Genomic Insights into the Probiotic Functionality and Safety of Lactiplantibacillus pentosus Strain TBRC 20328 for Future Food Innovation

Abstract

Lactiplantibacillus species have been historically used for food applications. Although several species are regarded as safe according to their regulatory status, the safety issues and functional roles of these lactic acid bacteria have been given attention. A selected Lactiplantibacillus strain TBRC 20328, with probiotic properties isolated from fermented Isan-style pork sausage (Mam), was evaluated for its safety through whole-genome sequencing and analysis using integrative bioinformatics tools. The metabolic genes were assessed through comparative genome analysis among Lactiplantibacillus species. The genome of the strain TBRC 20328 consisted of one circular chromosome (3.49 Mb) and five plasmids (totaling 0.25 Mb), encoding 3056 and 284 protein-coding genes, respectively. It exhibited an average nucleotide identity (ANI) with other Lactiplantibacillus pentosus strains of over 95%. Whole-genome analysis confirmed the absence of virulence and antimicrobial resistance genes, supporting its safety for food applications. Functional annotation revealed clusters for bacteriocins (plantaricin EF and pediocin) and polyketides, indicating potential roles in biopreservation and host interactions. Genes involved in the biosynthesis of some short-chain fatty acids and exopolysaccharides were also identified. Comparative genomic analysis across 33 other Lactiplantibacillus strains identified 2380 orthogroups, with 94 unique to the Lp. pentosus group. These included gene clusters involved in malonate decarboxylation, leucine biosynthesis, and 5-oxoprolinase activity. Such distinct genomic features emphasize the sustainable biotechnological potential and safety of Lp. pentosus TBRC 23028. Together, the findings highlight its promise as a safe and functional probiotic candidate with broad applications in functional food development and precision fermentation technologies.

1. Introduction

Probiotic bacteria are live microbes that, when carefully administered, contribute to host well-being. These microorganisms typically survive harsh conditions such as stomach acid and bile salts, adhere to the intestinal lining, and influence immune and barrier functions. Such characteristics made them invaluable in preventing gastrointestinal disorders, maintaining microbial balance, and supporting health beyond digestion. Among probiotic genera, Lactobacillus species, which are lactic acid bacteria (LAB), are widely known for their roles in food preservation and health promotion, which are achieved through traditional and precision fermentation technologies. The genus Lactiplantibacillus, such as Lactiplantibacillus plantarum, Lactiplantibacillus paraplantarum, and Lactiplantibacillus pentosus, is particularly notable for its broad applications in starter cultures, probiotics, and functional ingredients that offer significant health benefits [1,2]. These species are commonly isolated from a variety of fermented foods, such as olives, sourdough, and pickled vegetables, where they play essential biological functions [3,4]. Their metabolic activities enhance the complex flavors by producing organic acids and aroma compounds, and improve texture through structural and enzymatic modifications. Additionally, their growth enriches the nutritional value of foods by synthesizing bioactive compounds, including vitamins, exopolysaccharides, and antimicrobial peptides [5,6].

Among these species, Lp. pentosus is known for its exceptional ability to survive and thrive in harsh environmental conditions, such as high salinity, acidic pH level, and nutrient scarcity [7]. The cell resilience makes it especially valuable in industrial applications, particularly in the production process of fermented foods, where it ensures reliable performance under variable conditions [1,4]. It has been reported that certain Lp. pentosus strains possess significant potential for innovative biotechnological applications, including the development of functional foods, waste valorization, and the production of natural preservatives [8]. They can produce a variety of bioactive compounds, including antimicrobial peptides and exopolysaccharides (EPSs), that contribute to food preservation, enhance texture, and provide functional benefits in food applications [9]. These biomolecules contribute collectively to the functional dynamics of microbial consortia in waste fermentation systems. EPS supports the formation of structured biofilms that promote the retention and proximity of degradative enzymes and microbial populations, while AMPs selectively suppress competing or harmful microbes, thereby improving microbial efficiency, ecological balance, and system resilience during organic waste processing [10,11]. Moreover, the ability of Lp. pentosus to degrade certain toxins and inhibit the growth of harmful pathogens further strengthens its role in biopreservation [7]. Additionally, Lp. pentosus is renowned for its probiotic properties, which support gut health and boost the immune system. With its multifunctional properties and industrial significance, Lp. pentosus remains a central focus of fundamental and applied research in food biotechnology and related fields. However, Lp. pentosus strains exhibit diverse traits, with each strain demonstrating distinct functional capabilities and environmental adaptability, largely influenced by their genetic backgrounds, which can influence phenotypic traits, such as probiotic potency, metabolic pathways, antimicrobial activity, and stress tolerance capacity. With the advancement of next-generation sequencing technologies, exploring the genetic diversity of Lactiplantibacillus strains provides comprehensive insights into strain-specific characteristics by identifying genetic barcodes associated with desired traits and revealing novel metabolic pathways. Not only used for investigating evolutionary relationships and functional diversity among closely related species, comparative omics studies also facilitate the optimization of bioprocesses and uncover the full potential of Lactiplantibacillus strains, enabling the development of tailored solutions across various applications.

Previously, we identified an Lp. pentosus isolate (TBRC 20328) from fermented Isan-style pork sausage (Mam). The experimental investigations of fundamental probiotic properties were conducted, demonstrating the strain’s promising potential, including acid and bile tolerance, and adhesion to Caco-2 intestinal cells. The DPPH assay [12] and ELISA targeting tumor necrosis factor-alpha (TNF-α) secretion in THP-1 macrophages [13] showed that the strain possesses antioxidant and anti-inflammatory activities, respectively. Using the agar well diffusion method [14], the TBRC 20328 strain also exhibited antimicrobial activity against both Gram-positive bacteria (Streptococcus gordonii, S. pyogenes, S. mutans, Staphylococcus aureus, Listeria monocytogenes, and Propionibacterium acnes) and Gram-negative bacteria (Salmonella Typhimurium and Helicobacter pylori). These multifunctional properties highlight the strain’s technological relevance, supporting its potential application in the development of functional foods, natural preservatives, and probiotic formulations. Nevertheless, further investigation is needed to determine its safety and to explore its additional functional properties. In this study, the genome sequencing and characterization of the probiotic Lp. pentosus TBRC 20328 was conducted. The safety profile of the selected strain, TBRC 20328, was assessed using computational analysis, ensuring its suitability for industrial applications. The metabolic genes and pathways associated with growth behaviors, as well as additional probiotic functions, including bacteriocin, short-chain fatty acid (SCFA), and EPS biosynthesis, were systematically analyzed through a comparative genomic study. The findings offer valuable insights into the systematic development of this strain and its production process, driving the innovative creation of functional food ingredients, healthy foods, and other bioproducts. This study suggests beneficial possibilities for utilizing precision fermentation and synthetic biology approaches to enhance the efficiency and sustainability of targeted production.

