Distribution of C30 carotenoid biosynthesis genes suggests habitat adaptation function in insect-adapted and nomadic Lactobacillaceae

Distribution of C30 carotenoid biosynthesis genes suggests habitat adaptation function in insect-adapted and nomadic Lactobacillaceae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Distribution of C30 carotenoid biosynthesis genes suggests habitat adaptation function in insect-adapted and nomadic Lactobacillaceae Sarah Lebeer, Marie Legein, Tom Eilers, Jari Temmermans, Jelle Dillen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4637278/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Carotenoids are membrane-bound pigments that are essential for photosynthesizing plants and algae. These specialized metabolites are widely applied in food, feed and cosmetics because of their antioxidant and anti-inflammatory properties. The production of carotenoids, particularly C30 forms, has also been documented in a few non-photosynthetic prokaryotes. However, our understanding of the function, distribution and ecology of these compounds beyond photosynthesizing organisms is limited. In this study, we performed an eco-evolutionary analysis of terpenoid biosynthetic gene clusters in the Lactobacillaceae family, an important family of beneficial bacteria with diverse lifestyles and habitats. Hereto, 4203 dereplicated genomes were screened for terpenoid biosynthesis genes, resulting in detection of crtMN genes in 28/361 (7.7%) species in 14/34 (41.2%) genera. These genes encode the key enzymes that transform two farnesyl pyrophosphate molecules into the C30 carotenoid 4,4’-diaponeurosporene. These crtMN genes appeared to be convergently gained within Fructilactobacillus , and horizontally transferred across species and genera, e.g. from Lactiplantibacillus to Levilactobacillus . Subsequently, in our in-house Lactobacillaceae culture collection from various habitats (n = 575), the phenotype was confirmed in 87% of the isolates predicted to have the crtMN genes (27/31). Nomadic and insect-adapted species, particularly those isolated from vegetable fermentations, such as Lactiplantibacillus , and floral habitats, such as Fructilactobacillus , were capable of C30 carotenoid biosynthesis, while vertebrate-associated species, such as those from the vagina, lacked this trait. This habitat association aligned with the observations that C30 carotenoid-producing strains were more resistant to oxidative and UV-stress. Taken together, this study revealed that C30 carotenoid biosynthesis plays a role in habitat adaptation and is scattered across Lactobacillaceae in line with this habitat adaptation. Biological sciences/Microbiology/Environmental microbiology Biological sciences/Ecology/Microbial ecology Biological sciences/Microbiology/Microbial genetics/Bacterial genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Carotenoids are membrane-bound specialized metabolites found in all photosynthesizing organisms, such as plants, algae and cyanobacteria. They play vital roles in protecting producer cells against excess light energy and scavenging reactive oxygen species 1–3 . However, these molecules also perform functions that are unrelated to photosynthesis, such as being precursors to phytohormones 4,5 or attracting pollinators and promoting seed dispersal by accumulating in flowers and fruits 6 . Moreover, carotenoids are found in non-photosynthetic organisms, for example in the pathogenic bacterium Staphylococcus aureus , where the carotenoid staphyloxanthin increases virulence by protecting the bacteria against reactive oxygen species and inactivating host neutrophils 7 . In leaf-dwelling non-photosynthetic bacteria, such as Clavibacter , Pseudomonas and Methylobacteria , carotenoids protect bacterial cells and, in some cases, the plant host from UV radiation 8,9 . Animals require but are generally unable to synthesize carotenoids and consequently supplementation depends on food intake, with the notable exception of aphids, which have acquired the ability to produce carotenoids as fungal carotenoid biosynthetic genes have been taken up into their genome 10 . In humans, dietary intake of carotenoids is essential for vitamin A biosynthesis 11 and is associated with a reduction in inflammatory markers 12 and a reduced risk of prostate cancer and breast cancer 13 , although also opposite associations have been found such as for lung cancer 14 . Because mostly positive health benefits have been found, carotenoids are increasingly used as functional ingredients in food and feed, where they can also have additional technological benefits, for instance as natural pigments and antioxidants 1 . The best-studied carotenoids are those found in photosynthetic organisms. These specialized metabolites are mostly C40-carotenoids, which have 40 carbon atoms within their backbone 15 . More recently, the full scope of carotenoid diversity and evolution has gained increasing attention 15,16 . Phylogenetic analyses revealed that carotenoid biosynthesis is an ancient process that evolved prior to photosynthesis, likely under increased UV radiation conditions 16–18 . According to Santana-Molina et al. 19 , the earliest evolved group of carotenoids might be composed of 30 carbon atoms, or triterpenoids. These specialized compounds are found in plants under various forms, such as triterpenoid saponins 20 or oleanolic acids 21 , and they are often involved in the plant’s defense system. However, C30 carotenoid biosynthesis originated in prokaryotes 16,18 and has been detected in the photosynthesizing bacteria Heliobacter 22 , and several nonphotosynthetic bacteria such as Lactiplantibacillus plantarum 23 , Bacillus subtilis 24 , Enterococcus faecium (formerly Streptococcus faecium ) 25 , Staphylococcus aureus 26 and Methylobacterium rhodinum (formerly Pseudomonas rhodos ) 27 . In addition to these phenotypic observations, Santana and colleagues 18 showed that both 4,4’-diapophytoene (synthesis encoded by the crtM gene) as well as squalene (encoded by Sqs or the HpnCDE cluster) can be precursors to C30 carotenoids, and that these pathways are now found scattered across prokaryotes, ranging from Firmicutes to Planctomycetes to Archaea and found to be mobile via horizontal gene transfer. While carotenoid biosynthesis originated in, and is now scattered across nonphotosynthesizing prokaryotes, the function and ecological role of these specialized metabolites in these organisms have not been systematically studied. Within the Firmicutes, Lactobacillaceae are an interesting family of nonphotosynthesizing bacteria, with great potential for use in food, feed and pharma 28,29 . Two phenotypical screenings of Lactobacillaceae , focusing on isolates from fermented foods, identified Lp. plantarum as a C30 carotenoid producer via the 4,4'-diapophytoene pathway 23,30 . However, the genomic and ecological diversity of the biosynthetic and phenotypic potential of this family has not yet been investigated. In this study, an in-depth pangenome and evolutionary analysis of the presence of terpenoid biosynthetic gene clusters was performed on a large dataset of 4203 unique genomes of the Lactobacillaceae family. These genomic analyses were complemented with phenotypic confirmation using a biobank of Lactobacillaceae from various habitats by high-throughput screening based on absorbance maxima and high-performance liquid chromatography (HPLC) and high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF MS). Additionally, the role of C30 carotenoids in UV and oxidative stress resilience was assessed for selected strains. Finally, associations between the presence of carotenoid biosynthesis genes in a species and its lifestyle were established, using assigned lifestyles from literature 31,32 , and new isolation and metabolic analyses from our in-house library of strains. Materials and methods Pangenome and phylogenetic analysis of the terpenoid biosynthetic gene clusters. All publicly available Lactobacillaceae genomes were downloaded from the Genome Taxonomy Database (GTDB, gtdb.ecogenomic.org, version r214). With checkM, any incomplete ( 5%) genomes were excluded 33 . This resulted in a dataset of 6259 public genomes. To avoid pseudoreplication, the sample function of SCARAP (github.com/SWittouck/SCARAP) was used with an average nucleotide identity (ANI) cutoff of 99.99%, retaining only genomes with ANI values below this cutoff. Second, the pan genomes of this family were inferred using the SCARAP tool 34 . As a start, orthogroups of detected genes, i.e.4,4’-diapophytoene desaturase (encoded by the crtN gene) and of 4,4’-diapophytoene synthase (encoded by the crtM gene) were determined (Table 1 ). 33 For reference, the genes from two experimentally confirmed species, Lp. plantarum WCFS1 23 and Latilactobacillus fragifolii A MBP162 (experimentally confirmed in this study), were used as queries. Based on the results of this pangenome analysis, 37 in-house isolates were selected for whole genome sequencing, and were included in the subsequent analysis. Finally, all genomes were checked for other terpene biosynthesis genes using Antismash 6.0 35 focusing on terpenes and terpene pathways. Table 1 References to experimentally confirmed crtMN genes used to identify similar sequences in the pangenome. Strain crtM 4,4’-diapophytoene synthase (882 nt) crtN 4,4’-diapophytoene desaturase (1497 nt) Reference Lactiplantibacillus plantarum WCFS1 NC_004567.2_2720 NC_004567.2_2719 23 Latilactobacillus fragifolii AMBP162 NZ_CAJOCF010000002.1_110 NZ_CAJOCF010000002.1_111 Carotenoid biosynthesis was first observed during the description of the species 36 and further validated in this study The core tree of all Lactobacillaceae was constructed following the method described in Eilers et al. 37 . The core genome was inferred using SCARAP, which consists of 296 core genes. Protein sequences were extracted, aligned with MAFFT and trimmed with trimAL with a gap threshold of 10%. Aligned and trimmed core proteins were subsequently inferred with IQ-TREE using LG + F + G4. First, the prevalence of crtMN genes within all species of the family Lactobacillaceae was calculated and integrated with lifestyle data from Zheng et al ., 2020 32 , and metadata from the GTDB using tidygenomes (github.com/SWittouck/tidygenomes) based on ggtree packages in R. Genera were collapsed when no species of this genus contained the crtMN genes. Second, to examine the phylogeny of the crtM and crtN within the Lactobacillaceae family, a gene tree was inferred at the amino acid level using a similar procedure to the species tree. Since the crtM and crtN tree were highly similar, the crtN tree was used as a model due to its larger size and thus resolution. To reduce the number of branches, sequences were first clustered with cd-hit 38 with a 95% similarity threshold. In instances where sequences from different species were present in the same CD-hit cluster, they are shown in the adjacent table. The biosynthetic gene clusters obtained from antiSMASH were visualized using Bigscape 39 based on dereplicated genomes, and the clusters were mapped onto the crtN tree. Strains used in this study Phenotypic characterization started from the crtMN carriers within 575 in-house Lactobacillaceae isolates and three publicly available strains (Supplementary Table S1 ). These strains were previously isolated from vegetable fermentations 39 ; the human vagina 40 ; the human respiratory tract 41 , the phyllosphere 36 ; anthosphere 42 ; and liquid compost fermentations 43 . Forty-eight isolates, taxonomically related to carotenoid producers based on the pangenome analysis, were phenotypically screened. To complement the publicly available data, the genomes of 25 crtMN and 17 non- crtMN containing in-house isolates were sequenced and included in the pangenome analysis ( Supplementary Table S1 , study number PRJEB57255 ). UV and oxidative stress assays were performed using nine Lp. plantarum strains, as indicated in Supplementary Table S1 . C30 carotenoid extraction and identification Lipophilic compounds were extracted, and their absorption spectra were measured based on the methods of Garrido-Fernández and colleagues 23 . In brief, cells were harvested from a 50 ml overnight culture in Weissella Medium Broth (WMB) by centrifugation for 15 min at 2000 × g. The cells were subsequently washed with 50 ml of sterile distilled water. Afterwards, 10 ml of N,N-dimethylformamide was added to the washed cells, which were incubated for 15 min at 65°C. The cell debris was separated by centrifugation at 3000 × g for 10 min, after which