Lactobacillus Plantarum LRCC5314 Includes a Gene for a Substance that Stimulates the Secretion of Serotonin

Background: As the functions of probiotics within the same species may not be shared, it is important to analyze the genetic characteristics of strains to determine their safety and usefulness before industrial applications. Results: The complete genome of Lactobacillus plantarum LRCC5314 isolated from kimchi was acquired through PacBio sequencing to conrm probiotic functions and genetic characteristics. Phylogenetic and comparative genomic analyses were performed between L. plantarum LRCC5314 and L. plantarum ATCC 14917 T , and the results showed that two strains were highly similar. However, there were some genetic differences. The characteristics of carbon source metabolism were also determined based on the KEGG database and physiological properties. L. plantarum LRCC5314 could metabolize hexoses through homofermentation, which produces only lactic acid. According to gene annotation, L. plantarum LRCC5314 could encode almost complete biosynthetic pathway to produce tryptophan, which can be used as a precursor of serotonin. The serotonin ELISA test using Caco-2 and HT-22 cells treated with a supernatant of the bacterial culture showed that more serotonin was produced by treated cells than by untreated cells. Conclusion: L. plantarum LRCC5314 could provide a source for serotonin production, and L. plantarum LRCC5314 could be used as a functional probiotic for stress regulation.

not be shared among strains, even among those of the same species [14]. For this reason, the genomic characterization of probiotics is important for the study of bene cial functions or mechanisms. Moreover, genome-based analysis is required to investigate the stability and safety of probiotics for industrial applications. Probiotics also exert negative effects through undisclosed genes. Virulence or antibiotic resistance genes can be transferred to other bacteria from probiotics via horizontal gene transfer [15]. Whole-genome analysis of probiotic strains can prevent potential risks by predicting them and thus increasing utilization e ciency.
L. plantarum LRCC5314 was isolated from kimchi as a probiotic candidate. In this study, we investigated the whole genome of L. plantarum LRCC5314 to characterize the strain via comparative, genome content, and metabolic pathway analyses. In addition, we identi ed the functional factors of L. plantarum LRCC5314 through gene annotation and provided the molecular biological evidence for its practical use as a probiotic strain.

Materials And Methods
Phylogenetic and physiological characteristics of L. plantarum LRCC5314 The genomic DNA of L. plantarum LRCC5314 was extracted using the genomic G-spin DNA extraction kit for bacteria (Intron, Seongnam, Korea). The sequence of 16s rRNA was ampli ed by polymerase chain reaction (PCR) with the universal primers 8F and 1525R [33] and puri ed using the Accuprep PCR puri cation kit (Bioneer, Seoul, Korea). The puri ed gene was sequenced directly, and the sequence similarity levels among closely related strains were calculated with EzBioCloud (https://www.ezbiocloud.net/identify) [34]. The phylogenetic distance between L. plantarum LRCC5314 and related strains of the genus Lactobacillus was calculated using CLUSTAL-X 2.1 software [35]. The phylogenetic trees were constructed using the neighbor-joining, maximum-parsimony, and maxi-mumlikelihood methods with MEGA 7 software [35][36][37][38]. To determine the physiological characteristics of L. plantarum LRCC5314, API 50CH, API 20E, and API ZYM (Biomerieux, Marcy-l'Étoile, France) tests were performed according to the manufacturer's instructions.

Whole-genome sequencing and annotation
The whole genome of L. plantarum LRCC5314 was sequenced using the PacBio RS platform with the 20kb SMRTbell TPK library kit. The sequences were assembled de novo with RS HGAP Assembly (version 3.0) in PacBio SMRT Analysis software (version 2.3). Genomic annotation was performed using the RAST server (version 2.0) [39], and the National Center for Biotechnology Information (NCBI)'s Prokaryotic Genomes Annotation Pipeline 4.1 [40] was used to combine the results. WebMGA on-line tools (version 2.2.15; http://weizhong-lab.ucsd.edu/webMGA/) [41] were used to group protein functions with the COG database. Antimicrobial resistance genes were identi ed using the program ResFinder (version 4.1; https://cge.cbs.dtu.dk/services/ResFinder/) [42]. The carbon source metabolic pathway was constructed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [43]. The sequences generated in this study were deposited in the DDBJ/EMBL/GenBank International Nucleotide Sequence Database under the BioProject ID PRJNA716786.

Metabolic pathway analysis of carbon sources
Based on KEGG pathway and protein BLAST [44] analyses, the carbon metabolic pathways of L. plantarum LRCC5314 were constructed. The pyruvate, pentose phosphate, sucrose, lactose, and galactose metabolism pathways of the strain were mapped according to the KEGG pathway. To construct a pathway, only mapped genes were used. Protein BLAST was used to recon rm the functions of the characteristic genes.
Comparative genomic analysis of L. plantarum LRCC5314 The whole-genome sequence of L. plantarum ATCC 14917 T (GCF_000143745) was obtained from NCBI GenBank for comparative genomic analysis with L. plantarum LRCC5314. The digital DNA-DNA hybridization (dDDH) value with L. plantarum ATCC 14917T was calculated using the Genome-to-Genome Distance Calculator (GGDC; version 2.1; http://ggdc.dsmz.de/ggdc.php) [45]. OrthoANI values between L. plantarum LRCC5314 and related strains in the genus Lactobacillus were calculated using Orthologous Average Nucleotide Identity Tool (OAT) software [46]. A circular map to compare the genome sequence between L. plantarum LRCC5314 and L. plantarum ATCC 14917 T was created using Blast Ring Image Generator (BRIG) software [47]. CRISPRFinder Basel, Switzerland) supplemented with 10% fetal bovine serum and 1% penicillin. The cell lines were incubated at 37°C with 5% CO 2 .

