Carotenoid productivity in human intestinal bacteria Eubacterium limosum and Leuconostoc mesenteroides with functional analysis of their carotenoid biosynthesis genes

Abstract

The human intestinal microbiota that comprise over 1,000 species thrive in dark and anaerobic environments. They are recognized for the production of diverse low-molecular-weight metabolites crucial to human health and diseases. Carotenoids, low-molecular-weight pigments known for their antioxidative activity, are delivered to humans through oral intake. However, it remains unclear whether human intestinal bacteria biosynthesize carotenoids as part of the in-situ microbiota. In this study, we investigated carotenoid synthesis genes in various human gut and probiotic bacteria. As a result, novel candidates, the crtM and crtN genes, were identified in the carbon monoxide-utilizing gut anaerobe Eubacterium limosum and the lactic acid bacterium Leuconostoc mesenteroides subsp. mesenteroides. These gene candidates were isolated, introduced into Escherichia coli, which synthesized a carotenoid substrate, and cultured aerobically. Structural analysis of the resulting carotenoids revealed that the crtM and crtN gene candidates of E. limosum and L. mesenteroides mediate the production of 4,4′-diaponeurosporene through 15-cis-4,4′-diapophytoene. Evaluation of the crtE-homologous genes in these bacteria indicated their non-functionality for C₄₀-carotenoid production. E. limosum and L. mesenteroides, along with the known carotenogenic lactic acid bacterium Lactiplantibacillus plantarum, were observed to produce no carotenoids under strictly anaerobic conditions. The two lactic acid bacteria synthesized detectable levels of 4,4′-diaponeurosporene under semi-aerobic conditions. The findings highlight that the obligate anaerobe E. limosum retains aerobically functional C₃₀-carotenoid biosynthesis genes, potentially with no immediate self-utility, suggesting an evolutionary direction in carotenoid biosynthesis. (229 words)

Keywords

C₃₀ carotenoid biosynthesis / Obligate anaerobe / Human gut bacterium / Eubacterium limosum / Leuconostoc mesenteroides / Lactiplantibacillus plantarum

Data Availability Statement

Data used in this study are available on request to the corresponding authors. Main plasmids are available from Riken BRC (https://web.brc.riken.jp/en/).

Declaration of Competing Interest

All the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this research article.

CRediT authorship contribution statement

Wataru Matsumoto: Investigation. Miho Takemura: Writing - original draft, Investigation. Haruka Nanaura: Investigation. Yuta Ami: Investigation. Takashi Maoka: Investigation. Kazutoshi Shindo: Writing - original draft, Investigation. Shin Kurihara: Validation, Resources. Norihiko Misawa: Writing - review & editing, Writing - original draft, Supervision, Conceptualization.

Acknowledgments

The authors are grateful to Chiharu Takagi and Yuka Sakemi, Japan Women's University, for their support in purification experiments and NMR analysis. We thank Ms. Chisako Fuchimoto for her technical support in molecular experiments. This research received no external funding.

Ethical Approval

This research article does not contain any studies with human participants or animals performed by any of the authors.

References

[1] J. Qin, R. Li, J. Raes, M. Arumugam, K.S. Burgdorf, C. Manichanh, T. Nielsen, N. Pons, F. Levenez, T. Yamada, D.R. Mende, J. Li, J. Xu, S. Li, D. Li, J. Cao, B. Wang, Liang, H. Zheng, Y. Xie, J. Tap, P. Lepage, M. Bertalan, J.M. Batto, T. H D. L. Paslier, A. Linneberg, H.B. Nielsen, E. Pelletier, P. Renault, T. Sicheritz-Ponten, K. Turner, H. Zhu, C. Yu, S. Li, M. Jian, Y. Zhou, Y. Li, X. Zhang, S. Li, N. Qin, H. Yang, J. Wang, S. Brunak, J. Doré, F. Guarner, K. Kristiansen, O. Pedersen, J. Parkhill, J. Weissenbach, M. Consortium, P. Bork, S.D. Ehrlich, J. Wang, A human gut mi- crobial gene catalogue established by metagenomic sequencing, Nature 464 (2010) 59-65.

[2] M. Matsumoto, R. Kibe, T. Ooga, Y. Aiba, S. Kurihara, E. Sawaki, Y. Koga, Y. Benno, Impact of intestinal microbiota on intestinal luminal metabolome, Sci. Rep. 2 (2012) 233, doi:10.1038/srep00233.