the supernatant was transferred to a separator funnel. The extraction of the remaining cell debris with N,N-dimethylformamide was repeated four times. All the extracts were pooled and mixed with 100 ml of diethyl ether, and 10% NaCl was added to aid in the separation of the liquid phases. The organic phase was dried with anhydrous Na 2 SO 4 , followed by solvent evaporation in a rotary evaporator. The resulting residue was dissolved in 2 ml of methanol/tert-butyl methyl ether (1:1 v:v) containing 1% BHT. The absorption spectrum was measured between 550 nm and 300 nm using a spectrophotometer to determine the characteristic absorption maxima. Carotenoids were further purified and identified using high-performance liquid chromatography (HPLC) coupled inline to a diode array detector (DAD) and high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF MS), a procedure conducted by the RIC group. For high-throughput identification, a similar procedure was used with different extraction solvents and without further purification. The washed cells were extracted with 2 ml of molecular biology grade ethanol, vortexed thoroughly and incubated for 20 min at 65°C. The cell debris was separated by centrifugation at 8603 × g for 5 min, after which the supernatant was transferred to a 15 ml tube. The ethanol extraction step was repeated once on the remaining cell debris. Both extracts were pooled, and 2 ml of heptane was added and mixed vigorously for 1 min. Next, 2 ml of distilled water was added and mixed. The hydrophilic and lipophilic phases were then separated by centrifuging the tubes for 5 min at 4000 × g. The top layer (lipophilic phase) was cautiously transferred to a clean 2 ml tube and dried with anhydrous Na 2 SO 4 . Finally, the absorption peaks between 300 and 550 nm were measured using a spectrophotometer to detect characteristic absorption peaks. Oxidative and UV stress resistance assays Cells were harvested by centrifugation (10 min at 1500 × g) from a 10 mL two-day culture, grown in MRS medium at 28°C with shaking, washed with sterile phosphate-buffered saline (PBS) and resuspended to an optical density (OD) of 0.16 at 600 nm. For oxidative stress, 100 µL of diluted culture was added to a suspension containing a final concentration of 0.03% H 2 O 2 and incubated while shaking for 10, 20, or 30 min. For UV stress, 500 µL of each suspension and 8 mL of sterile PBS were dispensed into small Petri plates and placed on an orbital shaker with a gentle swirling motion inside a laminar air flow cabinet, after which the lids were removed. The swirling Petri plates were exposed to UV treatment, and samples were collected at four time points: before UV exposure, after 10 sec, after 20 sec, and after 30 sec of UV exposure. The number of colony-forming units (CFUs) was determined for all the samples through serial dilution and plating out in triplicate on MRS agar. The tested strains were tested in triplicates and the entire assay was repeated three times. The rate of decrease in CFU counts was determined as a measure of susceptibility to oxidative and UV stress and involved transforming the CFU counts to a logarithmic scale and fitting a linear regression to infer the slope of the decrease over time. Association of C30 carotenoid biosynthesis capacity and habitat To further associate the carotenoids and habitats, multiple statistical analyses were performed on the dereplicated dataset in R with the packages dunn.test, caper, nlme, and geiger to assess the correlation between crtMN prevalence and lifestyle and genome size, taking phylogenetic effects into account. A gene was considered a core trait of a species when more than 95% of the genomes within a species had this trait and an accessory trait when the prevalence was between 5% and 95%. Data availability The publicly available Lactobacillaceae genomes were downloaded from the Genome Taxonomy Database (GTDB, version r214). All newly sequenced genomes from in-house isolates are available in the ENA database in project PRJEB57255 using the accession numbers given in Supplementary Table S1 . Results C30 carotenoid biosynthesis genes are scattered across the Lactobacillaceae family and show frequent horizontal transfer To explore the carotenoid biosynthetic gene cluster potential within the Lactobacillaceae family, the presence of all known enzymes involved in carotenoid biosynthesis was evaluated. For this purpose, 6297 publicly available genomes were screened. Due to the high similarity among genomes in this dataset, genomes were dereplicated based on pairwise ANI < 99.99%, resulting in 4179 unique genomes, from which a pangenome was constructed. Enzymes involved in the 4,4'-diapophytoene pathway for the production of C30 carotenoids were present; 4,4'-diapophytoene synthase (encoded by the crtM gene) and 4,4’-diapophytoene desaturase ( crtN ) (Fig. 1). The orthogroups to which each gene belonged were the same in the experimentally confirmed carotenoid-producers, Lp. plantarum WCFS1 23 and Lt. fragifolii AMBP162 T (phenotype first observed during the description of the species 36 and confirmed in this paper), validating the use of these orthogroups to detect crtMN genes across the Lactobacillaceae pangenome. Additionally, analysis with antiSMASH confirmed that no other known clusters for carotenoid biosynthesis, such as the squalene pathway, were present in the Lactobacillaceae . The crtMN C30 carotenoid biosynthesis gene families showed to be scattered across 28 species from 14 genera out of a total of 361 species and 34 genera analyzed within the Lactobacillaceae family (i.e., 7.7% species and 41.2% genus prevalence) (Fig. 2). We selected 37 in-house strains ( Supplementary Table S1 ) for genome sequencing to complement the public dataset based on the likelihood for presence of crtMN genes (29 strains) and underrepresented species (8 strains). After genomic dereplication, 24 remained ( Supplementary Table S1 ) and were included in the subsequent analysis (4203 unique genomes). The detected crtMN genes clustered into two clades (Fig. 3). One clade included Lactiplantibacillus , Fructilactobacillus, Latilactobacillus and Companilactobacillus , while the other exhibited broader diversity, including genera such as Leuconostoc , Oenococcus, Holzapfelia and other taxa within Fructilactobacillus . Notably, the gene trees for crtM and crtN were highly similar, indicating that these genes were typically transferred or inherited together. The crtN tree was preferred for visualization because it was more accurate due to the greater length of the gene (i.e. 499 amino acids). Notably, the Fructilactobacillus genus was present in both clades, indicating two independent acquisition events of the crtMN genes in this genus and showing that this trait had been gained convergently in this genus. The crtMN phylogenetic trees did not align with the species tree based on the core genome (Fig. 2), suggesting that these genes were not only inherited through vertical transmission but also horizontally transferred across species and even genera. Clear examples of recent horizontal gene transfer (HGT) events were observed within clusters of multiple species that contain nearly identical crtMN genes (up to 100% similarity at the protein level), for example amongst Lp. plantarum, Levilactobacillus buchneri , Levilactobacillus brevis, Lactiplantibacillus pentosus and Pediococcus pentosaceus , as well as amongst several Leuconostoc species. Of note, one cluster present in Apilactobacillus ozensis and Apilactobacillus xinyiensis seemed to contain two genes that were both annotated as crtN. Identification of C30 carotenoid-producing species in Lactobacillaceae biobank Having shown the taxonomic spread of C30 carotenoid biosynthesis genes within Lactobacillaceae , we subsequently aimed to phenotypically substantiate the carotenoid biosynthesis capacity in strains containing crtMN genes. First, C30 carotenoid biosynthesis was confirmed in Latilactobacillus fragifolii AMBP162 T , originally isolated from the phyllosphere of a strawberry plant 36 and compared to that of the known C30 carotenoid producer Lp. plantarum WCFS1 23 . The pellets of both strains appeared yellow, providing the first indication of carotenoid biosynthesis (Fig. 4A). The extract was purified using HPLC coupled inline to a diode-array detector (DAD) and high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF MS). The highest peak in the UV chromatogram of the HPLC eluent (Fig. 4B) had absorption maxima at 467, 438 and 414 nm (Fig. 4C), similar to the peaks prior to purification (465, 435 and 409 nm) and corresponding to the absorption maxima of 4,4’-diaponeurosporene 23 . The peak also had a mass-to-charge ratio ( m/z) of 403.3338 (Fig. 4D), corresponding to the formula C 30 H 42 , which is identical to that of 4,4’-diaponeurosporene. Subsequently, a high-throughput method was developed to phenotypically test C30 carotenoids, using less solvent and excluding unnecessary purification of the extract (see Methods). Starting from 575 in-house isolates and five publicly sourced strains ( Supplementary Table S1 ) based on taxonomy and isolation source, 45 in-house and 5 public Lactobacillaceae were tested phenotypically. This selection included 17 related noncarriers to assess the absence of phenotype. In total, the biosynthesis of 4,4’-diaponeurosporene-like carotenoids was substantiated in 27 strains belonging to the crtMN -harboring species Lt. fragifolii , Lp. plantarum, Leuconostoc citreum, Lc. pseudomesenteroides and Holzapfelia floricola , and absent in all tested noncarrier strains ( 17 ) (Fig. 3, Supplementary Table S1 ). For only six of the isolates tested, the observed phenotype did not match the genotype. Specifically, the two tested strains of the species Lactiplantibacillus mudanjiangensis (AMBF-0197 and AMBF-0209) and one strain of Lc. mesenteroides (LMG 6893) did not appear to express the phenotype under the tested conditions (Fig. 1C), whereas two Lp. plantarum strains (AMBP-0214 and AMBP-0424) appeared to harbor inactive 4,4’-diapophytoene desaturase caused by the deletion of 522 bp in the crtN gene. These latter strains were considered natural nonproducing mutants of carotenoid production in Lp. plantarum , a characteristic employed in subsequent physiological tests. Finally, the Apilactobacillus xinyiensis (AMBP-0461) containing the unusual cluster with the duplicated crtN did not appear to express the phenotype under the tested conditions. C30 carotenoids are associated with oxidative and UV stress resistance in Lp. plantarum strains To assess whether carotenoid biosynthesis in Lactobacillaceae plays a role in resistance to oxidative and UV stress, we compared seven carotenoid-producing Lp. plantarum strains to the natural nonproducing strains AMBP-0214 and AMBP-0424. A phylogenetic tree of the core genomes of all the strains tested showed that all the strains were closely related and that the nonproducers were not an outgroup ( Supplementary Figure S1 ). Carotenoid-negative strains were more susceptible to oxidative and UV-induced stress than carotenoid-positive strains, in line with the protective role of carotenoid biosynthesis in Lp. plantarum strains. The degree of oxidative and UV stress resistance varied among the strains tested (Fig. 5A). A linear model including time, different experiments and the production of carotenoids showed that the viability of the strains decreased with longer oxidative stress treatment (Est − 0.32, p < 0.001), with increased survival for the carotenoid-producing strains (indicated by a less negative slope) (Est. 0.06, p < 0.05). The same protection was observed for UV-induced stress, as phenotypic carotenoid biosynthesis in these strains was associated with increased survival in all trials (Est. 0.024, p < 0.05). C30 carotenoid biosynthesis is associated with nomadic and insect-adapted lifestyles We subsequently evaluated whether the C30 carotenoid biosynthesis genes were associated with a particular Lactobacillaceae lifestyle and habitat adaptation using the categories defined by Zheng et al.Click or tap here to enter text.as free-living, vertebrate-adapted, invertebrate-adapted or nomadic 32 . Such lifestyle assignments were available for 170 out of 361 species in our public genome datasets. The presence of C30 carotenoid biosynthesis genes ( crtMN) was most common in nomadic and insect-adapted lactobacilli, where they appeared as core (e.g. in nomadic Lp. plantarum , insect-adapted Fructilactobacillus lindneri and uncategorized Lc. citreum ) and accessory traits. In contrast, crtMN genes were