Functional genomic assessment in comparison to other strains
The L. brevis strain DY55bre genome incorporated 2,791 genes, 2,699 protein-coding sequences, 82 tRNA, three 5S rRNA, two 16S rRNA, two 23S rRNA, three non-coding RNA, four CRISPR array regions, and 155 pseudogenes on a circular chromosome of 2.485.670bp with a 45.72% GC ratio. For the genome set examined in the present study, the GC percent ranged from 45.26 to 46.06. Table S1 summarizes the descriptive features of additional L. brevis genomes utilized in this research for comparison. The BLAST ring alignment of all L. brevis genomes, including DY55bre, was depicted in Fig. 1, where variations between them can be recognized easily due to gaps in the genomes. The detection of genomic islands among these gaps is achievable due to the associated drop in GC content and the absence of similar islands in other strains. In general, integrases, insertion sequences, and transposases are found in and/or around these islands, indicating the presence of possibly horizontally transmitted genes (Yetiman, et al., 2023). Nucleotide statistics, including cumulative GC skew, GC% content, tetranucleotide frequencies, or codon use, provide more evidence of genomic islands (Juhas, et al., 2009, Mann and Chen, 2010). Additionally, lactobacilli are generally regarded as low-GC organisms (Brandt, et al., 2020), and it was reported in a study by Brandt and Barrangou (2018) that the mean GC content of lactobacilli was approximately 40.70%. It is possible to infer from these findings that L. brevis has encountered less genomic drift.
The findings of the FastANI comparison between the DY55bre genome and the other genomes that were analyzed are shown in Fig S1. The determination of whether the genomes of the two species are regarded as identical relies on the ANI value surpassing 96% (Yetiman, et al., 2022). The genome of L. brevis DY55bre has been observed to be similar to the genomes of KB290 (99.934%), NSMJ23 (99.038%), ATCC 367 (98.983%), and AG48 (98.862%), which were derived from commercial probiotic(fermented vegetable), makgeolli, silage fermentation, and sheep rumen, respectively (Fig S1). The genetic divergence or similarities that exist among the DY55bre genome and the above-mentioned genomes can be attributed to variations or resemblances in the strain's ecological niche, evolutionary trajectories, and the environmental circumstances that have been subjected (Duar, et al., 2017). On the other hand, through the hierarchical clustering of functions of clusters of orthologous groups (COG) of 49 distinct L. brevis genomes found on IMG/M (JGI), the DY55bre has been a stronger correlation with KMB620 (0.98), NCTC 13386 (0.96), BDGP6 (0.95), LMT 1–73 (0.95), NBRC3345 (0.95), and NCTC 13768 (0.94). The isolates KMB 620, BDGP6, LMT 1–73, and NBRC 3345 have been reported as cheese, fruit fly, kimchi, and green olive originated in the IMG/M database, respectively (Fig. 2). The origin of the remaining strains has been unknown. Lactobacilli that are considered 'allochthonous' originate from a different location and haven't any ecological or evolutionary connection to the habitat where they are found, unlike 'autochthonous' species (Tannock, 2004, Walter, 2008). However, The observed association and differences among the aforementioned different strains can be attributed to the free-living lifestyle of L. brevis, which facilitates the development of adaptive evolution in various ecological niches (Duar, et al., 2017). Furthermore, lactobacilli can utilize host animals and insects as vectors or transient shelters for dispersal into diverse habitats (Yetiman, et al., 2023). This also validates the liberated lifestyle of L. brevis.
Pangenome analysis has been performed on the aforementioned set of genomes to estimate the complete gene pool of L. brevis. The Pangenome analysis yielded a total of 4610 estimated genes (pangenome), which consisted of singletons, partials(flexible), and core-genome (Fig. 3. and 4.). Based on the results of pangenome and phylogenetic pangenome clustering analysis of the target genome set in this study, it has been observed that the number of pangenomes tends to increase depending on the ascending genome number (Fig. 3A). Furthermore, the size of the pangenome could keep growing indefinitely as new genomes are added. Thus, this pangenome can be classified as open (Bosi, et al., 2015). The number of singletons, representing genes with no sequence homology to genes in other genomes, ranges between 443 and 571, depending on the specific genome depicted in the flower plot (Fig. 3C). Partial genes are sets of orthologs that exist in multiple genomes, but not in all of them. It has been evident that the number of genomes analyzed decreases, resulting in a corresponding decrease in their number (Fig S2). The core genome set, consisting of 1765 genes, has been found in all genomes that were analyzed in this study. Many studies have demonstrated an inverse relationship between the number of core genomes and the overall number of pangenomes, indicating that as the genome count increases, the number of core genomes decreases (Domingo-Sananes and McInerney, 2021). Findings of this study also reveal a comparable pattern (Fig. 3A and B). Core genomes are frequently linked with primary metabolic processes and housekeeping functions. They can also have genes that distinguish the species from other species in the genus (Mosquera-Rendon, et al., 2016).