[3] P.V. Kirjavainen, T. Arvola, S.J. Salminen, E. Isolauri, Aberrant composition of gut microbiota of allergic infants: a target of bifidobacterial therapy at weaning? Gut 51 (2002) 51-55.

[4] J. Penders, E.E. Stobberingh, P.A. van den Brandt, C. Thijs, The role of the intestinal microbiota in the development of atopic disorders, Allergy 62 (2007) 1223-1236.

[5] G.W. Tannock, Molecular analysis of the intestinal microflora in IBD, Mucosal Im- munol. 1 (2008) S15-S18 1Suppl, doi:10.1038/mi.2008.54.

[6] I. Sobhani, J. Tap, F. Roudot-Thoraval, J.P. Roperch, S. Letulle, P. Langella, G. Cor- thier, J. Tran van Nhieu, J.P Furet, Microbial dysbiosis in colorectal cancer (CRC) patients, PLoS ONE 6 (2011) e16393, doi:10.1371/journal.pone.0016393.

[7] S. Fukuda, H. Ohno, Gut microbiome and metabolic diseases, Semin. Immunopathol. 36 (2014) 103-114.

[8] S. Schippa, M.P. Conte, Dysbiotic events in gut microbiota: impact on human health, Nutrients 6 (2014) 5786-5805.

[9] D. Lin, D.M. Medeiros, The microbiome as a major function of the gastrointestinal tract and its implication in micronutrient metabolism and chronic diseases, Nutrition Res. 112 (2023) 30-45 ISSN 0271-5317, doi:10.1016/j.nutres.2023.02.007.

[10] N.I. Krinsky, Antioxidant functions of carotenoids, Free Radic. Biol. Med. 7 (1989) 617-635.

[11] G. Britton, S. Liaaen-Jensen, H. Pfander, Carotenoids Handbook. Basel editors, Birkhäuser Verlag, Boston, Berlin, 2004.

[12] P.D. Fraser, P.M. Bramley, The biosynthesis and nutritional uses of carotenoids, Prog. Lipid Res. 43 (2004) 228-265.

[13] S. Takaichi, Carotenoids in algae: distributions, biosynthesis and functions, Mar. Drugs 9 (2011) 1101-1118.

[14] Alcaíno J., Baeza M., Cifuentes V. Carotenoid distribution in nature. In: Stange C, ed- itor. Carotenoids in Nature. Subcellular Biochemistry, vol 79. Switzerland: Springer; 2016, p. 3-33. 10.1007/978-3-319-39126-7_1

[15] Misawa N., editor. Carotenoids: biosynthetic and Biofunctional Approaches. Adv. Exp. Med. Biol. 1261. Singapore: Springer; 2021. 10.1007/978-981-15-7360-6

[16] B. Tian, Y. Hua, Carotenoid biosynthesis in extremophilic Deinococcus-Thermus bac- teria, Trends Microbiol. 18 (2010) 512-520.

[17] K. Shindo, N. Misawa, New and rare carotenoids isolated from marine bac- teria and their antioxidant activities, Mar. Drugs 12 (2014) 1690-1698, doi:10.3390/md12031690.

[18] N. Takatani, K. Nishida, T. Sawabe, T. Maoka, K. Miyashita, M. Hosokawa, Identifi- cation of a novel carotenoid, 2′ -isopentenylsaproxanthin, by Jejuia pallidilutea strain 11shimoA1 and its increased production under alkaline condition, Appl. Microbiol. Biotechnol. 98 (2014) 6633-6640, doi:10.1007/s00253-014-5702-y.

[19] E. Reboul, Mechanisms of carotenoid intestinal absorption: where do we stand? Nu- trients 11 (4) (2019) 838, doi:10.3390/nu11040838.

[20] W. Stahl, H. Site, Bioactivity and protective effects of natural carotenoids, Biochim. Biophys. Acta 1740 (2004) 101-107.

[21] H. Nishino, H. Tokuda, Y. Satomi, M. Masuda, P. Bu, M. Onozuka, S. Yamaguchi, Y. Okuda, J. Takayasu, J. Tsuruta, M. Okuda, E. Ichiishi, M. Murakoshi, T. Kato, N. Misawa, T. Narisawa, N. Takasuka, M. Yano, Cancer prevention by carotenoids, Pure Appl. Chem. 71 (1999) 2273-2278.