completely absent in vertebrate-associated lactobacilli such as Lactobacillus crispatus and Limosilactobacillus reuteri . They were also found in a few genomes representing free-living species such as Lv. buchneri (Fig. 5B). Consequently, a significant association between the proportion of genomes with C30 carotenoid biosynthesis and the lifestyle of a species (p < 0.001) was found when the Kruskal-Wallis test was used. Pairwise comparison with a Dunn test with Bonferroni correction for multiple testing ( Supplementary Figure S2 A ) revealed the strongest association with nomadic and insect-adapted lifestyles. When considering the phylogenetic background with phylogenetic generalized least squares (PGLS), the same trends were found, but only insect-adapted versus free living was near significant (p = 0.059) ( Supplementary Figure S2 B ). Additionally, nomadic and insect-associated crtMN -harboring strains differed in genome size, with the nomadic strains having large genomes, similar to other nomadic Lactobacillaceae , and the insect-adapted strains having the smallest genomes within the family. These findings point to positive selection of these genes after horizontal gain in species with nomadic and insect-adapted lifestyles (Fig. 5C). To further investigate the lifestyle association, we also screened the isolation sources of 575 in-house isolates and three publicly sourced isolates. We correlated this with the C30 carotenoid phenotype and genotype. This analysis pointed towards leaves and flowers carrying a high number of carotenoid producers belonging to the species Lt. fragifolii (n = 13), Lc. pseudomesenteroides (n = 1), Lc. citreum (n = 3) and H. floricola (n = 1) ( Supplementary Figure S3 ). A second major habitat of carotenoid producers showed to be plant-based fermentations, which harbored carotenoid-producing Lc. citreum (n = 2), Lp. plantarum (n = 6) and Lactiplantibacillus paraplantarum (n = 2). In contrast, in our laboratory, carotenoid-producing Lactobacillaceae strains were only sporadically isolated from vertebrate habitats (e.g., the human vagina 40 or respiratory tract 41 ) and were all nomadic Lp. plantarum strains. Discussion In this study, we investigated the biosynthesis of C30 carotenoids in the Lactobacillaceae family, the largest family of beneficial bacteria known to date, and linked the crtMN- positive genotype to their lifestyles. For this purpose, we used an integrated comparative genomic approach combined with a phenotypic screening of a diverse in-house Lactobacillaceae strain collection and an assessment of the functional role of carotenoids in stress resistance. Pangenome analysis of Lactobacillaceae revealed that c rtMN -mediated carotenoid biosynthesis was a rare and scattered trait within the family: it occurred in 41% of all the genera but only in a few species or strains within each genus. It was found to be a core property in some nomadic species, such as Lp. plantarum; insect-adapted species, such as Fl. lindneri; and accessory in many others. Phylogenetic analyses indicated that the biosynthetic crtMN genes are frequently transferred horizontally across species and genera, for example, from Lp. plantarum to Lv. brevis and within the Leuconostoc genus. This finding is consistent with the high mobility of carotenoid pathways observed at higher taxonomic levels 16–18 . Moreover, the trait appeared to have been gained convergently in Fructilactobacillus , with this genus having acquired crtMN genes from distinct donors during distinct events. Using a high-throughput extraction and analysis method, the crtMN genotype was matched with the synthesis of 4,4’-diaponeurosporene in five species. Functionally, we showed that Lp. plantarum strains that do not produce carotenoids were less resistant to UV and oxidative stress, in line with the general knowledge on carotenoids 16 and the previously observed protection against oxidative stress conferred by 4,4’-diaponeurosporene biosynthesis in Lp. plantarum 44 . Finally, the scattered distribution and mobility of this trait across the family, coupled with its advantages in UV and oxidative stress, prompted us to systematically investigate the link between carotenoid biosynthesis and the lifestyle and ecology of Lactobacillaceae . When testing for associations between two features associated with a set of species, as we have done here for carotenoids, phylogeny needs to be considered. This was done in our study by applying a phylogenetic generalized least squares (PGLS) approach 45 . Such an approach effectively takes into account that closely related species will likely share similarity in any two traits (such as crtMN prevalence and lifestyle studied here) because of ”phylogenetic inertia”, not necessarily because the traits are correlated 46 . Since the lifestyles of Lactobacillaceae species are mostly conserved at genus level, this implies that the independent units of information are in general the genera rather than the species, resulting in a lower effective sample size and limitations in our dataset concerning statistical power. In addition, another limitation of our work is that a lifestyle assessment has not yet be attributed by 31,32 to 191 of the 361 Lactobacillaceae species in our dataset. This is due to lack of sufficient data on the isolation sources, metabolic potential, and related properties for these species 31,32 . For many of these species, only a single strain has been isolated from a single source. Repetitive isolation of species from the same environment, as well as substantiation with specific metabolic and experimental validation is required to attribute lifestyles, as the environment of isolation does not reflect the environment of adaptation (niche) for various reasons, such as random dispersal events and increasing anthropogenic effects on the biosphere. Such detailed information will have to be collected for these 191 Lactobacillaceae species to also be able to attribute a lifestyle in the future and further substantiate our analyses. Despite these shortcomings in public data and taking into account the phylogeny, a near significant association was found between carotenoid biosynthesis genes and an insect-adapted lifestyle (p = 0.056). However, excluding phylogenetic relatedness, we found that crtMN genes were mostly absent in free-living species and completely absent in vertebrate-associated species, such as the L. crispatus , which is dominant in the human vagina 40 and Lm. reuteri which typically colonizes the vertebrate gut 31 . The complete absence of carotenoids in well-studied vertebrate associated Lactobacillaceae suggests that carotenoid production is not selected for in mucosal and low-oxygen habitats, such as the gut and vagina. In contrast, carotenoid biosynthesis genes were strongly associated with nomadic and insect-adapted Lactobacillaceae indicating that oxygen- and UV-rich environments encountered by nomadic and insect-adapted strains can select for this trait. The habitat-adaptation strategy of nomadic carotenoid producers appeared to differ from that of insect-adapted species. Nomadic Lactobacillaceae species typically have large genomes (between 2.4 and 3.6 Mbp), making them metabolically versatile and adaptable to various environments. In other studies, nomadic species have been found to typically occur in low numbers in oligotrophic environmental niches, such as plant surfaces 29,52 , and in high abundances once carbohydrates become more available, such as in vegetable fermentation products 53 . Such fermentations are characterized by intense microbial competition and high-salt concentration 53 , possibly leading to osmotic and oxidative stress. In such fermentations and outdoor oligotrophic environments, we speculate based on the data obtained here that C30 carotenoids could provide a fitness advantage to nomadic lactobacilli by reducing susceptibility to oxidative stress. Insect-adapted lactobacilli have smaller genomes (1.2–2.2 Mbp) with a concomitant decrease in carbohydrate metabolic capacity 31,32 . This difference was also observed in our present study among crtMN carriers, as the insect-adapted crtMN carriers had remarkably small genome sizes (between 1.6 and 2.1 Mbp), but they still contained crtMN genes , indicating an evolutionary advantage. Interestingly, the insect-adapted crtMN carriers were all part of one clade within the Fructilactobacillus genus, a genus known to be transferred between pollinators via the environment, with flowers serving as key hubs 31,47,48 . Our data presented here indicate that the biosynthesis of C30 carotenoids, and its associated protection against UV radiation and oxidative stress, could be a significant advantage for these environmentally dispersed, insect-adapted Lactobacillaceae species. Our hypothesis is in line with the adaptation strategy previously described for leaf-dwelling bacteria, such as Clavibacter and Pseudomonas , which produce C40 carotenoids and other pigments to increase their survival in this UV-stressed environment 9 . Notably, carotenoid biosynthesis was absent in Lactobacillaceae associated with social pollinators, such as Lactobacillus apis and Bombilactobacillus . These bacteria are vertically passed down to offspring within the hive 49 , where they are protected from UV- and oxidative stress. An environmental survival strategy does not seem required for these bacteria 49 . An exception to this is the Apilactobacillus genus, which is also known to be dispersed among solitary bees via flower 50 . This genus was shown in our study here to have an unusual putative terpenoid cluster, characterized by a duplicated crtN and an absent crtM gene. However, it remains to be substantiated whether this duplication is associated with a particular phenotype or pigment. Our hypothesis that Lactobacillaceae bacteria that are dispersed to plants via insects have a competitive advantage expressing C30 carotenoids was supported by our extensive culture approach and collection. A diverse array of carotenoid-producing Lactobacillaceae were isolated from flowers and leaves, with a relative high prevalence found for Lc. citreum in flowers. This species has – to the best of our knowledge - not yet been assigned to a certain lifestyle butour data presented here add support to an insect or flower-adapted lifestyle. In contrast, Lactobacillaceae isolates from vertebrate habitats studied here (mainly from the human vagina and respiratory tract), showed to be predominantly non-producers. Among the producing strains isolated, the nomadic Lp. plantarum was the most predominant species. In addition to the ecological role, the presence of crtMN genes in Lactobacillaceae is of interest from an applied perspective, especially considering the beneficial properties and lack of virulence factors in this family of bacteria. For example, incorporating carotenoid-producing bacteria into food fermentations could add additional functional properties to these foods. In fact, 4,4’-diaponeurosporene is already present in many vegetable fermentations, as Lp. plantarum , a core producer, dominates the later stages of most typical vegetable fermentations, and Leuconostoc species generally dominate in the early stages 53 . Although the added benefits of 4,4’-diaponeurosporene in these fermented food ecosystems have not yet been studied, this metabolite has been connected to health-promoting effects via immune modulation. For instance, the introduction of crtMN genes originating from Staphylococcus aureus into Bacillus subtilis has been shown to reduce colitis in mice 54 and increase resistance to Salmonella typhimurium infection 55 . Furthermore, in piglets, heterologously c rtMN expressing- B. subtilis bacteria have been shown to improve the mucosal immune system of the gut 56 and the respiratory tract 57 . These studies were carried out with genetically modified bacteria and are thus unlikely to reach large market applications, especially in Europe. In contrast, our results presented here indicate that natural carotenoid-producing Lactobacillaceae constitute an interesting alternative. Moreover, microbial biosynthesis can offer advantages over traditional production methods at it can be safer and less reliant on fossil resources than chemical synthesis and less influenced by seasonality or climate than plant-based biosynthesis 58 and applied to C30 carotenoids 59 . In summary, this study on the ecology and evolution of carotenoid biosynthesis in the Lactobacillaceae family revealed a scattered distribution of crtMN -mediated C30 carotenoid 4,4’-diaponeurosporene biosynthesis across 28 species and 14 genera and highlighted the mobility of this trait. C30 carotenoid biosynthesis appears to have emerged as a core property in several species, notably Lp. plantarum , Lc. citreum and Fl. lindneri . Furthermore, carotenoid biosynthesis was strongly associated with nomadic and insect-adapted lifestyles, where it offers an advantage via protection from UV and oxidative stress. Declarations Conflict of interest S.L. received funding from different probiotic companies that were not involved in this research. T.E. is partially funded through an industrial research VLAIO grant not related to this work. M.L. is employed part-time by Biobest Group NV, this company was not involved in this research. I.V. and K.S. are employed by the RIC and their work was compensated via a service contract. P.A.B. is an independent consultant for several companies in the food and pharmaceutical industry bound by confidentiality agreements. Funding This research has received funding from the following funding bodies: the European Research Council (ERC; starting grant Lacto-Be 852600 of S.L.), the Belgian Science Policy Office (BELSPO; BRAIN-be 2.0; B@SEBALL (B2/191/P3/B@SEBALL)), the Scientific Research Foundation – Flanders (FWO; doctoral grant of J.T. 1SC3623N and postdoctoral grant of S.W. 12AZ624N), the Industrial Research Fund of the University of Antwerp (IOF-SEP 48530), VLAIO (HBC.2022.1000), and from the revenue of our microbiome platform services. Acknowledgments The authors would like to thank Tim Van Rillaer for his help with the bioinformatic pipelines and Ines Tuyaerts, Nele Van de Vliet and Sam Bakelants for their help with the UV and oxidative stress tests. References Sun, T., Rao, S., Zhou, X., Li, L.: Plant carotenoids: recent advances and future perspectives. 