On the other hand, the number of clusters of orthologous groups (COG) of protein-coding sequences has been determined for each genome in the studied genome set and is shown in Table S2. The total number of annotated COGs ranges from 1760 to 2611. The total number of annotated COGs for DY55bre has been detected as 2360. Remarkably, The number of COGs for general function prediction only genes (R: 229.83 ± 39.82), transcription-related genes (K: 217.83 ± 31.55), carbohydrate transport and metabolism (G: 189.17 ± 19.32), amino acid transport and metabolism (E: 166.78 ± 22.57), function unknown genes (S:155.22 ± 24.62), inorganic ion transport and metabolism (P: 122.39 ± 26.22), cell wall/membrane/envelope biogenesis processes (M: 122.22 ± 15.31), replication, recombination and repair mechanisms (L: 105.50 ± 13.33) and mobilome: prophages, transposons (X: 75.11 ± 51.26) were shown to have fluctuations among the genomes studied in this study (Fig. 4). The numbers for aforecited COG categories for DY55bre can be sorted as follows: R:249, K:221, J:204, G:195, E:176, S:168, P:140, M:126, L:115, and X:51. In contrast to the aforementioned categories, the remaining COG categories exhibit not much variation when comparing the genomes (Table S2). Variations in COG categories might result from the species' adaptation and defense mechanisms in response to their environment. Lactobacilli are commonly believed to adapt to their environment by either losing genes or acquiring essential genes through horizontal gene transfer during the coevolution of lactic acid bacteria with the environment in which they inhabit (Makarova, et al., 2006, Makarova and Koonin, 2007).
CRISPR structures are extensively found in prokaryotes, serving as an adaptive immune system versus foreign invasive DNA (Barrangou and Marraffini, 2014). Especially for lactic acid bacteria, it is possible to engineer novel strains with better functional features using CRISPR-Cas systems and associated molecular machines, either natively or externally (Roberts and Barrangou, 2020). All genomes in the studied dataset have CRISPR spacers detected by the CRISPR structure screening. Cas genes were found in only three genomes: DY55bre, AG48, and EW. Based on previous studies, L.brevis strains generally tend to have more than one CAS-system and most common CAS-systems are Type-II variants (Panahi, et al., 2022, Goh, et al., 2023). Unlike prior research, The DY55bre genome harbors only one CAS system which is Type-1E. The components of this system are the type signature Cas3 protein, the Type 1-E cascade complex consisting of Cas6e, Cse1, Cse2, Cas5, and Cas7, as well as the spacer-acquisition machinery proteins Cas1 and Cas2. Type 1E CAS systems are commonly associated with viral defense mechanisms (Makarova, et al., 2020), but they have also been used in rare cases for strain engineering attempts to acquire functional traits (Hidalgo-Cantabrana, et al., 2019). On the other side, The DY55bre strain contains a total of 42 CRISPR spacers, Several of those have been found on different L. brevis genomes, including: AG48(25), ZLB004(31), KB290(1), and ATCC367 (10). Based on these findings, DY55bre has the potential to be exploited in strain engineering studies via its natural CAS system.
Prophages, related horizontal gene transfer, mobilome
In this study, a total of 78 phage areas have been detected in the aforecited genome set by phage scan results (Table S3). Out of them, 24 regions were found to be intact, 8 regions were considered questionable, and 46 regions were incomplete. The average GC% value of all analyzed phage regions was calculated to be 42.59 ± 1.96%, while the average GC% value of the phages of DY55bre was found to be 44.86 ± 1.36%. The GC% content of the phages has been below the typical GC% content of the bacterial genome (Yetiman, et al., 2023). The phage region that has been spotted most frequently among all detected phages has emerged as Lactob_LBR48_NC_027990. The total count of this specific region found in the identified phages has been 25, with 14 being intact, 4 being questionable, and 7 being incomplete. DY55bre also possesses the Lactob_LBR48_NC_027990, however, its state has been declared incomplete and its score has been determined to be 20 via PHASTER annotation (Table S3). The second most frequently observed phage site among the found phages has been Lactob_Lb_NC_047983, which is composed of 6 intact, 2 questionable, and 4 incomplete regions. This phage region has not been detected in the genome of DY55bre. The third most frequently detected phage site has been identified as Entero_vB_EfaS_AL2_NC_042127, which consists of 4 incomplete and 2 questionable phages. DY55bre has not possess this phage region in its genome. Out of all the identified intact phage regions, only the NSMJ23 strain possesses an endolysin-containing phage area