2. Materials and Methods

2.1. Bacterial Strain and Cultivation Condition

Lp. pentosus strain TBRC 20328, isolated from the fermented Isan-style pork sausage (Mam) and deposited in the Thailand Bioresource Research Center (TBRC), was used in this work. It was cultivated in MRS broth at 37 °C overnight. The cells were harvested by centrifugation at 8000–10,000 rpm for 15–20 min for further extractions of plasmid and genomic DNA.

2.2. Plasmid and Genomic DNA Extractions

The genomic DNA of Lp. pentosus was extracted using a Wizard Genomic DNA Purification kit (Promega Corporation, Madison, MI, USA) following the manual’s instructions. The DNA concentration and quality were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

The plasmid DNA of Lp. pentosus was extracted using a ZymoPURE Plasmid Miniprep kit (Zymo Research Corporation, Tustin, CA, USA) following the manual’s instructions. Then, the number and size of plasmids were analyzed by gel electrophoresis using a 0.5% agarose gel in TBE buffer.

2.3. Whole-Genome Sequencing and Genome Assembly

The genome of Lactiplantibacillus strain TBRC 20328 was sequenced using the PacBio RSII SMRT cell platform at McGill University and Génome Québec Innovation Centre, Canada. The sequencing reads were assembled de novo using the Celera Assembler within the hierarchical genome assembly process (HGAP) workflow [15].

2.4. Gene Prediction and Functional Annotation

Gene prediction and computational annotation of protein-coding genes were performed using Prokka (v.1.14.6) [16] with default parameter settings. To achieve functional annotation, the protein sequences generated by Prokka were utilized through precomputed orthology assignments, employing the EggNOG-mapper tool (v.2.1.12) [17]. Subsequently, these protein sequences were searched against the EggNOG database (version 5) [18] using the DIAMOND protein aligner [19]. Gene prediction and computational annotation of protein-coding genes were performed using Prokka [16] with default parameter settings. To achieve functional annotation, the protein sequences generated by Prokka were utilized through precomputed orthology assignments, employing the EggNOG-mapper tool (v.2.1.12) [17]. Subsequently, these protein sequences were searched against the EggNOG database (version 5) [18] using the DIAMOND protein aligner [19].

2.5. Species Identification

The initial identification of the strain’s species was implemented by analyzing the 16S rRNA gene sequences. A set of 16S rRNA gene copies was screened and extracted from the genomic data available in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST v.2.16.0). After confirming all sequences of Lactobacillaceae, these sequences were utilized to construct a phylogenetic tree using the Molecular Evolutionary Genetic Analysis (MEGA 11 v.11.0.11) software [20].

To further validate the species classification, average nucleotide identity (ANI) was computed against the type strains of the designated species using the OrthoANI method [21]. The species categorization was confirmed based on a threshold ANI value of ≥95–96%, adhering to the criteria established in the previous report [22].

2.6. Determination of Antimicrobial Resistance (AMR), Virulence Factors (VFs), Antibiotic Resistance Genes (ARG), and Undesirable Genes

In silico analyses of AMR, VFs, and ARG were performed. The protein-coding genes of the Lp. pentosus TBRC 20328 genome were searched for antimicrobial resistance and virulence factors using seven specialized databases. These included the Comprehensive Antibiotic Resistance Database (CARD) [23], Antibiotic Resistance Gene-ANNOTation (ARG-annot) [24], Virulence Factor Database (VFDB) [25], ResFinder [26], MEGARes [27], the NCBI-AMRFinder [28], and PlasmidFinder [29]. This comprehensive screening was performed using ABRicate (version 1.0.1) [30] to identify annotated replicons and genetic elements related to AMR, virulence factors, and undesirable genes.

2.7. Biosynthesis Gene Cluster Analysis

To identify gene clusters responsible for the biosynthesis of secondary metabolites across various chemical classes in the Laciplantibacillus genome, we performed in silico analysis using antiSMASH 7.1.0 (Antibiotics and Secondary Metabolite Analysis Shell) [31] with default parameters. PRISM 4 (Prediction Informatics for Secondary Metabolomes) [32], available at http://prism.adapsyn.com/ (accessed on 24 March 2024), was used with default settings to further identify biosynthetic gene clusters. Bacteriocin operons within the genome were also examined using the BActeriocin GEnome mining tooL (BAGEL4) [33].

The genome sequences were prepared in FASTA format and analyzed using OrthoFinder (version 2.5.5) [34] for predicting orthologous gene groups across multiple genomes. OrthoFinder, utilizing the DIAMOND alignment algorithm, was employed to conduct high-speed and accurate sequence comparisons, clustering similar proteins into orthogroups that represent putative gene families, thereby elucidating evolutionary and functional relationships among genes.

The OrthoFinder analysis was conducted using default parameters, enabling the identification and classification of orthologous genes in a dataset that included the genome of Lactiplantibacillus strain TBRC 20328 along with 32 additional genomes from closely related Lactiplantibacillus species. This dataset comprised 12 Lp. pentosus, 13 Lp. plantarum, and 7 Lp. paraplantarum genomes, all sourced from the NCBI database. By including such diverse genomes, this analysis elaborated a comprehensive framework for exploring genetic conservation and divergence within the genus, providing valuable insights into the evolutionary dynamics and functional diversity of Lactiplantibacillus species.

3. Results and Discussions

3.1. Genome Features and Species Identification of Lactiplantibacillus TBRC 20328

The genome of Lactiplantibacillus TBRC 20328 consisted of 3,491,384 base pairs (bp) and a GC content of 46.60%. It contained five plasmids labeled A to E, ranging in size from 70,426 bp to 6885 bp, as shown in

Table 1. Genome statistics of Lactiplantibacillus TBRC 20328.