[22] H. Tapiero, D.M. Townsend, K.D. Tew, The role of carotenoids in the prevention of human pathologies, Biomed. Pharmacother. 58 (2004) 100-110 e4ISSN 0753-3322, doi:10.1016/j.biopha.2003.12.006.

[23] M.M. Ciccone, F. Cortese, M. Gesualdo, S. Carbonara, A. Zito, G. Ricci, F.D. Pascalis, P. Scicchitano, G. Riccioni, Dietary intake of carotenoids and their antioxidant and anti-inflammatory effects in cardiovascular care, Mediators Inflamm. (2013) Article ID 782137, doi:10.1155/2013/782137.

[24] J. Fiedor, K. Burda, Potential role of carotenoids as antioxidants in human health and disease, Nutrients 6 (2014) 466-488.

[25] J. Garrido-Fernández, A. Maldonado-Barragán, B. Caballero-Guerrero, D. Hornero-Méndez, J.L. Ruiz-Barba, Carotenoid production in Lactobacillus plantarum, Int. J. Food Microbiol. 140 (2010) 34-39.

[26] T. Hagi, M. Kobayashi, M. Nomura, Whole-transcriptome analysis of oxidative stress response genes in carotenoid-producing Enterococcus gilvus, Biosci. Biotech- nol. Biochem. 82 (2018) 1053-1057.

[27] C. Santana-Molina, V. Henriques, D. Hornero-Méndez, D.P. Devos, E. Rivas-Marin, The squalene route to C30 carotenoid biosynthesis and the origins of carotenoid biosynthetic pathways, PNAS 119 (52) (2022) e2210081119, doi:10.1073/pnas.2210081119.

[28] A. Gotoh, M. Nara, Y. Sugiyama, M. Sakanaka, H. Yachi, A. Kitakata, A. Nakagawa, H. Minami, S. Okuda, T. Katoh, T. Katayama, S. Kurihara, Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles, Biosci. Biotechnol. Biochem. 81 (2017) 2009-2017.

[29] J. Zheng, S. Wittouck, E. Salvetti, C.M.A.P. Franz, H.M.B. Harris, P. Mattarelli, P.W. O’Toole, B. Pot, P. Vandamme, J. Walter, K. Watanabe, S. Wuyts, G.E. Felis, M. G. Gänzle, S Lebeer, A taxonomic note on the genus Lactobacillus: description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae, Int. J. Syst. Evol. Microbiol. 70 (2020), doi:10.1099/ijsem.0.004107.

[30] M. Takemura, A. Kubo, Y. Higuchi, T. Maoka, T. Sahara, K. Yaoi, K. Ohdan, D. Umeno, N. Misawa, Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia. coli, Appl. Microbiol. Biotechnol. 103 (2019) 9393-9399.

[31] Y. Nishida, K. Adachi, H. Kasai, Y. Shizuri, K. Shindo, A. Sawabe, S. Komemushi, W. Miki, N Misawa, Elucidation of a carotenoid biosynthesis gene cluster encod- ing a novel enzyme, 2,2’-𝛽 -hydroxylase, from Brevundimonas sp. strain SD212 and combinatorial biosynthesis of new or rare xanthophylls, Appl. Environ. Microbiol 71 (2005) 4286-4296.

[32] J. Huerta-Cepas, F. Serra, P. Bork, ETE 3: reconstruction, analysis, and visualization of phylogenomic data, Mol. Biol. Evol. 33 (2016) 1635-1638 Epub 2016 Feb 26. PMID: 26921390; PMCID: PMC4868116, doi:10.1093/molbev/msw046.

[33] L-T Nguyen, H.A. Schmidt A, von Haeseler, Minh BQ. IQ-TREE: a fast and effec- tive stochastic algorithm for estimating maximum-likelihood phylogenies, Mol. Biol. Evol 32 (2015) 268-274.

[34] S. Kajiwara, P.D. Fraser, K. Kondo, N. Misawa, Expression of an exogenous isopen- tenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli, Biochem. J. 324 (1997) 421-426.

[35] R.E. Rose, The nucleotide sequence of pACYC184, Nuc. Acids Res. 16 (1988) 355.

[36] N. Misawa, T. Maoka, M. Takemura, Carotenoids: carotenoid and apocarotenoid analysis-use of E. coli to produce carotenoid standards, Methods Enzymol 670 (2022) 87-137 PMID: 35871847, doi:10.1016/bs.mie.2022.03.019.