known as Lactob_Lb_NC_047983. Endolysins are phage proteins that efficiently degrade the bacterial cell wall, facilitating the release of new virus particles (Cahill and Young, 2019). Therefore, with the exception of NSMJ23, the entire intact phages that have been found are temperate for their bacterial genomes. Furthermore, it has been observed that the phage genomes of AG48, KB290, UCCLBBS124, UCCLB95, EW, BDGP6, ATCC_367, DSM 20054, ZLB004, TMW 1,2113, TMW 1,2113, UCCLBBS455, KB290, AG48, and EW contain intact phages that possess integrase (Table S3). The presence of the integrase in a bacterial genome typically serves as a functional indicator for pathogenicity islands, phages, and integrative plasmids (Juhas, et al., 2009). Besides, phage integrases are enzymes that promote one-way, targeted recombination between two specified DNA sequences: the phage attachment site and the bacterial attachment site (Groth and Calos, 2004). Specifically, DY55bre has 1 intact, 1 questionable, and 2 incomplete phage regions, that can be sorted as Escher_RCS47, Entero_vB_EfaS_AL2, Lactob_LBR48, and Strept_T12, resceptively (Table S3). On the other hand, the overall number of matching proteins discovered in phages of DY55bre is much lower compared to other members of the genome set in this research. DY55 bre also possesses endolysin-containing Lactob_LBR48, although the status of this phage has been incomplete since it lacks integrase, attachment sites, and most of the members of coat/tail/fiber/plate/portal proteins. The intact phage Escher_RCS47_NC_042128 has 18 coding sequences, with 10 of them being transposases. The other coding sequences include a putative baseplate hub protein, a putative resolvase, a putative UDP-galactopyranose mutase, and several hypothetical proteins. Strangely, UDP-galactopyranose mutase (UGM) is a crucial flavoenzyme involved in the production of galactofuranose. It is a prominent constituent of the cell wall and cell surface glycoconjugates found among various bacteria (Tanner, et al., 2014). It appears probable that CRISPR structures affect suppressing phage activity on the DY55bre genome.
Horizontal gene transfer (HGT) is typically done in bacteria via three separate methods. First, Transformation during which extracellular DNA from the environment is taken up and expressed by cells. Transduction occurs when a virus or bacteriophage inserts its genetic material into the genome of a host cell, whereas conjugation occurs when a gene present on a mobile genetic element (plasmid or conjugative transposon) is transferred to another cell via direct physical contact. Bacterial conjugation is the most prevalent of these activities and hence provides the most HGT markers in prokaryotes (Kleerebezem, et al., 2003, Toomey, et al., 2009). If the gene acquired horizontally is preserved and spread across a bacterial community, it is expected to provide a favorable benefit to the host species (Kurland, et al., 2003). In this study, protein BLAST analysis revealed that 0.481% (13 genes) of the DY55bre genome has been acquired through horizontal gene transfer (HGT) from phage-related proteins (Table S4). In particular, those genes have been gained from various bacteria, including Secundilactobacillus collinoides, Lentilactobacillus buchneri, Levilactobacillus parabrevis, Lentilactobacillus parabuchneri, Lactiplantibacillus plantarum, Lactobacillus delbrueckii subsp. bulgaricus, Pediococcus damnosus, Latilactobacillus sakei, Lentilactobacillus diolivorans, Pediococcus pentosaceus, and Limosilactobacillus fermentum. Furthermore, a total of 10 transposase sequences have been found in the only intact phage of DY55bre (Escher_RCS47_NC_042128). These sequences are as follows: PP_02477, PP_02478, PP_02482, PP_02484, PP_02487, PP_02489, PP_02490, PP_02492, PP_02493, and PP_02494. Besides, the search for transposases based on the whole genome has yielded the identification of individuals belonging to the ISL3, IS3, IS256, IS5, IS30, IS982, and IS3 families. These findings were obtained using IS Finder and are shown in Table S5. The transposase protein facilitates the process of transposition, which involves the removal or duplication of an IS from one region in the genome and its subsequent insertion at a different location (Vigil-Stenman, et al., 2017). The prevalence of transposase genes in prokaryotes exhibits significant variability, spanning from absence to more than a thousand copies per genome (Vigil-Stenman, et al., 2015). Transposases also migrate across genomes via horizontal gene transfer (HGT) routes (including prophages), facilitating the transmission of functional genes known as transposons. Therefore, they serve as effective mechanisms for adaptation, which accounts for their prevalence among prokaryotes that live in challenging or harsh environments (Frost, et al., 2005, Li, et al., 2014).