	Genomic	Plasmid A	Plasmid B	Plasmid C	Plasmid D	Plasmid E	Total
Total base (bp)	3,491,384	70,426	68,335	48,057	55,487	6885	3,740,574
A (bp)	932,443	20,620	20,892	15,051	14,869	2133	1,006,008
T (bp)	932,054	22,020	19,008	14,340	17,651	2112	1,007,185
G (bp)	813,600	13,556	14,549	9448	11,106	1306	863,565
C (bp)	813,287	14,230	13,886	9218	11,861	1334	863,816
GC content (%)	46.60	39.45	41.61	38.84	41.39	38.34	46.18
No. of protein-coding genes	3056	79	81	54	62	8	3340
Avg. gene length (bp)	919	708	691	649	798	328	900
No. of tRNA genes	63	-	-	-	-	-	63
No. of tmRNA genes	1	-	-	-	-	-	1
No. of rRNA genes	16	-	-	-	-	-	16
Repeat regions	3	-	-	-	-	-	3

and Figure 1. Genome annotation using Prokka [16] identified 3136 genes, of which 3056 protein-coding sequences were annotated. The computational analysis revealed 63 transfer RNA (tRNA) genes, 16 ribosomal RNA (rRNA) genes, and one transfer-messenger RNA (tmRNA). The rRNA genes contained five copies of each of the 16S and 23S rRNA genes and six copies of the 5S rRNA genes. Assembly completeness was assessed using Benchmarking Universal Single-Copy Orthologs (BUSCO) v.5 [35] through the gVolante server [36] and CheckM [37]. Using a reference gene set from Lactobacillales, BUSCO analysis showed 99.8% completeness. CheckM also estimated completeness at 99.38% based on Lactobacillales marker sets, with a contamination level of 2.01%, as detailed in Supplementary Data Table S1.

Furthermore, the annotated genes were compared against the Clusters of Orthologous Groups (COG) [38] and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases [39] to gain insight into their biological functions. The functional categorization of the putative protein-coding genes of Lactiplantibacillus TBRC 20328 is illustrated in Figure 2. The genes of the Lactiplantibacillus TBRC 20328 chromosome were categorized into 18 functional groups based on their assignment to Clusters of Orthologous Groups (COG) families and a “function unknown” category [S], which consisted of 533 genes. Most of the genes in Lactiplantibacillus TBRC 20328 were associated with essential cellular functions. Notably, 301 genes were classified under the transcription category [K], suggesting a significant reliance on gene regulation and expression in various cellular processes. A substantial number of genes, 287 in total, were associated with carbohydrate transport and metabolism [G], while 246 genes were involved in amino acid transport and metabolism [E]. Together, these categories [G and E] underscore the essential role of carbohydrate and amino acid pathways in cellular functions. Additionally, 173 genes were associated with translation, ribosomal structure, and biogenesis [J], emphasizing the importance of protein synthesis for growth and cellular function. A total of 169 genes related to the cell wall, membrane, and envelope biogenesis [M] were identified, suggesting that maintaining structural integrity is essential for Lactiplantibacillus TBRC 20328. Only 15 genes were functionally assigned to cell motility [N], indicating that this function is minimally represented in the genome.

Figure 1. A circular map of the Lactiplantibacillus TBRC 20328 genome was generated using GenoVi v04.3 [40]. The primary map (left-hand side) displays the chromosome and five plasmids (A–E) illustrated on the right-hand side. The genomic features and Clusters of Orthologous Groups (COGs) were identified through PROKKA annotation and are visualized on the genome (from outer to inner): circles 1 and 2 are color-coded based on the COG classification and represent coding sequences (CDSs) located on the forward strand; circles 3 and 4 are color-coded based on the RNA classification, representing features on the forward and reverse strands, respectively; circles 5 and 6 are color-coded based on the CDS and COG classification, representing features on the reverse strand; circles 7 and 8 display GC content and GC skew, respectively.

Figure 1. A circular map of the Lactiplantibacillus TBRC 20328 genome was generated using GenoVi v04.3 [40]. The primary map (left-hand side) displays the chromosome and five plasmids (A–E) illustrated on the right-hand side. The genomic features and Clusters of Orthologous Groups (COGs) were identified through PROKKA annotation and are visualized on the genome (from outer to inner): circles 1 and 2 are color-coded based on the COG classification and represent coding sequences (CDSs) located on the forward strand; circles 3 and 4 are color-coded based on the RNA classification, representing features on the forward and reverse strands, respectively; circles 5 and 6 are color-coded based on the CDS and COG classification, representing features on the reverse strand; circles 7 and 8 display GC content and GC skew, respectively.

Figure 2. Functional Category of putative protein-coding genes in the Lactiplantibacillus TBRC 20328 genome. Numbers indicate the number of genes in each category.

Figure 2. Functional Category of putative protein-coding genes in the Lactiplantibacillus TBRC 20328 genome. Numbers indicate the number of genes in each category.

Furthermore, the annotated genes were compared against the Clusters of Orthologous Groups (COG) [38] and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases [39] to gain insight into their biological functions. The functional categorization of putative protein-coding genes of Lactiplantibacillus TBRC 20328 is illustrated in Figure 2. The genes of the Lactiplantibacillus TBRC 20328 chromosome were categorized into 18 functional groups based on their assignment to Clusters of Orthologous Groups (COG) families and a “function unknown” category [S], which consisted of 533 genes. Most of the genes in Lactiplantibacillus TBRC 20328 were associated with essential cellular functions. Notably, 301 genes were classified under the transcription category [K], suggesting a significant reliance on gene regulation and expression in various cellular processes. A substantial number of genes, 287 in total, were associated with carbohydrate transport and metabolism [G], while 246 genes were involved in amino acid transport and metabolism [E]. Together, these categories [G and E] underscore the essential role of carbohydrate and amino acid pathways in cellular functions. Additionally, 173 genes were associated with translation, ribosomal structure, and biogenesis [J], emphasizing the importance of protein synthesis for growth and cellular function. A total of 169 genes related to cell wall, membrane, and envelope biogenesis [M] were identified, suggesting that maintaining structural integrity is essential for Lactiplantibacillus TBRC 20328. Only 15 genes were functionally assigned to cell motility [N], indicating that this function is minimally represented in the genome.