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Antioxidants 11, (2022) Additional Declarations Yes there is potential Competing Interest. S.L. received funding from different probiotic companies that were not involved in this research. T.E. is partially funded through an industrial research VLAIO grant not related to this work. M.L. is employed part-time by Biobest Group NV, this company was not involved in this research. I.V. and K.S. are employed by the RIC and their work was compensated via a service contract. P.A.B. is an independent consultant for several companies in the food and pharmaceutical industry bound by confidentiality agreements. 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Theoretical mass of the\u003cstrong\u003e \u003c/strong\u003efinal product 4,4’-Diaponeurosporene is shown (made with ChemDraw and inkskape based on KEGG).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/4691f4a18eb0cf2607ce3a9a.png"},{"id":62394866,"identity":"a235023d-7a7b-47c1-857a-ae39433d4d89","added_by":"auto","created_at":"2024-08-13 16:49:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":909833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFamily-wide occurrence of 4,4’-diapophytoene synthase (crtM gene) and 4,4’-diapophytoene desaturase (crtN) in Lactobacillaceae\u003c/strong\u003e. The tree was inferred with IQ-Tree via the LG+F+G4 method. An orange tip indicates that carotenoid biosynthesis genes were present in at least one genome in a species. The inner circle corresponds to the assigned lifestyle, as described in Zheng and Wittouck et al.; the second circle corresponds to the percentage of genomes that contain crtMN genes compared to all tested genomes of that species (n is given at the branch tip). The outer circle corresponds to genome size. Species for which the phenotype was experimentally confirmed are indicated in bold. The genera were collapsed when no crtMN genes were detected.\u003c/p\u003e\n\u003cp\u003e32\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/06129df8a016a34af044d1df.png"},{"id":62395406,"identity":"e8e00b4f-5789-4bc0-afaf-235e68c317b0","added_by":"auto","created_at":"2024-08-13 16:57:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":398111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of the 4,4’-diapophytoene desaturase (crtN) gene detected in Lactobacillaceae, which shows clustering into two clades.\u003c/strong\u003e The tree was inferred with IQtree via the LG+F+G4 method. To reduce the number of branches, sequences were first clustered with cd-hit with a 95% similarity threshold. When a cluster contained multiple species, the species were clustered into multi-species horizontal gene transfer (HGT) groups, as shown in a table for clarity. The numbers indicate the number of strains that collapsed for each cluster. For each tip, the biosynthetic cluster is also shown as predicted by Bigscape using one representative.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/1643a7d6397ed6da7347f087.png"},{"id":62394552,"identity":"3b6e39cc-483e-47dc-a02d-87444d5cbb5d","added_by":"auto","created_at":"2024-08-13 16:41:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":412095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePigment identification in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLatilactobacillus fragifolii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e AMBP162 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLactiplantibacillus plantarum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WCFS1. \u003c/strong\u003e\u003cem\u003eLt. graminis \u003c/em\u003eDSM20719\u003csup\u003eT\u003c/sup\u003e was used as a nonpigmented control.\u003cstrong\u003e A) Washed cell pellets (left) and unpurified extracts (right) \u003c/strong\u003eof 2-day cultures. \u003cstrong\u003eB) UV chromatogram of the HPLC eluent\u003c/strong\u003e of the extracted pigments. \u003cstrong\u003eC)\u003c/strong\u003e \u003cstrong\u003eUV‒visible absorption spectrum \u003c/strong\u003eof the highest peak in the HPLC chromatogram; absorption maxima (nm) are indicated above the peaks. \u003cstrong\u003eD)\u003c/strong\u003e \u003cstrong\u003eHigh-resolution quadrupole time-of-flight mass spectrometry\u003c/strong\u003e image of the highest peak in the HPLC eluent. The mass-to-charge ratios are indicated above the peaks.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/7c2f35c768e14fa537b3c868.png"},{"id":62394556,"identity":"24af59c0-a0ea-4b66-8eaf-38324806fb71","added_by":"auto","created_at":"2024-08-13 16:41:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":257399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional and ecological roles of C30 carotenoid biosynthesis.\u003c/strong\u003e \u003cstrong\u003eA) Survival rate of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLp. plantarum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains under oxidative and ultraviolet (UV) stress.\u003c/strong\u003e All the strains were tested in three separate trials. Seven producers and two natural knockout mutants that do not produce carotenoids were included (supplementary table S1). B) \u003cstrong\u003ePercentage of species within a lifestyle (based on Zheng and Wittouck et al.) harboring the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecrtMN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene as a core or accessory trait\u003c/strong\u003e. The total number of species analyzed is given for each lifestyle. \u003cstrong\u003eC) The genome sizes of species in relation to their lifestyle and the presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecrtMN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e32\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/6dde23f2ec838d30e314cc13.png"},{"id":70540123,"identity":"a64e0154-ea00-4366-94ba-10d3c77ada7d","added_by":"auto","created_at":"2024-12-04 08:06:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3028957,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/7bbe6fb7-66c0-4d1d-8400-3adbecaf1e0e.pdf"},{"id":62394867,"identity":"f0d28d01-2437-482d-a6e0-99f2da65ddd7","added_by":"auto","created_at":"2024-08-13 16:49:43","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":41177,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/a661f834bc6c3d47f5fae1dc.docx"},{"id":62394555,"identity":"65875834-dd06-402b-980b-816af468b701","added_by":"auto","created_at":"2024-08-13 16:41:43","extension":"ai","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":101867,"visible":true,"origin":"","legend":"","description":"","filename":"supfigS1.ai","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/ad86545edeea10f4f8ae44e5.ai"},{"id":62394869,"identity":"143f493c-6e63-49c6-b18b-ce442d5b4a6c","added_by":"auto","created_at":"2024-08-13 16:49:43","extension":"ai","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1336033,"visible":true,"origin":"","legend":"","description":"","filename":"supfigS2new2.ai","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/1155d24cfe1cc2a251755c9c.ai"},{"id":62394557,"identity":"a3966020-edb6-479d-aee4-d52a6721e13d","added_by":"auto","created_at":"2024-08-13 16:41:43","extension":"ai","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":182832,"visible":true,"origin":"","legend":"","description":"","filename":"supfigS3.ai","url":"https://assets-eu.researchsquare.com/files/rs-4637278/v1/969e1da893be9531f4482995.ai"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nS.L. received funding from different probiotic companies that were not involved in this research. T.E. is partially funded through an industrial research VLAIO grant not related to this work. M.L. is employed part-time by Biobest Group NV, this company was not involved in this research. I.V. and K.S. are employed by the RIC and their work was compensated via a service contract. P.A.B. is an independent consultant for several companies in the food and pharmaceutical industry bound by confidentiality agreements.","formattedTitle":"Distribution of C30 carotenoid biosynthesis genes suggests habitat adaptation function in insect-adapted and nomadic Lactobacillaceae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCarotenoids are membrane-bound specialized metabolites found in all photosynthesizing organisms, such as plants, algae and cyanobacteria. They play vital roles in protecting producer cells against excess light energy and scavenging reactive oxygen species\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. However, these molecules also perform functions that are unrelated to photosynthesis, such as being precursors to phytohormones\u003csup\u003e4,5\u003c/sup\u003e or attracting pollinators and promoting seed dispersal by accumulating in flowers and fruits\u003csup\u003e6\u003c/sup\u003e. Moreover, carotenoids are found in non-photosynthetic organisms, for example in the pathogenic bacterium \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, where the carotenoid staphyloxanthin increases virulence by protecting the bacteria against reactive oxygen species and inactivating host neutrophils\u003csup\u003e7\u003c/sup\u003e. In leaf-dwelling non-photosynthetic bacteria, such as \u003cem\u003eClavibacter\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eMethylobacteria\u003c/em\u003e, carotenoids protect bacterial cells and, in some cases, the plant host from UV radiation\u003csup\u003e8,9\u003c/sup\u003e. Animals require but are generally unable to synthesize carotenoids and consequently supplementation depends on food intake, with the notable exception of aphids, which have acquired the ability to produce carotenoids as fungal carotenoid biosynthetic genes have been taken up into their genome\u003csup\u003e10\u003c/sup\u003e. In humans, dietary intake of carotenoids is essential for vitamin A biosynthesis\u003csup\u003e11\u003c/sup\u003e and is associated with a reduction in inflammatory markers\u003csup\u003e12\u003c/sup\u003e and a reduced risk of prostate cancer and breast cancer\u003csup\u003e13\u003c/sup\u003e, although also opposite associations have been found such as for lung cancer\u003csup\u003e14\u003c/sup\u003e. Because mostly positive health benefits have been found, carotenoids are increasingly used as functional ingredients in food and feed, where they can also have additional technological benefits, for instance as natural pigments and antioxidants\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe best-studied carotenoids are those found in photosynthetic organisms. These specialized metabolites are mostly C40-carotenoids, which have 40 carbon atoms within their backbone\u003csup\u003e15\u003c/sup\u003e. More recently, the full scope of carotenoid diversity and evolution has gained increasing attention\u003csup\u003e15,16\u003c/sup\u003e. Phylogenetic analyses revealed that carotenoid biosynthesis is an ancient process that evolved prior to photosynthesis, likely under increased UV radiation conditions\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. According to Santana-Molina et al.