Safety-related genes assessment
Resistome screening has been conducted utilizing the CARD, BV-BRC, and KEGG databases. Pursuant to the results derived from the CARD database, no antibiotic-resistance genes have been detected in the genome of DY55bre. However, a total of forty-six genes associated with antibiotic resistance were identified utilizing the KEGG and BV-BRC databases. The observed genes have been connected with drug targets (16), vancomycin (6), beta-lactams (9), Nitroimidazole (1), streptomycin (1), macrolides and lincosamides (1), triclosan (1), cationic antimicrobial peptides (7), antibiotics that modify cell wall charge (2), and multidrug efflux pumps (1). At first glance, antibiotic target genes and their drugs have garnered significant interest and can be sorted as follows: Alr(D-cycloserine), Ddl(D-cycloserine), EF-G(fusidic acid), EF-Tu(kirromycin, enacyloxin IIa, pulvomycin), folA(trimethoprim, brodimorphim, tetroxoprim, iclaprim), gyrA(clofazimine, ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, nalidixic acid, ofloxacin, sparfloxacin, trovafloxacin), gyrB(clofazimine, gatifloxacin, ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, ofloxacin, sparfloxacin, novobiocin, coumermycin A1, clorobiocin, coumermycin, trovafloxacin), Iso-tRNA(mupirocin), inhA(isoniazid, ethionamide, triclosan), kasA(isoniazid, triclosan), MurA(fosfomycin), rpoB(rifamycin, daptomycin, rifabutin, rifampin), rpoC(daptomycin), S10p(tetracycline, tigecycline), and S12p(streptomycin). Likewise, the vancomycin-related resistance genes were identified as vanX, murG, murF, mraY, ddl, and alr, based on the KEGG annotation results. Lactobacillus species are widely recognized for their resistance to vancomycin due to the presence of intrinsic peptidoglycan precursors that contain D-lactate instead of D-alanine at the C-terminus. In addition, the VanX gene exhibits a high degree of specificity in its ability to hydrolyze D-alanine-D-alanine dipeptides. This gene also plays a crucial role as a precursor in the formation of the cell wall (Yetiman, et al., 2022, Yetiman, et al., 2023). In addition, the gidB gene, which is responsible for streptomycin resistance, has been identified. It generates the enzyme 7-methylguanosine methyltransferase, which specifically targets the 16S rRNA. Genetic mutations in this specific gene have been reported to be the source of resistance to streptomycin and other aminoglycosides (Okamoto, et al., 2007, Alcock, et al., 2023). Apart from this, a nitroimidazole class antibiotic resistance gene of NimB has been identified, which is responsible for the synthesis of nitroimidazole reductase enzyme. This enzyme deactivates antibiotics of the nitroimidazole group, such as metronidazole (Theron, et al., 2004). The RlmA(II) gene has been identified for resistance to mycinamicin, tylosin, and lincosamides. It has been reported that this resistance is caused by the methylation of the N1-position of nucleotide G748 in the 23s rRNA (Douthwaite, et al., 2008). Further, a bacterial 3-oxoacyl enoyl-acyl carrier reductase enzyme encoding the fabV gene has been detected, which confers resistance to the biocide triclosan (Zhu, et al., 2010). Meanwhile, the existence of the ykkCD gene has been verified, which encodes a multidrug efflux pump including two subunits of SMR (small multidrug resistance)-type proteins. It exhibits resistance to cationic medicines, namely chloramphenicol, streptomycin, and tetracycline (Jack, et al., 2000). The existence of cell wall charge-altering protein encoding GdpD, MprF, and PgsA genes, which provides antibiotic resistance against daptomycin and defensin, has been verified via BV-BRC (Friedman, et al., 2006, Yang, et al., 2009). Notably, we have detected the presence of genes encoding elements of the oligopeptide transport system associated with beta-Lactam resistance (oppA, oppB, oppC, oppD, oppF, and AbcA), as well as genes expressing penicillin-binding protein 1A (mrcA) and 2A (pbp2A), and beta-lactamase class A (PenP). Recently, various researchers have reported the occurrence of Penicillin G resistance and have confirmed the availability of the previously mentioned resistome associated with beta-lactams in certain strains of Lactiplantibacillus plantarum, Limosilactobacillus reuteri, and Lacticaseibacillus rhamnosus (Abriouel, et al., 2015, Zheng, et al., 2017, Yetiman, et al., 2022). These findings further support the results of this study. The existence of transferable antimicrobial resistance genes in probiotics, which are used for humans or animals, is undesirable due to concerns about different food niches and common bacteria that could potentially serve as reservoirs for the resistome. Lactobacilli possess inherent resistance to many antimicrobial substances, and it is widely recognized that this resistance is not associated with any specific safety issues. However, the inherent resistome on the bacterial chromosome mustn't be flanked by transposases or integrases. The protein BLAST analysis performed in this work did not identify any horizontally transferred genes in the target resistome.