The 16S rRNA gene sequences of Lactiplantibacillus TBRC 20328 exhibited 100% identity with those of other Lactiplantibacillus species, including Lp. plantarum SRCM100442, Lp. plantarum DMC-S1, Lp. pentosus ZFM222, and Lp. pentosus strain 68-1. All similarity values were observed to surpass the 98.7% cut-off threshold proposed for species-level identification [41]. To further clarify evolutionary relationships, a phylogenetic analysis was performed using the Molecular Evolutionary Genetics Analysis program (v. 11). The evolutionary tree constructed based on 16S rRNA sequences from members of the Lactobacillaceae family is illustrated in Figure 3. Using multiple sequence comparisons by ClustalW with the maximum composite likelihood method, the sequence alignment results indicate that the 16S rRNA gene analysis could not effectively distinguish between Lp. plantarum and Lp. pentosus within this bacterial group. To overcome the limitation and ensure accurate species assignment, whole-genome sequence data for the strain TBRC 20328 were intensively analyzed. The ANI value calculation revealed that the strain TBRC 20328 shared the highest ANI value of 99.89% with Lp. pentosus strain 68-1, which is shown in Figure 4. ANI values for other Lp. pentosus strains also exceeded the 95–96% species delineation threshold as proposed by Richter and Rosselló-Móra (2009) [22]. In contrast, ANI values of other Lp. plantarum and Lp. paraplantarum species were notably lower, falling below 81%. These findings, supported by genome-level data, strongly confirm that the strain TBRC 20328 belongs to the species Lactiplantibacillus pentosus. This whole-genome approach demonstrated superiority over 16S rRNA analysis for resolving closely related taxa within the Lactobacillaceae family.

3.2. Absence of Antimicrobial Resistance Genes (AMR), Virulence Factors (VFs), Antibiotic Resistance Genes (ARG), and Mobile Genetic Elements

Using ABRicate (version 1.0.1) [30] with default parameters, the Lp. pentosus TBRC 20328 genome was screened against databases for AMR, VFs, ARG, and undesirable genes. The analysis revealed no detectable AMR, VFs, ARG, or undesirable genetic elements. Notably, no such genes were identified within plasmids or near plasmid replicon regions. These results suggest that the strain TBRC 20328 poses minimal safety risks for food applications, as the absence of functional or transferable AMR genes supports regulatory compliance and reduces concerns about horizontal gene transfer or pathogenicity. By avoiding the pitfalls associated with antimicrobial resistance and pathogenicity, this strain holds promise for the development of sustainable and safe food solutions. Future studies should focus on the long-term evaluations of its functional properties across diverse food matrices, as well as its interactions with native gastrointestinal microbiota, to further substantiate its safety and efficacy.

Mobile genetic elements play a crucial role in organizing bacterial genomes by facilitating horizontal gene transfer, which enables functional adaptation and contributes to genomic stability. Careful consideration of the safety and functionality of bacterial probiotics and starter strains is essential, as these are critical factors in functional food applications. Thus, the MGE analysis is mainly required for seeking genes associated with antimicrobial resistance, metabolic traits, or niche-specific adaptation. To discover prophage elements in the genome of Lp. pentosus TBRC 20328, we employed the PHAge Search Tool Enhanced Release (PHASTER), available at https://phastest.ca (accessed on 19 August 2024), to thoroughly examine potential prophage regions. This tool provided insights into their length, location, GC content, and annotated genes [42]. Despite using both ‘lite’ and ‘deep’ annotation modes, the analysis found no evidence of prophage fragments within the TBRC 20328 genome, indicating an absence of integrated viral sequences. Additionally, the PlasmidFinder tool could identify two plasmid replicons, rep38_2_repA (LBPp1) and rep38_1_rep (pLBUC03), in the TBRC 20328 strain, as shown in

Table 2. Plasmids in Lactiplantibacillus TBRC 20328 searched by PlasmidFinder.

Plasmid	Start	End	PlasmidFinder Annotation	Origin	Identity (%)
A	44,904	45,785	rep38_2_repA (LBPp1)	Lp. plantarum P-8	96.19
D	21,409	22,435	rep38_1_rep (pLBUC03)	L. buchneri NRRL B-30929	86.50

. These replicons have been previously annotated in Lp. plantarum P-8 and L. buchneri NRRL B-30929, respectively. The results highlight the presence of functional plasmid elements and confirm the absence of integrated prophage sequences in strain TBRC 20328, underscoring its genomic stability and suitability for industrial applications. Plasmids can confer adaptive advantages, such as metabolic versatility and stress resilience, thereby increasing strain robustness under industrial processing conditions and supporting its application in fermentation-based processes [43]. Moreover, the absence of prophages reduces the risk of horizontal gene transfer to other microorganisms, whether in food products or within the human gut microbiota, further enhancing the safety profile of Lp. pentosus TBRC 20328 and increasing consumer confidence in its use.

3.3. Biogenic Amine Biosynthesis Genes

LAB plays a pivotal role in producing biogenic amines (BAs) with the metabolic activity of amino acid decarboxylation. While BAs contribute to the distinctive flavors and characteristics of fermented products, they can also pose health risks. For some individuals, consuming foods high in biogenic amines may trigger adverse reactions, such as headaches, heart palpitations, vomiting, and diarrhea [44,45]. The severity of these symptoms can vary widely among individuals, emphasizing the importance of understanding the effects of LAB and their metabolic byproducts on food safety and human health. Selecting probiotic bacteria that do not produce BA is crucial for fermentation. In this study, we evaluated the presence of essential genes involved in the biosynthetic pathways of BAs in the genome of Lp. pentosus TBRC 20328 through amino acid sequence similarity searches. The analysis revealed the absence of several key genes associated with BAs production [46] in the TBRC 20328 genome, including lysine decarboxylase (EC: 4.1.1.18), ornithine/lysine decarboxylase (EC: 4.1.1.116), arginine decarboxylase (EC: 4.1.1.19), agmatinase (EC: 3.5.3.11), spermidine synthase (EC: 2.5.1.16), spermine synthase (EC: 2.5.1.22), arginase (EC: 3.5.3.1), ornithine decarboxylase (EC: 4.1.1.17), histidine decarboxylase (EC: 4.1.1.22), tyrosine decarboxylase (EC: 4.1.1.25), and tryptophan decarboxylase (EC: 4.1.1.28). This finding suggests the favorable safety profile of Lp. pentosus TBRC 20328 for consumption due to the genomic absence of critical genes involved in biogenic amine synthesis, highlighting its suitability for food-related applications. This genetic feature reduces concerns about toxic metabolite accumulation, reinforcing its promise as a stable and safe microbial resource for fermentation-based innovations.