\u003csup\u003e19\u003c/sup\u003e, the earliest evolved group of carotenoids might be composed of 30 carbon atoms, or triterpenoids. These specialized compounds are found in plants under various forms, such as triterpenoid saponins\u003csup\u003e20\u003c/sup\u003e or oleanolic acids\u003csup\u003e21\u003c/sup\u003e, and they are often involved in the plant\u0026rsquo;s defense system. However, C30 carotenoid biosynthesis originated in prokaryotes\u003csup\u003e16,18\u003c/sup\u003e and has been detected in the photosynthesizing bacteria \u003cem\u003eHeliobacter\u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e, and several nonphotosynthetic bacteria such as \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003csup\u003e23\u003c/sup\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003csup\u003e24\u003c/sup\u003e, \u003cem\u003eEnterococcus faecium\u003c/em\u003e (formerly \u003cem\u003eStreptococcus faecium\u003c/em\u003e)\u003csup\u003e25\u003c/sup\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e and \u003cem\u003eMethylobacterium rhodinum\u003c/em\u003e (formerly \u003cem\u003ePseudomonas rhodos\u003c/em\u003e)\u003csup\u003e27\u003c/sup\u003e. In addition to these phenotypic observations, Santana and colleagues\u003csup\u003e18\u003c/sup\u003e showed that both 4,4\u0026rsquo;-diapophytoene (synthesis encoded by the \u003cem\u003ecrtM\u003c/em\u003e gene) as well as squalene (encoded by \u003cem\u003eSqs\u003c/em\u003e or the \u003cem\u003eHpnCDE\u003c/em\u003e cluster) can be precursors to C30 carotenoids, and that these pathways are now found scattered across prokaryotes, ranging from \u003cem\u003eFirmicutes\u003c/em\u003e to \u003cem\u003ePlanctomycetes\u003c/em\u003e to \u003cem\u003eArchaea\u003c/em\u003e and found to be mobile via horizontal gene transfer.\u003c/p\u003e \u003cp\u003eWhile carotenoid biosynthesis originated in, and is now scattered across nonphotosynthesizing prokaryotes, the function and ecological role of these specialized metabolites in these organisms have not been systematically studied. Within the \u003cem\u003eFirmicutes, Lactobacillaceae\u003c/em\u003e are an interesting family of nonphotosynthesizing bacteria, with great potential for use in food, feed and pharma\u003csup\u003e28,29\u003c/sup\u003e. Two phenotypical screenings of \u003cem\u003eLactobacillaceae\u003c/em\u003e, focusing on isolates from fermented foods, identified \u003cem\u003eLp. plantarum\u003c/em\u003e as a C30 carotenoid producer via the 4,4'-diapophytoene pathway\u003csup\u003e23,30\u003c/sup\u003e. However, the genomic and ecological diversity of the biosynthetic and phenotypic potential of this family has not yet been investigated.\u003c/p\u003e \u003cp\u003eIn this study, an in-depth pangenome and evolutionary analysis of the presence of terpenoid biosynthetic gene clusters was performed on a large dataset of 4203 unique genomes of the \u003cem\u003eLactobacillaceae\u003c/em\u003e family. These genomic analyses were complemented with phenotypic confirmation using a biobank of \u003cem\u003eLactobacillaceae\u003c/em\u003e from various habitats by high-throughput screening based on absorbance maxima and high-performance liquid chromatography (HPLC) and high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF MS). Additionally, the role of C30 carotenoids in UV and oxidative stress resilience was assessed for selected strains. Finally, associations between the presence of carotenoid biosynthesis genes in a species and its lifestyle were established, using assigned lifestyles from literature\u003csup\u003e31,32\u003c/sup\u003e, and new isolation and metabolic analyses from our in-house library of strains.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003ePangenome and phylogenetic analysis of the terpenoid biosynthetic gene clusters.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll publicly available \u003cem\u003eLactobacillaceae\u003c/em\u003e genomes were downloaded from the Genome Taxonomy Database (GTDB, gtdb.ecogenomic.org, version r214). With checkM, any incomplete (\u0026lt;\u0026thinsp;90%) or contaminated (\u0026gt;\u0026thinsp;5%) genomes were excluded\u003csup\u003e33\u003c/sup\u003e. This resulted in a dataset of 6259 public genomes. To avoid pseudoreplication, the sample function of SCARAP (github.com/SWittouck/SCARAP) was used with an average nucleotide identity (ANI) cutoff of 99.99%, retaining only genomes with ANI values below this cutoff. Second, the pan genomes of this family were inferred using the SCARAP tool\u003csup\u003e34\u003c/sup\u003e. As a start, orthogroups of detected genes, i.e.4,4\u0026rsquo;-diapophytoene desaturase (encoded by the \u003cem\u003ecrtN\u003c/em\u003e gene) and of 4,4\u0026rsquo;-diapophytoene synthase (encoded by the \u003cem\u003ecrtM\u003c/em\u003e gene) were determined (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003csup\u003e33\u003c/sup\u003e For reference, the genes from two experimentally confirmed species, \u003cem\u003eLp. plantarum\u003c/em\u003e WCFS1\u003csup\u003e23\u003c/sup\u003e and \u003cem\u003eLatilactobacillus fragifolii A\u003c/em\u003eMBP162 (experimentally confirmed in this study), were used as queries. Based on the results of this pangenome analysis, 37 in-house isolates were selected for whole genome sequencing, and were included in the subsequent analysis. Finally, all genomes were checked for other terpene biosynthesis genes using Antismash 6.0\u003csup\u003e35\u003c/sup\u003e focusing on terpenes and terpene pathways.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eReferences to experimentally confirmed\u003c/b\u003e \u003cb\u003ecrtMN\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e used to identify similar sequences in the pangenome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ecrtM\u003c/em\u003e \u003c/p\u003e \u003cp\u003e4,4\u0026rsquo;-diapophytoene synthase\u003c/p\u003e \u003cp\u003e(882 nt)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecrtN\u003c/em\u003e \u003c/p\u003e \u003cp\u003e4,4\u0026rsquo;-diapophytoene desaturase\u003c/p\u003e \u003cp\u003e(1497 nt)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e WCFS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_004567.2_2720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC_004567.2_2719\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e23\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLatilactobacillus fragifolii\u003c/em\u003e AMBP162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNZ_CAJOCF010000002.1_110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNZ_CAJOCF010000002.1_111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarotenoid biosynthesis was first observed during the description of the species\u003csup\u003e36\u003c/sup\u003e and further validated in this study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe core tree of all \u003cem\u003eLactobacillaceae\u003c/em\u003e was constructed following the method described in Eilers \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e37\u003c/sup\u003e. The core genome was inferred using SCARAP, which consists of 296 core genes. Protein sequences were extracted, aligned with MAFFT and trimmed with trimAL with a gap threshold of 10%. Aligned and trimmed core proteins were subsequently inferred with IQ-TREE using LG\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;G4.\u003c/p\u003e \u003cp\u003eFirst, the prevalence of \u003cem\u003ecrtMN\u003c/em\u003e genes within all species of the family \u003cem\u003eLactobacillaceae\u003c/em\u003e was calculated and integrated with lifestyle data from Zheng \u003cem\u003eet al\u003c/em\u003e., 2020\u003csup\u003e32\u003c/sup\u003e, and metadata from the GTDB using tidygenomes (github.com/SWittouck/tidygenomes) based on ggtree packages in R. Genera were collapsed when no species of this genus contained the \u003cem\u003ecrtMN\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eSecond, to examine the phylogeny of the \u003cem\u003ecrtM\u003c/em\u003e and \u003cem\u003ecrtN\u003c/em\u003e within the \u003cem\u003eLactobacillaceae\u003c/em\u003e family, a gene tree was inferred at the amino acid level using a similar procedure to the species tree. Since the \u003cem\u003ecrtM\u003c/em\u003e and \u003cem\u003ecrtN\u003c/em\u003e tree were highly similar, the \u003cem\u003ecrtN\u003c/em\u003e tree was used as a model due to its larger size and thus resolution. To reduce the number of branches, sequences were first clustered with cd-hit\u003csup\u003e38\u003c/sup\u003e with a 95% similarity threshold. In instances where sequences from different species were present in the same CD-hit cluster, they are shown in the adjacent table. The biosynthetic gene clusters obtained from antiSMASH were visualized using Bigscape\u003csup\u003e39\u003c/sup\u003e based on dereplicated genomes, and the clusters were mapped onto the \u003cem\u003ecrtN\u003c/em\u003e tree.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains used in this study\u003c/h2\u003e \u003cp\u003ePhenotypic characterization started from the \u003cem\u003ecrtMN\u003c/em\u003e carriers within 575 in-house \u003cem\u003eLactobacillaceae\u003c/em\u003e isolates and three publicly available strains (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These strains were previously isolated from vegetable fermentations\u003csup\u003e39\u003c/sup\u003e; the human vagina\u003csup\u003e40\u003c/sup\u003e; the human respiratory tract\u003csup\u003e41\u003c/sup\u003e, the phyllosphere\u003csup\u003e36\u003c/sup\u003e; anthosphere\u003csup\u003e42\u003c/sup\u003e; and liquid compost fermentations\u003csup\u003e43\u003c/sup\u003e. Forty-eight isolates, taxonomically related to carotenoid producers based on the pangenome analysis, were phenotypically screened. To complement the publicly available data, the genomes of 25 \u003cem\u003ecrtMN\u003c/em\u003e and 17 non-\u003cem\u003ecrtMN\u003c/em\u003e containing in-house isolates were sequenced and included in the pangenome analysis (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, study number PRJEB57255\u003c/b\u003e). UV and oxidative stress assays were performed using nine \u003cem\u003eLp. plantarum\u003c/em\u003e strains, as indicated in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eC30 carotenoid extraction and identification\u003c/h2\u003e \u003cp\u003eLipophilic compounds were extracted, and their absorption spectra were measured based on the methods of Garrido-Fern\u0026aacute;ndez and colleagues\u003csup\u003e23\u003c/sup\u003e. In brief, cells were harvested from a 50 ml overnight culture in Weissella Medium Broth (WMB) by centrifugation for 15 min at 2000 \u0026times; g. The cells were subsequently washed with 50 ml of sterile distilled water. Afterwards, 10 ml of N,N-dimethylformamide was added to the washed cells, which were incubated for 15 min at 65\u0026deg;C. The cell debris was separated by centrifugation at 3000 \u0026times; g for 10 min, after which the supernatant was transferred to a separator funnel. The extraction of the remaining cell debris with N,N-dimethylformamide was repeated four times. All the extracts were pooled and mixed with 100 ml of diethyl ether, and 10% NaCl was added to aid in the separation of the liquid phases. The organic phase was dried with anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, followed by solvent evaporation in a rotary evaporator. The resulting residue was dissolved in 2 ml of methanol/tert-butyl methyl ether (1:1 v:v) containing 1% BHT. The absorption spectrum was measured between 550 nm and 300 nm using a spectrophotometer to determine the characteristic absorption maxima. Carotenoids were further purified and identified using high-performance liquid chromatography (HPLC) coupled inline to a diode array detector (DAD) and high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF MS), a procedure conducted by the RIC group. For high-throughput identification, a similar procedure was used with different extraction solvents and without further purification. The washed cells were extracted with 2 ml of molecular biology grade ethanol, vortexed thoroughly and incubated for 20 min at 65\u0026deg;C. The cell debris was separated by centrifugation at 8603 \u0026times; g for 5 min, after which the supernatant was transferred to a 15 ml tube. The ethanol extraction step was repeated once on the remaining cell debris. Both