Carbohydrate metabolism
L. brevis obtains ATP via engaging in heterofermentative carbohydrate fermentation, which is dependent on the strain's particular affinity for sugars (Buron-Moles, et al., 2019). Identifying strain-specific metabolic characteristics is crucial for predicting their potential industrial application and probiotic benefits. L. brevis has an adaptive nature and is capable of fermenting a variety of carbohydrates. This status is directly associated with the availability of the genes responsible for transporters and CAZYmes. The majority of transporters involved in carbohydrate metabolism are located inside the phosphoenolpyruvate-dependent sugar phosphotransferase system(PTS) (Ganzle and Follador, 2012). The genome of strain DY55bre possesses the majority of phosphotransferase system (PTS) enzymes. This includes the PTS enzyme I (ptsI), the gene for the phosphocarrier protein HPr (ptsH, also known as a proton carrier), the gene for the fructose transporter-specific multiphosphoryl transfer protein (fruB, also known as a proton carrier), and the gene for fructose PTS system (fruA) and other substrate-specific transporter complexes which are summarized in Table S6. In addition, carbohydrate-specific ABC transporters were given in the same table. The KEGG annotation results suggest that the remaining carrier complexes are selective for various carbohydrates and sugar molecules, including maltose, maltodextrin, glucose, mannose, galactose oligomers, maltooligosaccharides, arabinooligosaccharides, raffinose, stachyose, melibiose, sorbitol, mannitol, alpha-glucoside, cellobiose, chitobiose, ribose, D-xylose, diaceytlchitobiose, and L-ascorbate. Nevertheless, it is well recognized that several sugar transporters can import more than one substrate (Kleerebezem, et al., 2003). Predicted carbohydrate metabolism related enzymes have been presented in Table S7.
Heterofermentative species have been frequently equipped with the araBAD operon, which includes L-ribulokinase (araB), L-arabinose isomerase (araA), and L-ribulose-5P-4-epimerase (araD), and has been associated with the breakdown of L-arabinose, a critical step in heterofermentation (Buron-Moles, et al., 2019). Similarly, the araBAD operon has been found in the DY55bre genome. Additionally, the DY55bre genome is missing 1-phosphofructokinase (pfK) and fructose-1,6-bisphosphate aldolase enzymes, hence it is unable to use hexoses via the EMP pathway due to the absence of the aforementioned enzymes. Instead, it breaks down the hexoses via the phosphogluconate pathway (Fig. 5), producing not only lactic acid but also considerable amounts of ethanol, acetate, and CO2 (Pessione, 2012, Salvetti, et al., 2018). This finding has confirmed that DY55bre has an obligatory heterofermentative metabolism. Moreover, Transketolase (tktA), phosphoketolase (xpf), and glucose-6P-isomerase (pgi) genes have also been existed in its genome, as previously reported for L. fermentum (Yetiman, et al., 2023). The presence of enzymes that degrade sucrose, maltose, and glucose, namely alpha-glucosidase, sucrose alpha-glucohydrolase, and beta-glucosidase, has been verified. Even though DY55bre is a plant-derived L. brevis strain, the presence of lacZ, galE, galM, galK, galU, lacA, galT, malZ, pgm, glf, galA, and glk genes of galactose metabolism was observed in its genome (Table S5). This might be attributed to the free-living and obligatory heterofermentative lifestyle of L.brevis (Duar, et al., 2017, Buron-Moles, et al., 2019). The identification of xylose isomerase (xylA) and xylulokinase (xylB) genes, responsible for catalyzing the conversion of xylose into xylulose-5P before entering the glycolysis as glyceraldehyde-3P, has also been fulfilled.
DY55bre is made up of 24 distinct genes encoding pyruvate metabolism enzymes, some of which have multiple copies, such as alcohol dehydrogenase (adh; 2) and acetate kinase (ackA;3). At first glance, the presence of pyruvate kinase (pyk), an enzyme responsible for the conversion of phosphoenolpyruvate to pyruvate, grabbed notice as well. Through the L-lactate dehydrogenase (ldh) and D-lactate dehydrogenase (ldhA) genes, DY55bre is capable of producing D- and L-isomers of lactic acid from pyruvate. In addition, it contains lactate racemase (larA), an enzyme responsible for the reversible conversion of D- and L- enantiomers of lactate. The malolactic enzyme, which is also part of pyruvate metabolism, catalyzes the conversion of malate into lactate and CO2. Apart from that, the existence of two copies of alcohol dehydrogenase (adh) and two copies of propanol-preferring alcohol dehydrogenase (adhP) on the DY55bre genome is noteworthy. The adh gene catalyzes the conversion of acetaldehyde into ethanol, while adhP is capable of reversibly producing aldehydes from primary alcohols (Thomas, et al., 2013). During the conversion of pyruvate to ethanol, pyruvate is first transformed into acetyl-CoA by the TCA cycle. Subsequently, acetyl-CoA is turned into acetaldehyde, and ultimately, acetaldehyde is further converted into ethanol by the enzymes acetaldehyde dehydrogenase(adhE) and alcohol dehydrogenase (adh), respectively. As an alternative, pyruvate can also be converted into acetyl phosphate and then acetate by the action of pyruvate oxidase (poxL) and acetate kinase (ackA), respectively, in order to synthesize ATP.