3.4. Bile Salt Deconjugations

The ability to hydrolyze bile salts is a key criterion for probiotic selection. However, selecting effective probiotic strains that can function optimally in the gastrointestinal tract remains a significant challenge. We identified two genes within the genome of Lp. pentosus TBRC 20328 that are homologous to the choloylglycine hydrolase family and linked explicitly to bile salt hydrolase (BSH; cholylglycine hydrolase; and EC 3.5.1.24). BSH is an enzyme produced by LAB, such as Lactiplantibacillus and Bifidobacterium strains, playing a crucial role in bile acid metabolism [47,48] by catalyzing the hydrolysis of bile salts conjugated with amino acids, including glycine and taurine, thereby disrupting the formation of cholesterol micelles necessary for intestinal cholesterol absorption. This function can reduce cholesterol uptake and improve lipid profiles [49]. Thus, bile salt hydrolase in the TBRC 20328 strain could enhance its tolerance to bile acids and facilitate bile salt hydrolysis, suggesting its potential for cholesterol management. Recent studies highlighted the potential of bile salt deconjugation by LAB as a therapeutic approach to lower serum cholesterol levels in hypercholesterolemic patients and to prevent hypercholesterolemia in individuals with normal cholesterol levels [50].

3.5. D-Lactic Acid Production

Using the KEGG database searching, the lactate racemase (Lar) and D-lactate dehydrogenase (LDHD) genes were identified in the genome of Lp. pentosus TBRC 20328. These enzymes catalyze the production of D-lactic acid, a key component of cell wall peptidoglycan in several Gram-positive bacteria, including the Lactiplantibacillus genus [51]. Since D-lactic acid production is an intrinsic property of these bacteria, precautions should be taken when consuming high amounts of D-lactic acid-producing strains, particularly for individuals at risk of D-lactic acidosis, such as patients with short bowel syndrome or carbohydrate malabsorption [52,53]. However, it is essential to note that these bacteria are commonly found in various food sources, including yogurt and other fermented products, which have been safely consumed for generations. Moreover, several Lactiplantibacillus probiotics have been classified as “generally recognized as safe” (GRAS) by the United States Food and Drug Administration (US FDA) [54]. Therefore, we suggest that Lp. pentosus TBRC 20328 presents a low risk and does not raise safety concerns regarding the production of D-lactic acid.

3.6. Biosynthesis Gene Clusters for Bacteriocin and Secondary Metabolite Production

Biosynthesis gene clusters (BGCs) typically consist of two or more components within a genome, each contributing to the production of specialized metabolites and chemical variants. These components include (i) backbone enzymes, which are responsible for the initial step in synthesizing the product, and (ii) tailoring enzymes, which further modify the molecule produced by the backbone enzymes [55]. To achieve a comprehensive identification of secondary metabolite BGCs, we conducted a combined analysis using BAGEL 4, AntiSMASH (v.7.1.0), and PRISM 4. These tools employ distinct databases and detection algorithms, offering complementary strengths that enhance the robustness of BGC prediction and reduce the likelihood of missing clusters of interest. The BAGEL 4 software [33] was used to identify potential bacteriocin clusters and genes associated with antimicrobial protein biosynthesis. The result shows two areas of interest (AOI) in the genome of Lp. pentosus TBRC 20328 containing bacteriocin-coding genes that were plantaricin EF and pediocin. Additionally, the AntiSMASH tool [31] was utilized to identify gene clusters responsible for the biosynthesis of secondary metabolites, including non-ribosomal peptide synthetases (NRPs), polyketide synthases (PKSs), ribosomally synthesized and post-translationally modified peptides (RiPPs), and other antimicrobial synthases. Using the AntiSMASH pipeline with the “strict” parameter, no gene clusters were identified. However, when the “relaxed” parameter was applied, two genomic regions of interest were identified: one corresponds to a class II bacteriocin gene encoding plantaricin EF, and the other is involved in the biosynthesis gene cluster of type 3 polyketide synthase (T3PKS).

Furthermore, the PRISM 4 algorithm [32] was utilized to identify and compare gene clusters for secondary metabolites in the Lp. pentosus TBRC 20328 genome. This analysis revealed two antimicrobial gene clusters: one encoding a class II bacteriocin (plantaricin EF) and the other associated with polyketide biosynthesis. Conclusively, the combinatorial exploitation of BAGEL 4 [33], antiSMASH [31], and PRISM 4 [32] led to the identification of four AOIs in the Lp. pentosus TBRC 20328 genome, each associated with different metabolites, as illustrated in Figure 5.

AOI region 1, spanning approximately 20 kb, was identified as the genomic region encoding the gene cluster responsible for plantaricin E and plantaricin F biosynthesis in the TBRC 20328 genome, which was consistently generated by all three computational tools. This gene cluster was associated with the biosynthesis of plantaricin EF (PlnEF), a type II bacteriocin comprising two functionally complementary peptides, PlnE and PlnF. To further investigate this gene cluster, we conducted a comparative genomic analysis of the Lp. pentosus TBRC 20328 genome alongside 32 other Lactiplantibacillus genomes, encompassing 7 Lp. paraplantarum, 12 Lp. pentosus, and 13 Lp. plantarum strains. The result reveals that the PlnEF-encoding gene sequences were highly conserved among the Lactiplantibacillus strains studied, which occupy diverse ecological niches. It has been reported that type II bacteriocin is found in LAB, which has antimicrobial activity against pathogenic bacteria [56,57].

The plantaricin EF gene cluster of the strain TBRC 20328 shared high similarity with the corresponding cluster in Lp. pentosus strain 68-1, exhibiting 99% sequence identity and 100% coverage. However, the strain 68-1 does not produce these bacteriocins [58]. Notably, there might be genetic discrimination in regulating or expressing bacteriocin gene clusters of different strains of Lp. pentosus. Further elucidation of these clusters governing genetic and regulatory mechanisms is necessary to comprehensively understand bacteriocin diversity and function in Lp. pentosus TBRC 20328. The existence of antimicrobial gene clusters suggests their ability to produce antimicrobial peptides with enhanced efficacy, positioning the TBRC 20328 strain as a promising candidate for developing natural alternatives to chemical preservatives and antibiotics [59]. Such insights may also inform broader applications of bacteriocins in food technology and the strategic development of natural antimicrobial agents.

The AOI region 2 was predicted in the genome of strain TBRC 20328 by using BAGEL 4, which corresponded to the pediocin-encoding gene, a type of class II bacteriocin gene cluster. A pediocin gene cluster typically consists of four open reading frames (ORFs) encoding the structural peptide, immunity protein, accessory protein, and ABC transporter [60]. A subsequent manual BLAST search at the amino acid level against UniProtKB and NCBI revealed that the putative pediocin gene of the TBRC 20328 strain was similar to the pediocin PA-1 immunity protein. No actual pediocin structural gene was detected within the predicted pediocin gene cluster in this genome.