Measurement of serotonin release
The cultured Caco-2 and HT-22 cells were seeded in 24-well plates (1 × 105 cells/well) and incubated until sub-con uent. Then, the cells were treated with the bacterial super-natant and incubated for 1 h at 37°C.
The supernatants of the treated wells were collected after incubation and centrifuged at 1,000 × g for 3 min. The serotonin content of the cell supernatants was quanti ed with the Serotonin High Sensitive ELISA kit (IBL, Hamburg, Germany) according to the manufacturer's instructions.

Statistical analysis
Multiple comparisons were performed by t-test and GraphPad Prism (v.6.0.1) (Graph Pad Software Inc., La Jolla, CA, USA). All data are presented as the mean ± standard error of the mean (SEM), and p values < 0.05 were considered signi cant.

Results
Phylogenetic and phenotypic features of the isolated LAB strain The 16S rRNA gene of L. plantarum LRCC5314 was obtained and compared with available reference sequences from the GenBank database and EzBioCloud (http://www.ezbiocloud.net/eztaxon). Phylogenetic analysis based on the 16S rRNA gene showed that L. plantarum LRCC5314 was clustered with the L. plantarum species (Fig. 1). L. plantarum LRCC5314 was the most closely related to L.

Comparative genomic analysis
OrthoANI analysis between L. plantarum LRCC5314 and closely related Lactobacillus strains showed similarities in the range of 75.14-99.65%. L. plantarum LRCC5314 was the most closely related to L.  Fig. 2A). The dDDH value with L. plantarum ATCC 14917 T was 97%. OrthoANI and dDDH values showed that the LRCC5314 strain belonged to the species L. plantarum. Furthermore, based on BLAST comparison, the whole-genome sequences of L. plantarum LRCC5314 and L. plantarum ATCC 14917 T showed high similarity (Fig. 2B). However, a circular comparison of the genomes of L. plantarum LRCC5314 and L. plantarum ATCC 14917 T showed differences in the GC content and genomic similarities between the two strains. Furthermore, L. plantarum LRCC5314 contained a CRISPR in its genome. According to CRISPRFinder, L. plantarum LRCC5314 had a CRISPR without Cas genes between 1,536,174 bp and 1,536,259 bp, whereas no con rmed CRISPRs were detected in L. plantarum ATCC 14917 T . These results demonstrated that the LRCC5314 strain belonged to the same species (L. plantarum) as the ATCC 14917 T strain but had genetic differences.

Carbon source metabolic pathway
The metabolic pathways of carbon sources related to the genes of L. plantarum LRCC5314 were combined and mapped in one synthesis pathway (Fig. 3). For carbo-hydrate metabolism, 228 genes of L. plantarum LRCC5314 were mapped to the KEGG pathway, and only the metabolic pathways of carbon sources with a positive result in the API 50CH test were combined. L. plantarum LRCC5314 could utilize various carbon sources, including glucose, sucrose, maltose, and lactose. According to the carbon source metabolic pathway, L. plantarum LRCC5314 could produce pyruvate from glucose via glycolysis and could not produce alcohol. These ndings revealed the homofermentative activity L. plantarum LRCC5314, which could metabolize hexoses via the Embden-Meyerhof-Parnas pathway.

Tryptophan metabolism and serotonin production
According to gene annotation, the L. plantarum LRCC5314 genome contained a complete biosynthetic pathway for producing tryptophan from chorismate ( Fig. 4 and Table S2). Tryptophan is an essential amino acid for humans because it is not synthesized in the human body, and humans need to consume it. However, only 1% of the available tryptophan is used for protein synthesis, and 99% is used as a precursor of tryptamine, melatonin, and serotonin.