Following glycolysis, pyruvate is transformed to (S)-2-acetolactate and subsequently to diacetyl and acetoin by the enzymes acetolactate synthase (ilvB), acetolactate decarboxylase (alsD), and diacetyl reductase (budC) (Garcia-Quintans, et al., 2008). Subsequently, both enantiomers of (S)-2-acetoin and (R)-2-acetoin may be transformed into (S,S)-butane-2,3-diol or (R,R)-butane-2,3-diol, respectively, by the reactions of butanediol dehydrogenase (butA) (Takusagawa, et al., 2001). Except from those, it has been revealed that there is a deficiency in the members of the pdu operon, which is responsible for the metabolic conversion of glycerol into 1,2-propanediol, pronanal, 1-propanol, or propanoate. The enzymes involved in the conversion of glycerol to glycerone-P and then to methylglyoxal were not detected. But, enzymes like glycerol dehydrogenase and B12-dependent propanediol dehydratase (pduCDE) are available; the former converts hydroxyacetone to 1,2-propanediol, and the latter transforms 1,2-propanediol to propanal (Morita, et al., 2008). Surprisingly, the cbi operon (cobalamin, B12-biosynthesis), which is crucial for pduCDE functioning, has been absent from the DY55bre genome. Conversely, in the presence of exogenous cobalamin, the strain can synthesize 1-propanol from propanol via 1-propanol dehydrogenase (pduQ) (Bobik, et al., 1999, Dishisha, et al., 2014).
As determined by KEGG mapper reports, the DY55bre genome encodes a total of 190 carbohydrate metabolism-related genes. These genes are distributed as follows; 14 starch and sucrose metabolism genes, 9 fructose and mannose metabolism genes, 12 galactose metabolism genes, 19 pentose phosphate pathway genes, 17 pentose, and glucuronate interconversions associated genes, 19 glycolysis-related genes, 24 amino sugar, and nucleotide sugar metabolism genes, 7 ascorbate and aldarate metabolism genes, 24 pyruvate metabolism genes, 5 TCA cycle-related genes, 10 glyoxylate, and dicarboxylate metabolism genes, 16 propanoate metabolism genes, 10 butanoate metabolism genes, 2 inositol phosphate metabolism genes, and 2 C5-branched dibasic acid metabolism genes. Furthermore, 47 glycoside hydrolases, 37 glycosyltransferases, 12 carbohydrate-binding modules, 1 auxiliary activity, and 2 carbohydrate esterases encoded in the DY55bre genome were found as a result of DBCAN3 research. In summary, evaluation results revealed that DY55bre not only has the potential to produce industrially important plate-from chemicals like lactic acid, acetic acid, ethanol, 1-propanol, propanal, and butane-2,3-diol, but also some food grade aroma compounds such as acetaldehyde, acetoin, and diacetyl as an outcome of carbohydrate metabolism.
Aminoacid metabolism
Aminoacid metabolism of Lactobacilli has attracted substantial interest due to implications on food safety and quality since amino acids are precursors of compounds that considerably contribute to the specific flavor of fermented foods (Wang, et al., 2021). On the other side, amino acids are associated with many markets. In order to meet the demand for human and animal nutrition, food and feed amino acids like L-glutamate and L-lysine have been produced on an annual million-ton scale (Wendisch, 2020). Therefore, revealing the metabolic characteristics of a newly isolated Lactobacilli strain is critical for potential strain engineering studies. The putative model of amino acid metabolism has been constructed and depicted in Fig. 7 based on KEGG metabolic pathways. The genome of DY55bre has encoded ABC transporter proteins that are responsible for Aspartate, Glutamate, Glutamine, Methionine, L-cysteine, and oligopeptides transport, as shown in Fig. 9. Predicted amino acid metabolism related transporters and enzymes have been displayed in Table S8 and S9.