In Figure 5, the AOI region 3 in the Lp. pentosus TBRC 20328 was identified using the antiSMASH tool. This cluster was the largest AOI, comprising 46 genes, which were classified as the Type III Polyketide Synthase (T3PKS) family. T3PKS proteins are typically small, dimeric molecules with a molecular weight between 80 and 90 kDa. These enzymes are involved in the production of polyketides, complex secondary metabolites that serve as the backbone for various bioactive substances, such as antibiotics, antifungals, parasiticides, and immunomodulators [61]. Type III PKSs are among the most common biosynthetic gene clusters found in LAB, indicating their widespread role in secondary metabolism across these microorganisms [62,63]. The presence of the T3PKS gene cluster in Lp. pentosus TBRC 20328 suggests that the polyketides synthesized by T3PKS enzymes may contribute to the antimicrobial activity.

Using PRISM 4, the AOI region 4, containing the PKS gene cluster, was identified in the Lp. pentosus TBRC 20328 genome. This cluster included a gene encoding an acyltransferase (AT) domain, a crucial component in the synthesis of polyketides. The AT domain is a monomeric unit responsible for producing secondary metabolites. In addition, the analysis revealed several other genes located both upstream and downstream of the AT gene, including additional biosynthetic genes, transporter genes, and the genes encoding key PKS domains, such as ketosynthase (KS) and ketoreductase (KR). The domains of the PKS enzymes of Lp. pentosus displayed mechanistic similarities to those of fatty acid synthases (FAS). PKS and FAS share a similar biosynthetic framework, utilizing the same precursors and cofactors in their respective pathways [64,65]. Despite these similarities, the PKS gene cluster of Lp. pentosus TBRC 20328 appeared to overlap with gene clusters typically associated with fatty acid synthesis identified through genome annotation, presuming that the pathways for fatty acid and polyketide biosynthesis in this strain may not be as distinctly separated as previously thought. The apparent integration between primary (fatty acids) and secondary (polyketides) metabolic pathways highlights the need for further research to clarify the specific roles and interactions of these biosynthetic pathways in this strain.

The detection of gene clusters associated with bacteriocin and polyketide biosynthesis in Lp. pentosus TBRC 20328 postulates its capacity to produce bioactive metabolites with antimicrobial and potentially therapeutic properties. Bacteriocins, ribosomally synthesized antimicrobial peptides produced by LAB, are effective natural preservatives and alternatives to chemical additives in food products [66,67]. Likewise, polyketides are a diverse class of microbial metabolites with well-documented antimicrobial, anti-inflammatory, and antioxidant activities, supporting their use in functional foods and health-promoting formulations [55,68]. These genomic features may support their functional role in modulating microbial communities and enhancing host interactions. From a technological perspective, such genetic traits position the strain as a promising candidate for use in natural preservation and the development of next-generation probiotic and fermented food products, reducing reliance on synthetic additives.

3.7. The Phosphotransferase System (PTS) in Carbohydrate Uptake of Lp. pentosus TBRC 20328

The PTS is a complex, multi-component mechanism in bacteria that facilitates the uptake of various sugar substrates, including monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. This system operates by transporting specific saccharides across the bacterial inner membrane through a series of sequential steps for transferring a phosphate group from phosphoenolpyruvate (PEP) to the incoming sugar [69,70]. The PTS comprises various proteins, including soluble phosphotransferases and an integral membrane protein that directly mediates the translocation of sugars into the cytoplasm [71]. Of LAB, particularly the Lactiplantibacillus genus, PTS transporters are the primary pathways for carbohydrate transport [72]. Previous studies revealed notable diversity in PTS transporter proteins among different strains; for example, Lp. plantarum WCSF1, which harbors 25 PTS transporters [73], Lp. pentosus strain 68-1, containing 46 PTS transporters [58], and Lp. plantarum MC5, possessing 61 distinct PTS transporters [74]. The variation in PTS transporter abundance strongly correlates with the ability of these strains to utilize diverse sugar substrates, highlighting their metabolic flexibility.

Based on homology with annotated PTS protein-encoding genes, 65 PTS-type sugar transporter proteins associated with various sugars were functionally annotated in the Lp. pentosus TBRC 20328 genome (Supplementary Data Table S2). These included six lactose/cellobiose family transporters, six beta-glucosidase transporters, five glucose transporters, four fructose transporters, four galactitol transporters, and four glucitol/sorbitol transporters. It also possessed other transporters for galactosamine (3 transporters), mannose/fructose/sorbose family (3 transporters), sucrose (2 transporters), trehalose (2 transporters), mannitol (1 transporter), N-acetylglucosamine (1 transporter), and 17 unspecified sugar transporters. These findings suggest that Lp. pentosus TBRC 20328 could utilize a broad range of carbon sources for cell growth and function, which helps develop culture conditions for specific applications. Additionally, several genes associated with the oligosaccharide phosphate transfer system were found in multiple copies in the TBRC 20328 genome. This genetic redundancy could enhance its capability to utilize these carbon sources efficiently. These functional proteins may confer metabolic flexibility, allowing Lp. pentosus TBRC 20328 to thrive and survive in diverse and complex environments, which helps design fermentation processes for functional food production.

3.8. Short-Chain Fatty Acid Biosynthesis Genes in Lp. pentosus TBRC 20328

Some LABs have been identified for their capacity to produce SCFAs [75,76]. The investigation of SCFA production by LAB is of particular interest due to its potential health benefits for human hosts [77,78]. Through a functional analysis of the Lp. pentosus TBRC 20328 genome, we identified four genes that significantly contribute to acetate production, two genes involved in formate synthesis, and six genes associated with lactate biosynthesis. Our analysis also revealed two thioesterase genes, which may be linked to SCFA production. Previous studies demonstrated that E. coli can produce butyrate by expressing heterologous thioesterases, suggesting the potential of these identified genes for heterologous production of butyrate and other SCFAs in a microbial host system of interest [79,80]. However, further investigation is needed to elucidate the functional roles of the thioesterase genes in Lp. pentosus TBRC 20328 and their potential contributions to SCFA biosynthetic pathways. Putative genes and enzymes involved in SCFA and lactate synthesis are listed in Supplementary Data Table S3.

The presence of SCFA biosynthetic genes in Lp. pentosus TBRC 20328 suggests its potential role in promoting gut microbial balance and supporting host metabolic functions. These metabolites are known to influence intestinal barrier maintenance and immune modulation [81,82]. From an industrial innovation standpoint, such genetic capabilities position the strain as a valuable ingredient for next-generation functional foods and probiotic formulations that target digestive and systemic health, offering a naturally derived alternative to conventional supplements.