extracts were pooled, and 2 ml of heptane was added and mixed vigorously for 1 min. Next, 2 ml of distilled water was added and mixed. The hydrophilic and lipophilic phases were then separated by centrifuging the tubes for 5 min at 4000 \u0026times; g. The top layer (lipophilic phase) was cautiously transferred to a clean 2 ml tube and dried with anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Finally, the absorption peaks between 300 and 550 nm were measured using a spectrophotometer to detect characteristic absorption peaks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOxidative and UV stress resistance assays\u003c/h2\u003e \u003cp\u003eCells were harvested by centrifugation (10 min at 1500 \u0026times; g) from a 10 mL two-day culture, grown in MRS medium at 28\u0026deg;C with shaking, washed with sterile phosphate-buffered saline (PBS) and resuspended to an optical density (OD) of 0.16 at 600 nm. For oxidative stress, 100 \u0026micro;L of diluted culture was added to a suspension containing a final concentration of 0.03% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and incubated while shaking for 10, 20, or 30 min. For UV stress, 500 \u0026micro;L of each suspension and 8 mL of sterile PBS were dispensed into small Petri plates and placed on an orbital shaker with a gentle swirling motion inside a laminar air flow cabinet, after which the lids were removed. The swirling Petri plates were exposed to UV treatment, and samples were collected at four time points: before UV exposure, after 10 sec, after 20 sec, and after 30 sec of UV exposure. The number of colony-forming units (CFUs) was determined for all the samples through serial dilution and plating out in triplicate on MRS agar. The tested strains were tested in triplicates and the entire assay was repeated three times. The rate of decrease in CFU counts was determined as a measure of susceptibility to oxidative and UV stress and involved transforming the CFU counts to a logarithmic scale and fitting a linear regression to infer the slope of the decrease over time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAssociation of C30 carotenoid biosynthesis capacity and habitat\u003c/h2\u003e \u003cp\u003eTo further associate the carotenoids and habitats, multiple statistical analyses were performed on the dereplicated dataset in R with the packages dunn.test, caper, nlme, and geiger to assess the correlation between \u003cem\u003ecrtMN\u003c/em\u003e prevalence and lifestyle and genome size, taking phylogenetic effects into account. A gene was considered a core trait of a species when more than 95% of the genomes within a species had this trait and an accessory trait when the prevalence was between 5% and 95%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe publicly available \u003cem\u003eLactobacillaceae\u003c/em\u003e genomes were downloaded from the Genome Taxonomy Database (GTDB, version r214). All newly sequenced genomes from in-house isolates are available in the ENA database in project PRJEB57255 using the accession numbers given in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eC30 carotenoid biosynthesis genes are scattered across the\u003c/b\u003e \u003cb\u003eLactobacillaceae\u003c/b\u003e \u003cb\u003efamily and show frequent horizontal transfer\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the carotenoid biosynthetic gene cluster potential within the \u003cem\u003eLactobacillaceae\u003c/em\u003e family, the presence of all known enzymes involved in carotenoid biosynthesis was evaluated. For this purpose, 6297 publicly available genomes were screened. Due to the high similarity among genomes in this dataset, genomes were dereplicated based on pairwise ANI\u0026thinsp;\u0026lt;\u0026thinsp;99.99%, resulting in 4179 unique genomes, from which a pangenome was constructed. Enzymes involved in the 4,4'-diapophytoene pathway for the production of C30 carotenoids were present; 4,4'-diapophytoene synthase (encoded by the \u003cem\u003ecrtM\u003c/em\u003e gene) and 4,4\u0026rsquo;-diapophytoene desaturase (\u003cem\u003ecrtN\u003c/em\u003e) (Fig.\u0026nbsp;1). The orthogroups to which each gene belonged were the same in the experimentally confirmed carotenoid-producers, \u003cem\u003eLp. plantarum\u003c/em\u003e WCFS1\u003csup\u003e23\u003c/sup\u003e and \u003cem\u003eLt. fragifolii\u003c/em\u003e AMBP162\u003csup\u003eT\u003c/sup\u003e (phenotype first observed during the description of the species\u003csup\u003e36\u003c/sup\u003e and confirmed in this paper), validating the use of these orthogroups to detect \u003cem\u003ecrtMN\u003c/em\u003e genes across the \u003cem\u003eLactobacillaceae\u003c/em\u003e pangenome. Additionally, analysis with antiSMASH confirmed that no other known clusters for carotenoid biosynthesis, such as the squalene pathway, were present in the \u003cem\u003eLactobacillaceae\u003c/em\u003e. The \u003cem\u003ecrtMN\u003c/em\u003e C30 carotenoid biosynthesis gene families showed to be scattered across 28 species from 14 genera out of a total of 361 species and 34 genera analyzed within the \u003cem\u003eLactobacillaceae\u003c/em\u003e family (i.e., 7.7% species and 41.2% genus prevalence) (Fig.\u0026nbsp;2). We selected 37 in-house strains (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) for genome sequencing to complement the public dataset based on the likelihood for presence of \u003cem\u003ecrtMN\u003c/em\u003e genes (29 strains) and underrepresented species (8 strains). After genomic dereplication, 24 remained (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e and were included in the subsequent analysis (4203 unique genomes). The detected \u003cem\u003ecrtMN\u003c/em\u003e genes clustered into two clades (Fig.\u0026nbsp;3). One clade included \u003cem\u003eLactiplantibacillus\u003c/em\u003e, \u003cem\u003eFructilactobacillus, Latilactobacillus and Companilactobacillus\u003c/em\u003e, while the other exhibited broader diversity, including genera such as \u003cem\u003eLeuconostoc\u003c/em\u003e, \u003cem\u003eOenococcus, Holzapfelia\u003c/em\u003e and other taxa within \u003cem\u003eFructilactobacillus\u003c/em\u003e. Notably, the gene trees for \u003cem\u003ecrtM\u003c/em\u003e and \u003cem\u003ecrtN\u003c/em\u003e were highly similar, indicating that these genes were typically transferred or inherited together. The \u003cem\u003ecrtN\u003c/em\u003e tree was preferred for visualization because it was more accurate due to the greater length of the gene (i.e. 499 amino acids). Notably, the \u003cem\u003eFructilactobacillus\u003c/em\u003e genus was present in both clades, indicating two independent acquisition events of the \u003cem\u003ecrtMN\u003c/em\u003e genes in this genus and showing that this trait had been gained convergently in this genus. The \u003cem\u003ecrtMN\u003c/em\u003e phylogenetic trees did not align with the species tree based on the core genome (Fig.\u0026nbsp;2), suggesting that these genes were not only inherited through vertical transmission but also horizontally transferred across species and even genera. Clear examples of recent horizontal gene transfer (HGT) events were observed within clusters of multiple species that contain nearly identical \u003cem\u003ecrtMN\u003c/em\u003e genes (up to 100% similarity at the protein level), for example amongst \u003cem\u003eLp. plantarum, Levilactobacillus buchneri\u003c/em\u003e, \u003cem\u003eLevilactobacillus brevis, Lactiplantibacillus pentosus\u003c/em\u003e and \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, as well as amongst several \u003cem\u003eLeuconostoc\u003c/em\u003e species. Of note, one cluster present in \u003cem\u003eApilactobacillus ozensis and Apilactobacillus xinyiensis\u003c/em\u003e seemed to contain two genes that were both annotated as \u003cem\u003ecrtN.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of C30 carotenoid-producing species in\u003c/b\u003e \u003cb\u003eLactobacillaceae\u003c/b\u003e \u003cb\u003ebiobank\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHaving shown the taxonomic spread of C30 carotenoid biosynthesis genes within \u003cem\u003eLactobacillaceae\u003c/em\u003e, we subsequently aimed to phenotypically substantiate the carotenoid biosynthesis capacity in strains containing \u003cem\u003ecrtMN\u003c/em\u003e genes. First, C30 carotenoid biosynthesis was confirmed in \u003cem\u003eLatilactobacillus fragifolii\u003c/em\u003e AMBP162\u003csup\u003eT\u003c/sup\u003e, originally isolated from the phyllosphere of a strawberry plant\u003csup\u003e36\u003c/sup\u003e and compared to that of the known C30 carotenoid producer \u003cem\u003eLp. plantarum\u003c/em\u003e WCFS1\u003csup\u003e23\u003c/sup\u003e. The pellets of both strains appeared yellow, providing the first indication of carotenoid biosynthesis (Fig.\u0026nbsp;4A). The extract was purified using HPLC coupled inline to a diode-array detector (DAD) and high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF MS). The highest peak in the UV chromatogram of the HPLC eluent (Fig.\u0026nbsp;4B) had absorption maxima at 467, 438 and 414 nm (Fig.\u0026nbsp;4C), similar to the peaks prior to purification (465, 435 and 409 nm) and corresponding to the absorption maxima of 4,4\u0026rsquo;-diaponeurosporene\u003csup\u003e23\u003c/sup\u003e. The peak also had a mass-to-charge ratio (\u003cem\u003em/z)\u003c/em\u003e of 403.3338 (Fig.\u0026nbsp;4D), corresponding to the formula C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e42\u003c/sub\u003e, which is identical to that of 4,4\u0026rsquo;-diaponeurosporene.\u003c/p\u003e \u003cp\u003eSubsequently, a high-throughput method was developed to phenotypically test C30 carotenoids, using less solvent and excluding unnecessary purification of the extract (see Methods). Starting from 575 in-house isolates and five publicly sourced strains (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) based on taxonomy and isolation source, 45 in-house and 5 public \u003cem\u003eLactobacillaceae\u003c/em\u003e were tested phenotypically. This selection included 17 related noncarriers to assess the absence of phenotype. In total, the biosynthesis of 4,4\u0026rsquo;-diaponeurosporene-like carotenoids was substantiated in 27 strains belonging to the \u003cem\u003ecrtMN\u003c/em\u003e-harboring species \u003cem\u003eLt. fragifolii\u003c/em\u003e, \u003cem\u003eLp. plantarum, Leuconostoc citreum, Lc. pseudomesenteroides\u003c/em\u003e and \u003cem\u003eHolzapfelia floricola\u003c/em\u003e, and absent in all tested noncarrier strains (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) (Fig.\u0026nbsp;3, \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). For only six of the isolates tested, the observed phenotype did not match the genotype. Specifically, the two tested strains of the species \u003cem\u003eLactiplantibacillus mudanjiangensis\u003c/em\u003e (AMBF-0197 and AMBF-0209) and one strain of \u003cem\u003eLc. mesenteroides\u003c/em\u003e (LMG 6893) did not appear to express the phenotype under the tested conditions (Fig.\u0026nbsp;1C), whereas two \u003cem\u003eLp. plantarum\u003c/em\u003e strains (AMBP-0214 and AMBP-0424) appeared to harbor inactive 4,4\u0026rsquo;-diapophytoene desaturase caused by the deletion of 522 bp in the \u003cem\u003ecrtN\u003c/em\u003e gene. These latter strains were considered natural nonproducing mutants of carotenoid production in \u003cem\u003eLp. plantarum\u003c/em\u003e, a characteristic employed in subsequent physiological tests. Finally, the \u003cem\u003eApilactobacillus xinyiensis\u003c/em\u003e(AMBP-0461) containing the unusual cluster with the duplicated \u003cem\u003ecrtN\u003c/em\u003e did not appear to express the phenotype under the tested conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eC30 carotenoids are associated with oxidative and UV stress resistance in\u003c/b\u003e \u003cb\u003eLp. plantarum\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess whether carotenoid biosynthesis in \u003cem\u003eLactobacillaceae\u003c/em\u003e plays a role in resistance to oxidative and UV stress, we compared seven carotenoid-producing \u003cem\u003eLp. plantarum\u003c/em\u003e strains to the natural nonproducing strains AMBP-0214 and AMBP-0424. A phylogenetic tree of the core genomes of all the strains tested showed that all the strains were closely related and that the nonproducers were not an outgroup (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Carotenoid-negative strains were more susceptible to oxidative and UV-induced stress than carotenoid-positive strains, in line with the protective role of carotenoid biosynthesis in \u003cem\u003eLp. plantarum\u003c/em\u003e strains. The degree of oxidative and UV stress resistance varied among the strains tested (Fig.