Discussion
L. plantarum is one of the probiotics commonly found in various fermented dairy products or traditional fermented foods such as kimchi or sausage [3], and its safety and bene cial effects on human health have been established. This bacterium is a versatile species that can be isolated from various environments, including fermented vegetables [16]. OrthoANI analysis showed that L. plantarum LRCC5314 was the most closely related to L. plantarum ATCC 14917T with a similarity of 99.65%. The G+C content range of the L. plantarum species has been reported to be 44.37-44.71 mol% [17], and L. plantarum LRCC5314 had a numerical value coinciding with the average [18]. These results indicated the LRCC5314 strain belonged to the L. plantarum species.
According to the API test, the LRCC5314 strain showed a positive result for β-galactosidase. βgalactosidase, also known as lactase, is an enzyme that catalyzes lactose into glucose and galactose. People who lack this enzyme can suffer from lactose intolerance and may be unable to consume dairy products [19]. However, probiotic strains with β-galactosidase can support lactose digestion in the small intestine. Bile salts from the gallbladder stimulate the lactase activity of probiotics and mitigate lactose intolerance symptoms [20]. L. plantarum LRCC5314 was positive for the activation of the β-galactosidase enzyme in the API test, suggesting that the strain can be used as a probiotic to alleviate lactose intolerance in the human intestine.
Hexoses are monosaccharides that contain six carbon atoms, such as mannose, glucose, and fructose. LAB can be classi ed as homofermentative and heterofermentative based on how they metabolize hexoses. Heterofermentative LAB metabolize hexoses via the acetyl-phosphate and phosphoketolase pathway, whereas homofermentative LAB metabolize hexoses via the Embden-Meyerhof-Parnas pathway [21]. L. plantarum species, including L. plantarum LRCC5314, are homofermentative LAB and metabolize hexoses to pyruvate, lactate, carbon dioxide, and ethanol through the Embden-Meyerhof-Parnas pathway. L. plantarum LRCC5314 could utilize D-galactose, D-glucose, D-fructose, and D-mannose according to the API 50CH test and could metabolize them via glycolysis, which was veri ed by in silico analysis.
The European Food Safety Authority (EFSA) recommends that bacterial strains with antibiotic resistance genes should not be used as probiotics for humans or as an additive to animal feeds [22]. The drug resistance genes of probiotic strains have been a focus of re-search because of their risk of transfer to other microbial species in the human intestine [23]. ResFinder was used to detect genes conferring antibiotic resistance in the L. plantarum LRCC5314 genome, and none were detected. The gene search results showed that L. plantarum LRCC5314 may be safe as a potential probiotic strain without risk of antibiotic gene transfer.
In our in vitro experiment, the bacterial supernatant promoted the secretion of serotonin in Caco-2 and HT-22 cells. 5-HT regulates the digestive process at several levels within the human gastrointestinal system. When foods enter the gastrointestinal tract, digestion is carried out through peristaltic waves, which are modulated by 5-HT [24]. The HT-22 cell line is an immortalized mouse hippocampal cell line and is used to identify the release of 5-HT at the brain level. The hippocampus is part of the limbic system and plays an important role in the conversion of short-term memories to long-term memories and emotional regulation [25]. Furthermore, hippocampal cells have the 5-HT receptor, the 5-HT transporter (5-HTT), and tryptophan hydroxylase (the rate-limiting enzyme in 5-HT pro-duction), which were discovered in these cells previously [26].
Serotonin, also known as 5-HT, is synthesized via a short pathway from L-tryptophan, in which tryptophan hydroxylase and aromatic L-amino acid decarboxylase are involved [27]. This monoamine neurotransmitter has multifaceted and complex biological functions. 5-HT has been reported to play functional roles in cognition, mood modulation, learning, memory, and various physiological processes [28]. Around 90% of 5-HT is found in the gastrointestinal tract, especially in enterochroma n cells. 5-HT secreted from enterochroma n cells acts in synergy with other digestive hormones to control intestinal motility. However, 1-2% of serotonin is produced in nerve cells, such as neurons with the 5-HT receptor. 5-HT produced at the end of neurons is released to the synapse and serves to transmit signals to other neurons [29]. This process is also known to be involved in the regulation of stress hormones such as cortisol, and a de ciency in 5-HT can cause depression and bipolar disorder. Therefore, several antidepressants function to prevent the absorption of 5-HT [30].
However, 5-HT cannot directly cross the blood-brain barrier (BBB). For the biosynthesis of serotonin in the brain, a precursor needs to be transported into the BBB. Tryptophan enters the brain through a common transporter protein and competes with other neutral amino acids for transport [31]. The amount of tryptophan entering the brain through the transporter protein is proportional to the concentration of tryptophan in the plasma. Several studies have reported that tryptophan-enriched diets could increase 5-HT levels in the brain and decrease cortisol levels [32].

Conclusion
We analyzed the complete genome of L. plantarum LRCC5314 and shed light on its genomic features, carbon source metabolic pathway, and functional genes. Genomic analysis con rmed that L. plantarum LRCC5314 had the tryptophan biosynthetic gene, which could produce tryptophan, a precursor that can be used in 5-HT synthesis. Moreover, in vitro analysis demonstrated that L. plantarum LRCC5314 could stimulate 5-HT se-creation in intestinal epithelial cells and serotonergic neurons. These results suggest that L. plantarum LRCC5314 may be used as a bene cial probiotic to alleviate mental stress and possibly to improve mental health.

Declarations
Availability of data and materials The data set of this study have been deposited to DDBJ/EMBL/GenBank International Nucleotide Sequence Database under the BioProject ID PRJNA716786.

Consent for publication
Not Applicable.

Competing interests
The authors declare that they do not have any competing interests.