L-alanine can be synthesized from pyruvate and has been employed in bacterial D-aminoacid metabolism. L-alanine has been converted to D-steroisomer by activity of alanine racemase (alr) and then used for peptidoglycan synthesis (Feng, et al., 2002). D-alanine can also be converted into D-alanyl-poly-ribitol-P for lipoteichoic acid synthesis via the D-alanine-poly(phosphoribitol) ligase (dltA/B) (Perego, et al., 1995). L-aspartate can be acquired extrinsically or metabolized reversibly to L-asparagine with aspartate-ammonia ligase (asnA) and asparagine synthase (asnB) enzymes (Sugiyama, et al., 1992, Larsen, et al., 1999). Because of the presence of the aspartate racemase (racD) gene, it is possible to conversion of the L-aspartate to D-stereoisomer (Yamashita, et al., 2004). If required, the enzymes of the TCA cycle can convert L-aspartate to S-malate. The fate of L-glutamate differs depending on cell requirements or growth phase. L-glutamate also has been used for the construction of peptidoglycan cell wall component synthesis as UDP-MurNAc-L-Ala-D-Glu. Differently, L-glutamate has been produced intrinsically from N-Acetyl-L-glutamine or L-glutamine. Similarly, L-glutamine has been generated from L-glutamate or it can be obtained from the exterior. Besides, L-Glutamine can be metabolized to G-glucosamine-6P for utilization in Aminosugar and nucleotide sugar metabolism. DY55bre has the capacity to produce 4-aminobutanoate (GABA) from L-glutamate by using the Glutamate decarboxylase enzyme. GABA has various physiological roles, including acting as a primary inhibitory neurotransmitter that sends chemical messages in mammalian central nervous systems and playing an important role in stress response, cognition, and behavior (Yetiman, et al., 2023). Moreover, the supplementation of GABA has a beneficial impact on various conditions such as depression, insomnia, anxiety, blood pressure management, immune function, visual cortex function, and menopausal syndrome (Okada, et al., 2000, Abdou, et al., 2006).
Glycine biosynthesis in DY55bre has been performed by the conversion of L-serine that originated from Methane metabolism. Glycine and serine can be converted into each other reversibly depending on the needs of the cell. Conversely, Proline biosynthesis has been conducted through the degradation of peptides by the reaction of proline iminopeptidase(pip) (Zhang, et al., 2012). Meanwhile, the presence of carbamoyl-P and L-ornithine is required for arginine production. L-ornithine is generally derived from D-aminoacid metabolism. Carbamoyl-P can be obtained in two ways: it could be obtained by L-glutamine degradation or through carbamate kinase (arcC) enzyme catalysis (Durbecq, et al., 1997). Citruline was then synthesized using the ornithine carbamoyltransferase (argF) enzyme and carbamoyl-P and L-ornithine. Arginine has been created from citrulline reversibly by the action of arginine deiminase (arcA), depending on the metabolic requirements of the cell (Ruepp and Soppa, 1996). Arginine supplementation in humans has an important effect on the health and function of cardiac and muscular tissues, as well as on exercise capacity maintenance and stimulation. However, citrulline can also be converted to arginine in various cell types as aforecited, and can effectively restore arginine and nitric oxide shortages in both pathological and healthy situations. In addition, citrulline has a significant role in the elimination of ammonia, resulting in less fatigue and tension and enhanced exercise performance. When combined, arginine and citrulline create an essential component for boosting and improving human sports performance and exercise (Allerton, et al., 2018, Park, et al., 2023).
Methionine has been obtained from the exterior or produced intrinsically from L-methionie-S-oxide or S-adenosyl-L-methionine. Conversely, S-adenosyl-L-methionine (SAM) has also been generated by the reaction of the S-adenosylmethionine synthetase (metK) enzyme(Horikawa and Tsukada, 1991). SAM is a molecule that has therapeutic significance (especially for liver disease) and plays a role in various biological activities. It serves as a precursor for the production of glutathione and acts as a primary methyl donor for the methylation of nucleic acids, phospholipids, histones, biogenic amines, and proteins(Anstee and Day, 2012). Although DY55bre cannot generate L-cysteine, it can metabolize itself to thiosulphate, pyruvate, and ammonia through the catalysis of L-cystine thiocysteine-lyase (CCBL). Furthermore, L-cysteine degradation products or L-cysteine itself have been employed in glutathione metabolism for antioxidant defense. Overall, assessment results revealed that the DY55bre strain has a potential for L-asparagine, L-aspartate, L-Glutamine, L-glutamate, S-Adenosyl-L-methionine, Citruline, Arginine, Serine, Glycine and GABA. Genome editing techniques can be employed to enhance the manufacturing capacity of certain amino acids if needed. These findings further demonstrate that DY55bre exhibits an adaptive character to the competitive and harsh environment of the gastrointestinal tract.
