Phylogenetic and phenotypic features of the isolated LAB strain
The bacterial strain, designated WiKim0068, was isolated from a Korean fermented food, kimchi. In order to identify the phylogenetic similarity of the strain, 16S rRNA gene based phylogenetic analysis of strain WiKim0068 was performed and the closely related strains were found to be L. raffinolactis NBRC 100932T with a similarity of 99.9% (Fig. 1). This result indicated that strain WiKim0068 belongs to L. raffinolactis species. Sugar assimilation/acid formation test conducted using API 50CH revealed positive results for galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, esculin, ferric citrate, salicin, cellobiose, maltose, melibiose, saccharose, trehalose, raffinose, and turanose, whereas H2S production and urease were negative. Enzyme detection performed with an API ZYM kit indicated esterase, leucine arylamidase, and naphthol-AS-BI-phosphohydrolase activities.
General genomic features of L. raffinolactis WiKim0068
The PacBio RS II sequencing system generated 74,558 reads, with an average read length of 8,212 bp. The complete genome of the strain WiKim0068 consisted of a circular 2.22 Mb chromosome and two circular plasmids, with a total size of 2.29 Mb. The chromosome contained 2,060 predicted protein-coding genes (CDSs), 13 rRNA genes (5S rRNA, 5; 16S rRNA, 4; 23S rRNA, 4), 55 tRNAs, and 3 other RNAs. The WiKim0068 genome was found to contain 39.7 mol% G+C content (Table 1), which is within the range of 35.5–46.4% reported for Lactococcus species , and similar to the 39.25 mol% observed in two L. raffinolactis strains, 4877 (CALL00000000) and NBRC 100932T (BCVN00000000). For functional classification, WiKim0068 genome was analyzed using the clusters of orthologous genes (COG) database (http://www.ncbi.nlm.nih.gov/COG/), and 2,000 genes were annotated. The annotated genes were associated with the following categories: general function prediction only (R; 237 genes), carbohydrate transport and metabolism (G; 210 genes), function unknown (S; 190 genes), amino acid transport and metabolism (E; 180 genes), coenzyme transport and metabolism (H; 70 genes), defense mechanisms (V; 63 genes), and secondary metabolites biosynthesis, transport, and catabolism (Q; 15 genes; Supplementary Table S1). In addition, Rapid Annotation using Subsystem Technology analysis revealed genes related to the following categories: stress response (2.62%), cofactors, vitamins, prosthetic groups, pigments (5.30%), and virulence, disease, and defense (3.39%) (Supplementary Fig. S1). Stress response-related genes category included: “osmotic stress” (5 genes), “oxidative stress” (17 genes), “cold shock” (1 gene), “heat shock” (15 genes), “detoxification” (9 genes), and “no subcategory” (1 gene). The category of cofactors, vitamins, prosthetic groups, pigments included those related to “biotin” (15 genes), “NAD and NADP” (14 genes), “riboflavin, FMN, FAD” (8 genes), and “folate and pterines” (33 genes). The category of virulence, disease, and defense included mainly those involved in “bacteriocins, ribosomally synthesized antibacterial peptides” (8 genes), “resistance to antibiotics and toxic compounds” (29 genes), and “invasion and intracellular resistance” (15 genes). Of these, biotin, riboflavin, and folate are related to human health and digestion and cause various symptoms when deficient . Bacteriocins are antimicrobial peptides produced by bacteria  and an alternative to treat antibiotic resistant bacteria. Significantly, bacteriocins production have been regarded as an important feature in the selection of probiotic strains. These genes were associated with the presence of useful probiotic characteristics, which play important roles in the food and pharmaceutical industries [15-17].
Comparative genomic analysis
Analysis of the orthologous average nucleotide identity (orthoANI) values among Lactococcus genome sequences showed that strain WiKim0068 had 68.55–98.73% genome sequence similarities with other species and subspecies. Its genome was most closely related to that of L. raffinolactis NBRC 100932T (98.73%), followed by L. raffinolactis 4877 (87.02%), L. piscium MKFS47 (76.57%), L. lactis subsp. lactis IL 1403 (69.41%), L. fujiensis JCM 16395 (68.12%), and L. garvieae ATCC 49156 (68.55%; Fig. 2). Thus, the comparative whole genome sequence analysis indicated that the strain WiKim0068 belongs to the species L. raffinolactis . Its similarity to its two closest relative strains (L. raffinolactis NBRC 100932T and L. raffinolactis 4877), based on BLAST comparison, is shown in Fig. 3. This figure describes the difference in GC contents and similarities between strain WiKim0068 and two closest relative strains. Furthermore, we searched for clustered regularly interspaced short palindromic repeats (CRISPRs) using the CRISPRFinder platform, but no confirmed CRISPRs were found in the WiKim0068 genome.