\u0026nbsp;5A). A linear model including time, different experiments and the production of carotenoids showed that the viability of the strains decreased with longer oxidative stress treatment (Est \u0026minus;\u0026thinsp;0.32, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with increased survival for the carotenoid-producing strains (indicated by a less negative slope) (Est. 0.06, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The same protection was observed for UV-induced stress, as phenotypic carotenoid biosynthesis in these strains was associated with increased survival in all trials (Est. 0.024, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eC30 carotenoid biosynthesis is associated with nomadic and insect-adapted lifestyles\u003c/h2\u003e \u003cp\u003eWe subsequently evaluated whether the C30 carotenoid biosynthesis genes were associated with a particular \u003cem\u003eLactobacillaceae\u003c/em\u003e lifestyle and habitat adaptation using the categories defined by Zheng et al.Click or tap here to enter text.as free-living, vertebrate-adapted, invertebrate-adapted or nomadic \u003csup\u003e32\u003c/sup\u003e. Such lifestyle assignments were available for 170 out of 361 species in our public genome datasets. The presence of C30 carotenoid biosynthesis genes (\u003cem\u003ecrtMN)\u003c/em\u003e was most common in nomadic and insect-adapted lactobacilli, where they appeared as core (e.g. in nomadic \u003cem\u003eLp. plantarum\u003c/em\u003e, insect-adapted \u003cem\u003eFructilactobacillus lindneri\u003c/em\u003e and uncategorized \u003cem\u003eLc. citreum\u003c/em\u003e) and accessory traits. In contrast, \u003cem\u003ecrtMN\u003c/em\u003e genes were completely absent in vertebrate-associated lactobacilli such as \u003cem\u003eLactobacillus crispatus\u003c/em\u003e and \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e. They were also found in a few genomes representing free-living species such as \u003cem\u003eLv. buchneri\u003c/em\u003e (Fig.\u0026nbsp;5B). Consequently, a significant association between the proportion of genomes with C30 carotenoid biosynthesis and the lifestyle of a species (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was found when the Kruskal-Wallis test was used. Pairwise comparison with a Dunn test with Bonferroni correction for multiple testing (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003c/b\u003e) revealed the strongest association with nomadic and insect-adapted lifestyles. When considering the phylogenetic background with phylogenetic generalized least squares (PGLS), the same trends were found, but only insect-adapted versus free living was near significant (p\u0026thinsp;=\u0026thinsp;0.059) (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, nomadic and insect-associated \u003cem\u003ecrtMN\u003c/em\u003e-harboring strains differed in genome size, with the nomadic strains having large genomes, similar to other nomadic \u003cem\u003eLactobacillaceae\u003c/em\u003e, and the insect-adapted strains having the smallest genomes within the family. These findings point to positive selection of these genes after horizontal gain in species with nomadic and insect-adapted lifestyles (Fig.\u0026nbsp;5C). To further investigate the lifestyle association, we also screened the isolation sources of 575 in-house isolates and three publicly sourced isolates. We correlated this with the C30 carotenoid phenotype and genotype. This analysis pointed towards leaves and flowers carrying a high number of carotenoid producers belonging to the species \u003cem\u003eLt. fragifolii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;13), \u003cem\u003eLc. pseudomesenteroides\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eLc. citreum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3) and \u003cem\u003eH. floricola\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1) (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). A second major habitat of carotenoid producers showed to be plant-based fermentations, which harbored carotenoid-producing \u003cem\u003eLc. citreum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eLp. plantarum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;6) and \u003cem\u003eLactiplantibacillus paraplantarum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2). In contrast, in our laboratory, carotenoid-producing \u003cem\u003eLactobacillaceae\u003c/em\u003e strains were only sporadically isolated from vertebrate habitats (e.g., the human vagina\u003csup\u003e40\u003c/sup\u003e or respiratory tract\u003csup\u003e41\u003c/sup\u003e) and were all nomadic \u003cem\u003eLp. plantarum\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the biosynthesis of C30 carotenoids in the \u003cem\u003eLactobacillaceae\u003c/em\u003e family, the largest family of beneficial bacteria known to date, and linked the \u003cem\u003ecrtMN-\u003c/em\u003epositive genotype to their lifestyles. For this purpose, we used an integrated comparative genomic approach combined with a phenotypic screening of a diverse in-house \u003cem\u003eLactobacillaceae\u003c/em\u003e strain collection and an assessment of the functional role of carotenoids in stress resistance.\u003c/p\u003e \u003cp\u003ePangenome analysis of \u003cem\u003eLactobacillaceae\u003c/em\u003e revealed that c\u003cem\u003ertMN\u003c/em\u003e-mediated carotenoid biosynthesis was a rare and scattered trait within the family: it occurred in 41% of all the genera but only in a few species or strains within each genus. It was found to be a core property in some nomadic species, such as \u003cem\u003eLp. plantarum;\u003c/em\u003e insect-adapted species, such as \u003cem\u003eFl. lindneri;\u003c/em\u003e and accessory in many others. Phylogenetic analyses indicated that the biosynthetic \u003cem\u003ecrtMN\u003c/em\u003e genes are frequently transferred horizontally across species and genera, for example, from \u003cem\u003eLp. plantarum\u003c/em\u003e to \u003cem\u003eLv. brevis\u003c/em\u003e and within the \u003cem\u003eLeuconostoc\u003c/em\u003e genus. This finding is consistent with the high mobility of carotenoid pathways observed at higher taxonomic levels\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. Moreover, the trait appeared to have been gained convergently in \u003cem\u003eFructilactobacillus\u003c/em\u003e, with this genus having acquired \u003cem\u003ecrtMN\u003c/em\u003e genes from distinct donors during distinct events. Using a high-throughput extraction and analysis method, the \u003cem\u003ecrtMN\u003c/em\u003e genotype was matched with the synthesis of 4,4\u0026rsquo;-diaponeurosporene in five species. Functionally, we showed that \u003cem\u003eLp. plantarum\u003c/em\u003e strains that do not produce carotenoids were less resistant to UV and oxidative stress, in line with the general knowledge on carotenoids\u003csup\u003e16\u003c/sup\u003e and the previously observed protection against oxidative stress conferred by 4,4\u0026rsquo;-diaponeurosporene biosynthesis in \u003cem\u003eLp. plantarum\u003c/em\u003e\u003csup\u003e44\u003c/sup\u003e. Finally, the scattered distribution and mobility of this trait across the family, coupled with its advantages in UV and oxidative stress, prompted us to systematically investigate the link between carotenoid biosynthesis and the lifestyle and ecology of \u003cem\u003eLactobacillaceae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWhen testing for associations between two features associated with a set of species, as we have done here for carotenoids, phylogeny needs to be considered. This was done in our study by applying a phylogenetic generalized least squares (PGLS) approach\u003csup\u003e45\u003c/sup\u003e. Such an approach effectively takes into account that closely related species will likely share similarity in any two traits (such as \u003cem\u003ecrtMN\u003c/em\u003e prevalence and lifestyle studied here) because of \u0026rdquo;phylogenetic inertia\u0026rdquo;, not necessarily because the traits are correlated\u003csup\u003e46\u003c/sup\u003e. Since the lifestyles of \u003cem\u003eLactobacillaceae\u003c/em\u003e species are mostly conserved at genus level, this implies that the independent units of information are in general the genera rather than the species, resulting in a lower effective sample size and limitations in our dataset concerning statistical power. In addition, another limitation of our work is that a lifestyle assessment has not yet be attributed by \u003csup\u003e31,32\u003c/sup\u003e to 191 of the 361 \u003cem\u003eLactobacillaceae\u003c/em\u003e species in our dataset. This is due to lack of sufficient data on the isolation sources, metabolic potential, and related properties for these species\u003csup\u003e31,32\u003c/sup\u003e. For many of these species, only a single strain has been isolated from a single source. Repetitive isolation of species from the same environment, as well as substantiation with specific metabolic and experimental validation is required to attribute lifestyles, as the environment of isolation does not reflect the environment of adaptation (niche) for various reasons, such as random dispersal events and increasing anthropogenic effects on the biosphere. Such detailed information will have to be collected for these 191 \u003cem\u003eLactobacillaceae\u003c/em\u003e species to also be able to attribute a lifestyle in the future and further substantiate our analyses.\u003c/p\u003e \u003cp\u003eDespite these shortcomings in public data and taking into account the phylogeny, a near significant association was found between carotenoid biosynthesis genes and an insect-adapted lifestyle (p\u0026thinsp;=\u0026thinsp;0.056). However, excluding phylogenetic relatedness, we found that \u003cem\u003ecrtMN\u003c/em\u003e genes were mostly absent in free-living species and completely absent in vertebrate-associated species, such as the \u003cem\u003eL. crispatus\u003c/em\u003e, which is dominant in the human vagina\u003csup\u003e40\u003c/sup\u003e and \u003cem\u003eLm. reuteri\u003c/em\u003e which typically colonizes the vertebrate gut\u003csup\u003e31\u003c/sup\u003e. The complete absence of carotenoids in well-studied vertebrate associated \u003cem\u003eLactobacillaceae\u003c/em\u003e suggests that carotenoid production is not selected for in mucosal and low-oxygen habitats, such as the gut and vagina. In contrast, carotenoid biosynthesis genes were strongly associated with nomadic and insect-adapted \u003cem\u003eLactobacillaceae\u003c/em\u003e indicating that oxygen- and UV-rich environments encountered by nomadic and insect-adapted strains can select for this trait. The habitat-adaptation strategy of nomadic carotenoid producers appeared to differ from that of insect-adapted species. Nomadic \u003cem\u003eLactobacillaceae\u003c/em\u003e species typically have large genomes (between 2.4 and 3.6 Mbp), making them metabolically versatile and adaptable to various environments. In other studies, nomadic species have been found to typically occur in low numbers in oligotrophic environmental niches, such as plant surfaces\u003csup\u003e29,52\u003c/sup\u003e, and in high abundances once carbohydrates become more available, such as in vegetable fermentation products\u003csup\u003e53\u003c/sup\u003e. Such fermentations are characterized by intense microbial competition and high-salt concentration\u003csup\u003e53\u003c/sup\u003e, possibly leading to osmotic and oxidative stress. In such fermentations and outdoor oligotrophic environments, we speculate based on the data obtained here that C30 carotenoids could provide a fitness advantage to nomadic lactobacilli by reducing susceptibility to oxidative stress.