The genus Sphingomonas was classified into the family Sphingomonadaceae, the order Sphingomonadales and the class Alphaproteobacteria. Members of the genus Sphingomonas are Gram-negative, strictly aerobic and rod-shaped bacteria (Yabuuchi et al., 1990). The genus Sphingomonas is widely distributed in soil, phyllosphere and rhizosphere of plant, air, water, desert, and more (Shi et al., 2001; An et al., 2011; Kim et al., 2014; Liu et al., 2015; Zhu et al., 2015; Lee et al., 2016; Cha et al., 2019). Some of the members within the order Sphingomonadales produce carotenoid pigments (Siddaramappa et al., 2018).

Strain NP2-R2 was isolated from morning glory flower in Jangseong, Republic of Korea. The flower sample was serially diluted with 0.85% (w/v) NaCl and appropriate 10-fold dilutions were plated on R2A agar (Difco). The plates were incubated at 28°C for 5 days. Strain NP2-R2 was one of the isolates that appeared on the R2A medium which could be distinguished in terms of yellow color colony. 16S rRNA sequence (1,475 bp) was extracted from the whole-genome sequences. The sequence similarity values were calculated between NP2-R2 and the closely related species using EzBioCloud blast (http://www. ezbiocloud.net/identify) (Yoon et al., 2017). The phylogenetic trees based on the 16S rRNA gene sequence alignment were reconstructed by the neighbor-joining (Saitou and Nei, 1987) algorithms using the software package MEGA (version 7) (Kumar et al., 2016). The tree topology was evaluated by bootstrapping with 1,500 replicates. NP2-R2 showed the highest sequence similarities in 16S rRNA sequences with Sphingomonas sanguinis NBRC 13937^T (99.86%), S. pseudosanguinis G1-2^T (99.35%), S. yabuuchiae GTC 868^T (99.08%), and S. parapaucimobilis NBRC 15100^T (99.01%). Phylogenetic analysis using the neighbor-joining method based on the 16S rRNA sequences showed that strain NP2-R2 was clustered with S. sanguinis NBRC 13937^T, S. pseudosanguinis G1-2^T, S. yabuuchiae GTC 868^T, S. parapaucimobilis NBRC 15100^T, and S. roseiflava NMK341^T (Fig. 1).

Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of strains NP2-R2. Bootstrap percentages (based on 1,500 replicates) > 70% are shown at branching points. Bar, 0.02 accumulated changes per nucleotide.

To identify genes related to carotenoid production, we conducted whole-genome sequencing. The genome of NP2-R2 was sequenced using PacBio RS II sequencer (Pacific Biosciences) and Illumina HiSeq platform data (Illumina) at Macrogen Inc. The genome annotation was conducted using the NCBI prokaryotic genome annotation pipeline (Tatusova et al., 2016). As a result, two contigs were assembled as one circular chromosome and one plasmid. The genome size of NP2-R2 is 4,372,860 bp. 3,920 protein-coding genes (CDS) were annotated, and 65 and 12 genes were annotated as tRNA and rRNA, respectively (Table 1). The DNA G + C content was 66.06% in two contigs. One circularized contig with 4,275,167 bp is the chromosome, and the other small contig with 97,693 bp may be a plasmid.

Table 1

General genomic features of the strain NP2-R2

Genome features	Contig 1	Contig 2	Total
Genome size (bp)	4,275,167	97,693	4,372,860
G + C content (%)	66.11	64.08	66.06
Protein-coding genes	3,831	89	3,920
Transfer-RNA genes	65	-	65
Ribosomal-RNA genes	12	-	12

Based on the functional categories specified using EggNOG database, 3734 genes were annotated with transport and metabolism of amino acid (216), inorganic ion (201), lipid (97), carbohydrate (197), coenzyme (102), and nucleotide (56), biosynthesis, transport and catabolism of secondary metabolites (43), energy production and conversion (174), etc. The biosynthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) which are carotenoid precursors occurs via two different pathways, methylerythritol 4-phosphate (MEP) pathway and mevalonate (MVA) pathway (Cheng et al., 2020; Ram et al., 2020). In the case of the strain NP2-R2, the genes involved in the MEP pathway were found, such as 1-deoxy-D-xylulose 5-phosphate synthase (dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr). The MEP pathway is typically found in most bacteria and plant plastids while the MVA pathway is mostly present in plants and archaea (Cheng et al., 2020). The genome contains carotenoid biosynthesis genes coding for proteins, 15-cis-phytoene synthase (crtB, NP2-R2_1_02095), phytoene desaturase (crtI, NP2-R2_1_02087) and lycopene-beta-cyclase (crtY, NP2-R2_1_02086) (Table 2). This complete genome sequence data can be useful for comparative genome research, metabolic engineering of carotenoid biosynthesis and exploring the metabolism of Sphingomonas species.

Table 2

Carotenoid biosynthesis genes of the strain NP2-R2

Gene	EC number	Product	Length (bp)	Length (a.a.)
crtB	2.5.1.32	15-cis-phytoene synthase	951	316
crtI	1.3.99.31	Phytoene desaturase (lycopene-forming)	1,476	491
crtY	5.5.1.19	Lycopene beta-cyclase	1,173	390

Nucleotide sequence accession numbers

The complete genome sequence of S. sanguinis NP2-R2 has been deposited at NCBI under the GenBank accession no. CP079203 and CP079204. The strain has been deposited in the Korean Agricultural Culture Collection (KACC) under the accession number KACC 81176BP.

카로티노이드 생산 균주를 탐색하기 위하여 나팔꽃에서 분리한 노란색 색소를 생산하는 NP2-R2 균주를 선발하였다. 16S rRNA 염기서열 분석 결과, NP2-R2 균주는 Sphingomonas sanguinis에 가장 높은 유사도(99.86%)를 나타내었다. NP2-R2 균주의 총 염기서열은 4,372,860 bp이고 G + C content가 66.06%이었다. 유전체에서 단백질 유전자 3,920개, tRNA 유전자 65개, rRNA 유전자 12개를 확인하였다. 또한 카로티노이드 생합성과 관련된 유전자 crtB, crtI, crtY를 포함하고 있는 것을 확인하였다. 본 유전체 염기서열은 비교유전체 연구와 카로티노이드 생합성의 대사공학을 위한 유용한 정보를 제공할 것이다.

This study was carried out with the support (Project No. PJ01550601) of National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea.

The authors have no conflict of interest to report.