Phage and pathogenesis-related genes
PHAST analysis was performed to identify prophage contamination in the genome of WiKim0068. The chromosome contained two intact, one incomplete, and one questionable prophage. The first plasmid (pWiKim0068-1) contained only one incomplete prophage, while the second plasmid (pWiKim0068-2) contained none (Supplementary Fig. S2). Intact prophage regions were located between positions 57,319–90,123 and 1,524,268–1,563,900 bp of the chromosome.
Carbon metabolic pathway
Predicted metabolic pathways in the strain WiKim0068 were associated with diverse phosphotransferase (PTS) systems or permeases that transport various carbohydrates, including d-glucose, d-galactose, d-mannose, trehalose, sucrose, cellobiose, N-acetyl-glucosamine, fructose, maltose, mannitol, galactitol, and lactose. The presence of these transport genes suggested that the strain WiKim0068 uses various carbohydrates for fermentation (Fig. 4). Based on the metabolic pathways, it was confirmed that the strain WiKim0068 had heterofermentative pathways.
Hexoses (glucose, fructose, and mannose) are converted to lactate, ethanol, and carbon dioxide. Additionally, d- and l-Lactate are produced from the reduction of pyruvate by d-lactate dehydrogenase (d-LDH) (EC 22.214.171.124) and l-lactate dehydrogenase (l-LDH) (EC 126.96.36.199), respectively. However, strain WiKim0068 harbors only l-LDH (locus tag: CMV25_RS07125). Notably, as shown in a previous report, l-LDH was identified in Lactococcus lactis, which belongs to the same genus as the strain WiKim0068 . Since d-lactate produced by LAB may induce d-lactate acidosis in some individuals , it is important to develop LAB for the production of dairy products that produce only l-lactate. Therefore, the lack of d-LDH is an advantage that makes the strain WiKim0068 suitable for potential applications in the dairy industry.
Recently, interest in foods as mediators of antibiotics resistance has been increasing. LAB, which are widely used in probiotics and as starter cultures, have the potential to serve as hosts for antibiotic resistance genes, and present the risk of transferring genes from various LAB and bacterial pathogens . The strain WiKim0068 showed susceptibility to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, penicillin, rifampin, tetracycline, and vancomycin (Table 2). Although the strain WiKim0068 was predicted to have vancomycin resistant genes in the genome, antibiotics test confirmed that it was sensitive to vancomycin.
Amino acid and nicotinate and nicotinamide metabolism
The amino acid metabolism-related genes of strain WiKim0068 were annotated using the KEGG database. Among 163 genes involved in amino acid metabolism, strain WiKim0068 harbors the most genes involved in the amino acid metabolism of cysteine, methionine, alanine, aspartate, and glutamate (Fig. 5), suggesting that the strain biosynthesize and utilize various amino acids.
Vitamin B3, one of the eight B-vitamins, is also known as nicotinate or niacin. This endogenous metabolite is an effective antioxidant that prevents oxidative damage . In general, nicotinamide and nicotinate metabolites are frequently reported in Lactobacillus strains [23-25], while Lactococcus members were not known to produce these metabolites until now. Interestingly, in silico analysis of WiKim0068 genome predicted an almost complete complement metabolic pathway from the genes involved in the metabolism of nicotinate and nicotinamide (Fig. 6). Demonstrating this, 0.932 mg L-1 vitamin B3 was extracted from the cultured cells (Supplementary Fig. S3). These results indicated that nicotinate and nicotinamide metabolism occurs in strain WiKim0068. For comparison, studied 15 LAB isolated from kimchi; Leuconostoc spp. produced 0.837-1.05 mg L-1 vitamin B3, and Lactobacillus species, L. sakei, and L. curvatus produced 0.05-0.1 mg L-1.