Probiogenomic characterization
The adaptation of probiotic lactobacilli strains to the gastrointestinal tract is regulated by a large number of genes that encode proteins involved in response to stress. These stress responses include temperature, pH, bile, osmotic pressure, and oxidative stress. To determine its probiotic capabilities at genomic levels, the genome of DY55bre has been screened for the determination of a variety of genes linked to probiotic properties, including stress resistance, bile salt hydrolase activity, adhesion ability, and immunomodulatory activities (Kandasamy, et al., 2022, Yetiman, et al., 2023). Putative probiogenomic traits that exist in the genome of DY55bre have been summarized in Table S10. DY55bre has been found to carry eighteen genes linked to proteins associated with heat shock tolerance, such as the heat shock-related regulator protein (hrcA), molecular chaperones (dnaK, dnaJ, grpE, groEL, groES), and genes encoding proteases (clpB, clpC, clpE, clpP, clpX, hslU, hslV, htpX, lon, mecA, yabO, YidC). These genes have a crucial function in the formation of protein aggregates within cells and the maintenance of membrane integrity, enabling Lactobacillus strains to survive at higher temperatures (Li, et al., 2021). In addition, DY55bre harbors three copies of the cspA gene, which encodes cold shock proteins that are involved in enhancing survival under low-temperature conditions (Serror, et al., 2003). The DY55bre genome had twelve genes related to acidity tolerance. Eight of those genes encode a group of subunits (atp A/B/C/D/E/F/G/H) of the F0F1 ATPase. These subunits play an important role in maintaining the cytoplasm's pH, which improves acid tolerance (Yetiman and Ortakci, 2023). Moreover, the presence of Na+:H + antiporter (nhaC), glutamate: GABA antiporter (gadC), and glutamate decarboxylase chains A (gadA) and B (gadB) has been verified that is responsible for the maintenance of H + and Na + homeostasis (Yetiman, et al., 2023). Several genes have been detected that play key functions in bile salt resistance. These include cbh gene coding cholylglycine hydrolase, ppaC gene coding manganese-dependent-inorganic-pyrophosphatase, and cfa gene coding cyclopropane-fatty-acyl phospholipid synthase. A similar situation for bile resistance was reported for L. petauri LZys1, L. plantarum DJF10, and L. fermentum AGA52 through in vitro and in silico studies (Li, et al., 2021, Kandasamy, et al., 2022, Yetiman, et al., 2023). The adherence of probiotic strains to the host epithelium can be explained by the presence of cell surface proteins. DY55bre genome also has 19 genes that encode adhesion-related putative proteins such as maltose phosphorylase (mapA), lipoprotein-anchoring transpeptidase (efrK/sfrK), elongation factor Tu(ef-tu), triosephosphate isomerase(tpiA), glyceraldehyde 3-phosphate dehydrogenase(gapA), β-galactosidase(lacZ), LPXTG specific sortase A (srtA), isopeptide-forming pilin-related protein(spaA), LPXTG specific collagen adhesin (lmo0159), putative peptidoglycan bound protein (lmo0160), LPXTG motif-containing internalin like proteins (lmo0732 and lmo2396 homologs), tyrosine-protein kinase(epsD), tyrosine-protein kinase transmembrane modulator (epsC), UDP-N-acetylglucosamine–N-acetylmuramyl-(pentapeptide) pyrophosphorylase-undecaprenol N-acetylglucosamine transferase (murG), and enolase. The majority of those genes have been reported to be adhesion-related in previous studies (Sun, et al., 2022, Yetiman, et al., 2023, Yetiman and Ortakci, 2023). In addition to their function in carbohydrate metabolism, the xylose isomerase (xylA) and xylokinase (xylB) genes are involved in gut persistance (Kandasamy, et al., 2022, Yetiman, et al., 2023).
During oxidative stress, which is a major contributor to the development of various diseases, cellular oxidative and antioxidant processes are disrupted by a variety of external as well as internal stimuli such as inflammation or nutrition. Probiotic Lactobacillus strains have been proven to provide a variety of antioxidant, and immunomodulatory properties in the host (Zhao, et al., 2023). DY55bre had 13 genes linked to antioxidant activity, which are consisted of glutathione peroxidase (gpx), glutathione reductase (gsr), glutaredoxin like protein (nrdH), manganese transport protein (mntH), NADH dehydrogenase (ndh), NADH peroxidase (npx), pyruvate oxidase (poxL), Thiol peroxidase (tpx), thioredoxin (trxA), thioredoxin reductase (trxB), Peptide-methionine (S)-S-oxide reductases (msrA and msrB), and Arsenate reductase (arsC). Lactobacilli, having a whole thioredoxin system (tpx, trxA, trxB), can eliminate reactive-oxygen-species (ROS) and reactive-nitrogen-species at greater rates by providing electrons to thiol-dependent peroxidases (Lu and Holmgren, 2014). The glutathione system controls the protein dithiol/disulfide balance to detoxify radicals like hydrogen peroxide and lipid peroxyl (Pophaly, et al., 2012). The NADH oxidase/peroxidase, and pyruvate oxidase(poxL) have a direct role in the breakdown of hydrogen peroxide and ROS (Zhang, et al., 2018). Notably, the functions of msrA and msrB genes are associated with the restoration of ROS-damaged proteins containing oxidized methionine residues(Kandasamy, et al., 2022). It has also been confirmed that DY55bre's genome carries the genes critical to immunomodulatory (dltA, dltB, dltC, dltD, dltX) and anti-pathogenic (LuxS) effects. Based on those findings, it is conceivable to assume that L. brevis DY55bre possesses gut adaptation traits that enable it to endure a broad spectrum of stresses.