3.9. Exopolysaccharides (EPS) Gene Clusters in Lp. pentosus TBRC 20328

To investigate the genes related to the EPS production, we performed a comparative whole-genome analysis of Lp. pentosus TBRC 20328 and other previously characterized Lactiplantibacillus strains [74] downloaded from the NCBI database. The selected strains, including Lp. plantarum WCFS1, ST-III, JDM1, and SMB758, were chosen for this study due to the confirmed presence of eps genes in their genomes and the extensive characterization of their gene clusters. Lp. plantarum subsp. plantarum ST-III is an outlier, harboring three complete eps gene clusters: eps2ABCEF, eps3ABDEFHIJ, and eps4ABCEFGHIJ. Our analysis revealed that the genome of Lp. pentosus TBRC 20328 also contained three eps gene clusters, showing high similarity to those identified in Lp. plantarum ST-III. Furthermore, A distinct epsF gene was identified which shared a high degree of sequence similarity with the corresponding genes found in Lp. plantarum JDM1 and SMB758 (

Table 3. The eps gene clusters and their homologous sequences in Lp. pentosus TBRC 20328.

GeneID	Length (aa)	NCBI Accession	Species	Length (aa)	Gene	Gene Function	Homology (%)
EPS gene Cluster 1
Orf00270	376	ADN98210	Lp. plantarum ST-III	359	eps3I	O-acetyltransferase	267/360 (74%)
Orf00271	367	ADN98209	Lp. plantarum ST-III	369	eps3H	polysaccharide biosynthesis protein	287/367 (78%)
Orf00272	393	ADN98208	Lp. plantarum ST-III	406	eps3F	polysaccharide polymerase	301/388 (78%)
Orf00273	207	ADN98207	Lp. plantarum ST-III	210	eps3E	polysaccharide biosynthesis protein	141/207 (68%)
Orf00274	375	ADN98206	Lp. plantarum ST-III	377	eps3D	polysaccharide biosynthesis protein	248/375 (66%)
Orf00276	310	ADN98204	Lp. plantarum ST-III	310	eps3B	glycosyltransferase	308/310 (99%)
Orf00277	303	ADN98203	Lp. plantarum ST-III	303	eps3A	glycosyltransferase	294/303 (97%)
EPS gene Cluster 2
Orf00292	225	ADN98183	Lp. plantarum ST-III	200	eps2E	priming glycosyltransferase	200/200 (100%)
Orf00294	271	ADN98181	Lp. plantarum ST-III	257	eps2C	exopolysaccharide biosynthesis protein	252/257 (98%)
Orf00295	242	ADN98180	Lp. plantarum ST-III	242	eps2B	exopolysaccharide biosynthesis protein	241/242 (99%)
Orf00296	256	ADN98179	Lp. plantarum ST-III	256	eps2A	exopolysaccharide biosynthesis protein	254/256 (99%)
EPS gene Cluster 3
Orf00286	460	ADN98188	Lp. plantarum ST-III	460	wxz	exopolysacharide protein Wzx	451/460 (98%)
Orf00288	319	ADN98187	Lp. plantarum ST-III	319	eps4I	glycosyltransferase	314/319 (98%)
Orf00289	424	ADN98186	Lp. plantarum ST-III	424	eps4H	polysaccharide polymerase	421/424 (99%)
Orf00290	343	ADN98185	Lp. plantarum ST-III	345	eps4G	glycosyltransferase	341/343 (99%)
Orf00291	364	ADN98184	Lp. plantarum ST-III	364	eps4F	glycosyltransferase	359/364 (99%)
Orf02621	251	ADN98956	Lp. plantarum ST-III	252	eps4A	exopolysaccharide biosynthesis protein	194/253 (77%)
Orf02622	238	ADN98955	Lp. plantarum ST-III	235	eps4B	capsular exopolysaccharide family protein	193/234 (82%)
		WP_015640554	Lp. plantarum JDM1	235	epsD/ epsB/ epsF	CpsD/CapB family tyrosine-protein kinase	192/234 (82%)
		WDQ20187	Lp. plantarum SMB758	235	epsD/epsB	CpsD/CapB family tyrosine-protein kinase	193/234 (82%)
Orf02623	259	ADN98954	Lp. plantarum ST-III	273	eps4C	phosphotyrosine-protein phosphatase	184/259 (71%)
Orf02625	221	ADN98952	Lp. plantarum ST-III	223	eps4E	priming glycosyltransferase	171/221 (77%)
Orf02630	481	ADN98947	Lp. plantarum ST-III	483	eps4J	repeat unit transporter	353/481 (73%)
Specific EPS gene
Orf00813	245	WP_003643865	Lp. plantarum JDM1	245	epsF	WecB/TagA/CpsF family glycosyltransferase	225/245 (92%)
		WDQ21455	Lp. plantarum SMB758	245	epsF	WecB/TagA/CpsF family glycosyltransferase	225/245 (92%)

). However, we did not find any eps gene similarity between our strain and Lp. plantarum WCFS1. Cluster 3 of the eps genes in Lp. pentosus TBRC 20328 notably encoded a wzx gene, which plays a critical role in the transport of glycosyl units during exopolysaccharide (EPS) biosynthesis [83]. The presence of this gene indicates a strong capacity for repeat unit transport within this cluster. Based on genomic similarity, we propose that TBRC 20328 is a close genetic relative of Lp. plantarum subsp. plantarum ST-III. The presence of multiple eps gene clusters in both strains may contribute to their enhanced EPS production capabilities.

EPSs produced by Lactiplantibacillus species are increasingly recognized for their diverse health-promoting properties. Studies have shown that LAB-derived EPSs can exhibit immunomodulatory activity [84], enhance gut barrier integrity [85], and promote the growth of beneficial gut microbiota [11]. Additionally, EPSs may contribute to antioxidant effects [86], cholesterol-lowering potential [87], and inhibition of pathogen adhesion in the gastrointestinal tract [88]. The genomic features observed in Lp. pentosus TBRC 20328 suggest its potential to produce functionally relevant EPSs with similar bioactivities. However, it is important to note that these conclusions are based solely on in silico analysis, and further experimental validation is required to confirm the strain’s EPS production capacity and its associated health benefits.