[37] N. Misawa, Y. Satomi, K. Kondo, A. Yokoyama, S. Kajiwara, T. Saito, T. Ohtani, W. Miki, Structure and functional analysis of a marine bacterial carotenoid biosyn- thesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level, J. Bacteriol. 177 (1995) 6575-6584.

[38] M. Albrecht, N. Misawa, G. Sandmann, Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids 𝛽-carotene and zeaxanthin, Biotechnol. Lett. 21 (1999) 791-795.

[39] M. Kim, D.H. Jung, D.H. Seo, W.H. Chung, M.J. Seo, Genome analysis of Lactobacil- lus plantarum subsp. plantarum KCCP11226 reveals a well-conserved C30 carotenoid biosynthetic pathway, 3 Biotech. 10 (2020) 150, doi:10.1007/s13205-020-2149-y.

[40] B. Wieland, C. Feil, E. Gloria-Maercker, G. Thumm, M. Lechner, J.M. Bravo, K. Po- ralla, F. Götz, Genetic and biochemical analyses of the biosynthesis of the yellow carotenoid 4,4′ -diaponeurosporene of Staphylococcus aureus, J. Bacteriol. 176 (1994) 7719-7726.

[41] S. Tiziani, S.J. Schwartz, Y. Vodovotz, Profiling of carotenoids in tomato juice by one- and two-dimensional NMR, J. Agric. Food Chem. 54 (2006) 6094-6100.

[42] M. Inomata, N. Hirai, R. Yoshida, H. Ohigashi, Biosynthesis of abscisic acid by the direct pathway via ionylideneethane in a fungus, Cercospora cruenta, Biosci. Biotech- nol. Biochem 68 (2004) 2571-2580.

[43] W. Turpin, C. Renaud, S. Avallone, A. Hammoumi, J.P. Guyot, C. Humblot, PCR of crtNM combined with analytical biochemistry: an efficient way to identify carotenoid producing lactic acid bacteria, Syst. Appl. Microbiol. 39 (2016) 115-121.

[44] M. Kim, D-H Seo, Y.S. Park, I.T. Cha, M.J. Seo, Isolation of Lactobacillus plantarum subsp. plantarum producing C30 carotenoid 4,4′ -diaponeurosporene and the assess- ment of its antioxidant activity, J. Microbiol. Biotechnol. 29 (2019) 1925-1930.

[45] D. Umeno, A.V. Tobias, F.H. Arnold, Evolution of the C30 carotenoid syn- thase CrtM for function in a C40 pathway, J. Bacteriol. 184 (2002) 6690-6699, doi:10.1128/jb.184.23.6690-6699.2002.

[46] J. Harada, K.V.P. Nagashima, S. Takaichi, M. Misawa, K. Matsuura, K. Shimada, Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelati- nosus, produces both neurosporene and lycopene, Plant Cell Physiol. 42 (2001) 1112-1118, doi:10.1093/pcp/pce140.

[47] G.A. Armstrong, M. Alberti, F. Leach, J.E. Hearst, Nucleotide sequence, or- ganization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus, Mol. Gen. Genet. 216 (1989) 254-268, doi:10.1007/BF00334364.

[48] L. Tao, A. Schenzle, J.M. Odom, Q. Cheng, Novel carotenoid oxidase involved in biosynthesis of 4,4’-diapophytoene dialdehyde, Appl. Envion. Microbiol. 71 (2005) 3294-3301.

[49] S. Steiger, L. Perez Fons, S.M. Cutting, P.D. Fraser, G Sandmann, Annotation and functional assignment of the genes for the C30 carotenoid pathways from the genomes of two bacteria: bacillus indicus and Bacillus firmus, Microbiology 161 (2015) 194-202.

[50] B.R.S. Genthner, M.P. Bryant, Growth of Eubacterium limosum with carbon monoxide as the energy source, Appl. Environ. Microbiol. 43 (1982) 70-74.

[51] D. Litty, V. Müller, Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source, Microb. Biotechnol. 14 (2021) 2686-2692.

[52] H.G. Hur, F. Rafii, Biotransformation of the isoflavonoids biochanin A, for- mononetin, and glycitein by Eubacterium limosum, FEMS Microbiol. Lett. 192 (2000) 21-25, doi:10.1111/j.1574-6968.2000.tb09353.x.