\u003c/p\u003e \u003cp\u003eInsect-adapted lactobacilli have smaller genomes (1.2\u0026ndash;2.2 Mbp) with a concomitant decrease in carbohydrate metabolic capacity \u003csup\u003e31,32\u003c/sup\u003e. This difference was also observed in our present study among \u003cem\u003ecrtMN\u003c/em\u003e carriers, as the insect-adapted \u003cem\u003ecrtMN\u003c/em\u003e carriers had remarkably small genome sizes (between 1.6 and 2.1 Mbp), but they still contained \u003cem\u003ecrtMN genes\u003c/em\u003e, indicating an evolutionary advantage. Interestingly, the insect-adapted \u003cem\u003ecrtMN\u003c/em\u003e carriers were all part of one clade within the \u003cem\u003eFructilactobacillus\u003c/em\u003e genus, a genus known to be transferred between pollinators via the environment, with flowers serving as key hubs\u003csup\u003e31,47,48\u003c/sup\u003e. Our data presented here indicate that the biosynthesis of C30 carotenoids, and its associated protection against UV radiation and oxidative stress, could be a significant advantage for these environmentally dispersed, insect-adapted \u003cem\u003eLactobacillaceae\u003c/em\u003e species. Our hypothesis is in line with the adaptation strategy previously described for leaf-dwelling bacteria, such as \u003cem\u003eClavibacter\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, which produce C40 carotenoids and other pigments to increase their survival in this UV-stressed environment\u003csup\u003e9\u003c/sup\u003e. Notably, carotenoid biosynthesis was absent in \u003cem\u003eLactobacillaceae\u003c/em\u003e associated with social pollinators, such as \u003cem\u003eLactobacillus apis\u003c/em\u003e and \u003cem\u003eBombilactobacillus\u003c/em\u003e. These bacteria are vertically passed down to offspring within the hive\u003csup\u003e49\u003c/sup\u003e, where they are protected from UV- and oxidative stress. An environmental survival strategy does not seem required for these bacteria\u003csup\u003e49\u003c/sup\u003e. An exception to this is the \u003cem\u003eApilactobacillus\u003c/em\u003e genus, which is also known to be dispersed among solitary bees via flower\u003csup\u003e50\u003c/sup\u003e. This genus was shown in our study here to have an unusual putative terpenoid cluster, characterized by a duplicated \u003cem\u003ecrtN\u003c/em\u003e and an absent \u003cem\u003ecrtM\u003c/em\u003e gene. However, it remains to be substantiated whether this duplication is associated with a particular phenotype or pigment.\u003c/p\u003e \u003cp\u003eOur hypothesis that \u003cem\u003eLactobacillaceae\u003c/em\u003e bacteria that are dispersed to plants via insects have a competitive advantage expressing C30 carotenoids was supported by our extensive culture approach and collection. A diverse array of carotenoid-producing \u003cem\u003eLactobacillaceae\u003c/em\u003e were isolated from flowers and leaves, with a relative high prevalence found for \u003cem\u003eLc. citreum\u003c/em\u003e in flowers. This species has \u0026ndash; to the best of our knowledge - not yet been assigned to a certain lifestyle butour data presented here add support to an insect or flower-adapted lifestyle. In contrast, \u003cem\u003eLactobacillaceae\u003c/em\u003e isolates from vertebrate habitats studied here (mainly from the human vagina and respiratory tract), showed to be predominantly non-producers. Among the producing strains isolated, the nomadic \u003cem\u003eLp. plantarum\u003c/em\u003e was the most predominant species.\u003c/p\u003e \u003cp\u003eIn addition to the ecological role, the presence of \u003cem\u003ecrtMN\u003c/em\u003e genes in \u003cem\u003eLactobacillaceae\u003c/em\u003e is of interest from an applied perspective, especially considering the beneficial properties and lack of virulence factors in this family of bacteria. For example, incorporating carotenoid-producing bacteria into food fermentations could add additional functional properties to these foods. In fact, 4,4\u0026rsquo;-diaponeurosporene is already present in many vegetable fermentations, as \u003cem\u003eLp. plantarum\u003c/em\u003e, a core producer, dominates the later stages of most typical vegetable fermentations, and \u003cem\u003eLeuconostoc\u003c/em\u003e species generally dominate in the early stages\u003csup\u003e53\u003c/sup\u003e. Although the added benefits of 4,4\u0026rsquo;-diaponeurosporene in these fermented food ecosystems have not yet been studied, this metabolite has been connected to health-promoting effects via immune modulation. For instance, the introduction of \u003cem\u003ecrtMN\u003c/em\u003e genes originating from \u003cem\u003eStaphylococcus aureus\u003c/em\u003e into \u003cem\u003eBacillus subtilis\u003c/em\u003e has been shown to reduce colitis in mice\u003csup\u003e54\u003c/sup\u003e and increase resistance to \u003cem\u003eSalmonella typhimurium\u003c/em\u003e infection\u003csup\u003e55\u003c/sup\u003e. Furthermore, in piglets, heterologously c\u003cem\u003ertMN\u003c/em\u003e expressing-\u003cem\u003eB. subtilis\u003c/em\u003e bacteria have been shown to improve the mucosal immune system of the gut\u003csup\u003e56\u003c/sup\u003e and the respiratory tract\u003csup\u003e57\u003c/sup\u003e. These studies were carried out with genetically modified bacteria and are thus unlikely to reach large market applications, especially in Europe. In contrast, our results presented here indicate that natural carotenoid-producing \u003cem\u003eLactobacillaceae\u003c/em\u003e constitute an interesting alternative. Moreover, microbial biosynthesis can offer advantages over traditional production methods at it can be safer and less reliant on fossil resources than chemical synthesis and less influenced by seasonality or climate than plant-based biosynthesis\u003csup\u003e58\u003c/sup\u003e and applied to C30 carotenoids\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, this study on the ecology and evolution of carotenoid biosynthesis in the \u003cem\u003eLactobacillaceae\u003c/em\u003e family revealed a scattered distribution of \u003cem\u003ecrtMN\u003c/em\u003e-mediated C30 carotenoid 4,4\u0026rsquo;-diaponeurosporene biosynthesis across 28 species and 14 genera and highlighted the mobility of this trait. C30 carotenoid biosynthesis appears to have emerged as a core property in several species, notably \u003cem\u003eLp. plantarum\u003c/em\u003e, \u003cem\u003eLc. citreum\u003c/em\u003e and \u003cem\u003eFl. lindneri\u003c/em\u003e. Furthermore, carotenoid biosynthesis was strongly associated with nomadic and insect-adapted lifestyles, where it offers an advantage via protection from UV and oxidative stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eS.L. received funding from different probiotic companies that were not involved in this research. T.E. is partially funded through an industrial research VLAIO grant not related to this work. M.L. is employed part-time by Biobest Group NV, this company was not involved in this research. I.V. and K.S. are employed by the RIC and their work was compensated via a service contract. P.A.B. is an independent consultant for several companies in the food and pharmaceutical industry bound by confidentiality agreements.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research has received funding from the following funding bodies: the European Research Council (ERC; starting grant Lacto-Be 852600 of S.L.), the Belgian Science Policy Office (BELSPO; BRAIN-be 2.0; B@SEBALL (B2/191/P3/B@SEBALL)), the Scientific Research Foundation \u0026ndash; Flanders (FWO; doctoral grant of J.T. 1SC3623N and postdoctoral grant of S.W. 12AZ624N), the Industrial Research Fund of the University of Antwerp (IOF-SEP 48530), VLAIO (HBC.2022.1000), and from the revenue of our microbiome platform services.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors would like to thank Tim Van Rillaer for his help with the bioinformatic pipelines and Ines Tuyaerts, Nele Van de Vliet and Sam Bakelants for their help with the UV and oxidative stress tests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun, T., Rao, S., Zhou, X., Li, L.: Plant carotenoids: recent advances and future perspectives. \u003cem\u003eMolecular Horticulture\u003c/em\u003e vol. 2 Preprint at (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43897-022-00023-2\u003c/span\u003e\u003cspan address=\"10.1186/s43897-022-00023-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHavaux, M., And, Niyogi, K.K.: \u003cem\u003eThe Violaxanthin Cycle Protects Plants from Photooxidative Damage by More than One Mechanism\u003c/em\u003e. \u003cem\u003ePlant Biology\u003c/em\u003e vol. 96 (1999). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.pnas.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFraser, N.J., Hashimoto, H., Cogdell, R.J.: Carotenoids and Bacterial Photosynthesis: The Story so Far. 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Sci. \u003cb\u003e136\u003c/b\u003e, 310\u0026ndash;317 (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCravens, A., Payne, J., Smolke, C.D.: Synthetic biology strategies for microbial biosynthesis of plant natural products. \u003cem\u003eNature Communications\u003c/em\u003e vol. 10 Preprint at (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-019-09848-w\u003c/span\u003e\u003cspan address=\"10.1038/s41467-019-09848-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiziya, I.N., Hwang, C.Y., Seo, M.J.: Antioxidant Potential and Capacity of Microorganism-Sourced C30 Carotenoids\u0026mdash;A Review. \u003cem\u003eAntioxidants\u003c/em\u003e 11, (2022)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4637278/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4637278/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarotenoids are membrane-bound pigments that are essential for photosynthesizing plants and algae. These specialized metabolites are widely applied in food, feed and cosmetics because of their antioxidant and anti-inflammatory properties. The production of carotenoids, particularly C30 forms, has also been documented in a few non-photosynthetic prokaryotes. However, our understanding of the function, distribution and ecology of these compounds beyond photosynthesizing organisms is limited. In this study, we performed an eco-evolutionary analysis of terpenoid biosynthetic gene clusters in the \u003cem\u003eLactobacillaceae\u003c/em\u003e family, an important family of beneficial bacteria with diverse lifestyles and habitats. Hereto, 4203 dereplicated genomes were screened for terpenoid biosynthesis genes, resulting in detection of \u003cem\u003ecrtMN\u003c/em\u003e genes in 28/361 (7.7%) species in 14/34 (41.2%) genera. These genes encode the key enzymes that transform two farnesyl pyrophosphate molecules into the C30 carotenoid 4,4\u0026rsquo;-diaponeurosporene. These \u003cem\u003ecrtMN\u003c/em\u003e genes appeared to be convergently gained within \u003cem\u003eFructilactobacillus\u003c/em\u003e, and horizontally transferred across species and genera, e.g. from \u003cem\u003eLactiplantibacillus\u003c/em\u003e to \u003cem\u003eLevilactobacillus\u003c/em\u003e. Subsequently, in our in-house \u003cem\u003eLactobacillaceae\u003c/em\u003e culture collection from various habitats (n\u0026thinsp;=\u0026thinsp;575), the phenotype was confirmed in 87% of the isolates predicted to have the \u003cem\u003ecrtMN\u003c/em\u003e genes (27/31). Nomadic and insect-adapted species, particularly those isolated from vegetable fermentations, such as \u003cem\u003eLactiplantibacillus\u003c/em\u003e, and floral habitats, such as \u003cem\u003eFructilactobacillus\u003c/em\u003e, were capable of C30 carotenoid biosynthesis, while vertebrate-associated species, such as those from the vagina, lacked this trait. This habitat association aligned with the observations that C30 carotenoid-producing strains were more resistant to oxidative and UV-stress. Taken together, this study revealed that C30 carotenoid biosynthesis plays a role in habitat adaptation and is scattered across \u003cem\u003eLactobacillaceae\u003c/em\u003e in line with this habitat adaptation.\u003c/p\u003e","manuscriptTitle":"Distribution of C30 carotenoid biosynthesis genes suggests habitat adaptation function in insect-adapted and nomadic Lactobacillaceae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-13 16:41:38","doi":"10.21203/rs.3.rs-4637278/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bc9a5a39-27cf-46a6-95b2-49c4ed2ed735","owner":[],"postedDate":"August 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34120514,"name":"Biological sciences/Microbiology/Environmental microbiology"},{"id":34120515,"name":"Biological sciences/Ecology/Microbial ecology"},{"id":34120516,"name":"Biological sciences/Microbiology/Microbial genetics/Bacterial genes"}],"tags":[],"updatedAt":"2024-12-04T08:06:35+00:00","versionOfRecord":{"articleIdentity":"rs-4637278","link":"https://doi.org/10.1038/s42003-024-07291-2","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2024-12-03 05:00:00","publishedOnDateReadable":"December 3rd, 2024"},"versionCreatedAt":"2024-08-13 16:41:38","video":"","vorDoi":"10.1038/s42003-024-07291-2","vorDoiUrl":"https://doi.org/10.1038/s42003-024-07291-2","workflowStages":[]},"version":"v1","identity":"rs-4637278","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4637278","identity":"rs-4637278","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}