3.10. Comparative Analysis of Lactiplantibacillus Genomes Exploring Unique Genetic Features of Lp. pentosus Strains

A comparative genomics analysis was performed across 33 Lactiplantibacillus, including 13 Lp. pentosus, 13 Lp. plantarum, and 7 Lp. paraplantarum, using an all-against-all genome comparison approach with OrthoFinder [34]. This analysis identified orthologous genes and orthogroups, thoroughly characterizing the genomic relationships, core genome, and species-specific content of Lp. pentosus strains. A comparative study of 13 Lp. pentosus genomes identified 2380 orthogroups of orthologous protein-coding genes, collectively referred to as the “consensus Lp. pentosus gene set”. Notably, variation in copy number was observed across these orthogroups, suggesting strain-specific genomic divergence within Lp. pentosus strains. Further analysis revealed 1880 orthogroups (78.99%) of the consensus Lp. pentosus gene set were conserved across the 33 Lactiplantibacillus genomes and were thus classified as the core gene set shared among these species. In contrast, 94 orthogroups (out of the identified 2380 orthogroups) were exclusive to the 13 Lp. pentosus genomes and were absent in the Lp. plantarum and Lp. paraplantarum genomes analyzed in this study. These proteins were designated “specific orthogroups” for Lp. pentosus. Based on genome annotation data of Lp. pentosus strain TBRC 20328, these specific orthogroups were classified into five functional categories: (i) protein-coding genes involved in metabolism (17 of total specific orthogroups), (ii) toxin–antitoxin system proteins (7 of total specific orthogroups), (iii) transcriptional regulators (6 of total specific orthogroups), (iv) transporter proteins (15 of total specific orthogroups), and (v) unclassified functions (49 of total specific orthogroups) (Supplementary Data Table S4). Among these, we focused on the protein-coding genes related to metabolism, which are likely critical to the species’ ecological role and metabolic adaptability.

One of the most notable findings was the discovery of the malonate decarboxylase (mdc) gene cluster, a unique genetic feature present exclusively in Lp. pentosus and absent in both Lp. plantarum and Lp. paraplantarum. The mdc gene cluster in Lp. pentosus TBRC 20328 consists of five distinct subunits: alpha (α), beta (β), gamma (γ), delta (δ), and epsilon (ε), which work together to form a functional enzymatic complex responsible for malonate decarboxylation, a process critical for carbon metabolism. In addition, we identified a LysR-family transcriptional regulator (LTTR) associated with the mdc operon. This regulator plays a crucial role in activating transcription of the gene cluster, a mechanism previously described for Lp. pentosus strain KCA1 [89], corresponding to tightly regulated expression of the malonate decarboxylase system in Lp. pentosus [90,91].

Malonate decarboxylase catalyzes the decarboxylation of malonate to acetate and CO₂, a reaction that is driven cyclically by acetyl-CoA. The α, β, and γ subunits are central to this process. In contrast, the δ and ε subunits function as an acyl-carrier protein (ACP) and a malonyl-CoA processing protein, respectively [92,93]. Our findings suggest that Lp. pentosus TBRC 20328 might be capable of utilizing malonate as a sole carbon source for growth and metabolic energy by incorporating the malonate decarboxylase system as a key component of its metabolic pathways, similar to several bacterial species [93].

Additionally, we identified four genes involved in the leucine biosynthesis pathway in Lp. pentosus TBRC 20328, including 2-isopropylmalate synthase (leuA), 3-isopropylmalate dehydrogenase (leuB), 3-isopropylmalate dehydratase large subunit (leuC), and 3-isopropylmalate dehydratase small subunit (leuD). These enzymes convert precursor molecules into leucine, an essential amino acid. These genes were found to be present exclusively in Lp. pentosus, which is consistent with the previous reports on other Lp. pentosus strains, including IG1 [94] and KCA1 [89], suggesting that there is a conserved mechanism for leucine biosynthesis in Lp. pentosus that might offer metabolic advantages related to amino acid production and ecological adaptability.

Furthermore, we identified an additional copy of the acetyl-CoA carboxylase (ACC) in Lp. pentosus compared to Lp. plantarum and Lp. paraplantarum. This extra gene copy included two components: the acetyl-CoA carboxylase biotin carboxyl carrier protein subunit (BCCP) and the acetyl-CoA carboxylase biotin carboxylase subunit (BC). ACCs are key enzymes that catalyze the conversion of acetyl-CoA to malonyl-CoA, a crucial intermediate in fatty acid biosynthesis and autotrophic carbon fixation. This process occurs in two steps: First, biotin is carboxylated in an ATP-dependent reaction catalyzed by the biotin carboxylase subunit of ACC. In the second step, the carboxyl transferase subunit of ACC transfers bicarbonate to acetyl-CoA, forming malonyl-CoA [95]. The additional ACC gene copy found in Lp. pentosus indicated that such metabolic evolution has advantages for growth and survival adaptation, particularly in lipid synthesis and energy metabolism processes.

Finally, our comparative genomic analysis revealed that putative genes associated with the γ-glutamyl cycle were uniquely present in Lp. pentosus but absent in Lp. plantarum and Lp. paraplantarum. 5-Oxoprolinase catalyzes the ATP-dependent conversion of 5-oxoproline to L-glutamate, a crucial step in the γ-glutamyl cycle, typically found in eukaryotes [96]. Although most prokaryotes lack homologs for this enzyme and do not exhibit the γ-glutamyl cycle [97], the previous studies linked prokaryotic 5-oxoprolinase to a conserved gene cluster comprising pxpA, pxpB, and pxpC. In this study, we identified only two conserved genes in the 5-oxoprolinase cluster of Lp. pentosus TBRC 20328, including the gene encoding 5-oxoprolinase subunit B (pxpB) and a biotin-dependent carboxyltransferase family protein (pxpC). These findings suggest that a unique gene composition of the 5-oxoprolinase cluster existed in Lp. pentosus, and further experimental investigation is required to elucidate the functional roles of subunits B and C in this species. A summary of the species-specific pathways of Lp. pentosus is illustrated in Figure 6.

4. Conclusions

This study presents comprehensive genomic insights into the safety and functional potential of Lp. pentosus TBRC 20328, supporting its application in food and biotechnological industries. The absence of antimicrobial resistance genes, virulence factors, and other undesirable genetic elements confirms its compliance with international safety standards when used under recommended conditions. The presence of genes encoding bacteriocins and PKS underscores its potential for the development of natural preservatives, functional food ingredients, and probiotic formulations. Comparative genomic analysis also revealed a metabolically versatile profile, including pathways for sugar transport, malonate decarboxylation, and leucine biosynthesis traits that contribute to its adaptability and efficiency in diverse fermentation environments. These characteristics make Lp. pentosus TBRC 20328 a promising candidate for large-scale probiotic production and the creation of innovative dietary supplements. Overall, this work provides a strong foundation for future omics- and AI-guided studies and highlights the strain’s potential as a chassis for genetic engineering and precision fermentation in industrial applications.