었다. Leuconostoc citreum SG255는 올리고당을 Among 36 enzymes, 5 enzymes belonged to glycoside hydrolase 합성할 수 있는 glycosyltransferase 중 alternansucrase를 가지 family (GH) 70. 고 있는 것으로 확인되었다. Among five GH70 enzymes, an enzyme which was encoded by a gene consisted of 6,174 bp, was supposed to be an Acknowledgments Table 1. General genomic features of L. citreum SG255

Features Chromosome This work was supported by the National Research Foundation Genome size (bp) 1,869,057 of Korea (NRF) grant funded by the Korea government (MSIT) Contigs 28 (No. 2019R1A2C1004950). GC content (%) 38.8 rRNA genes 3 tRNA genes 51 References Protein coding genes 1,829 Genes assigned to COGs 1,614 Côté GL, Dunlap CA, and Vermillion KE. 2009. Glucosylation of

미생물학회지 제57권 제1호 Genome sequence of Leuconostoc citreum SG255 ∙ 71

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Korean Journal of Microbiology, Vol. 57, No. 1