The human intestine hosts a complex bacterial community that plays a major role in nutrition and in maintaining human health. A functional metagenomic approach was used to explore the prebiotic breakdown potential of human gut bacteria, including non-cultivated ones. Two metagenomic libraries, constructed from ileum mucosa and fecal microbiota, were screened for hydrolytic activities on the prebiotic carbohydrates inulin, fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides and lactulose. The DNA inserts of 17 clones, selected from the 167 hits that were identified, were pyrosequenced in-depth, yielding in total 407, 420 bp of metagenomic DNA. From these sequences, we discovered novel prebiotic degradation pathways containing carbohydrate transporters and hydrolysing enzymes, for which we provided the first experimental proof of function. Twenty of these proteins are encoded by genes that are also present in the gut metagenome of at least 100 subjects, whatever are their ages or their geographical origin. The sequence taxonomic assignment indicated that still unknown bacteria, for which neither culture conditions nor genome sequence are available, possess the enzymatic machinery to hydrolyse the prebiotic carbohydrates tested. The results expand the vision on how prebiotics are metabolized along the intestine, and open new perspectives for the design of functional foods.
References
[1]
Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, et al. (2005) Diversity of the human intestinal microbial flora. Science 308: 1635–1638.
[2]
Wang M, Ahrné S, Jeppsson B, Molin G (2005) Comparison of bacterial diversity along the human intestinal tract by direct cloning and sequencing of 16S rRNA genes. FEMS Microbiol Ecol 54: 219–231.
[3]
Booijink CC, Zoetendal EG, Kleerebezem M, de Vos WM (2007) Microbial communities in the human small intestine: coupling diversity to metagenomics. Future Microbiol 2: 285–295.
[4]
Rajili？-Stojanovi？ M, Smidt H, de Vos WM (2007) Diversity of the human gastrointestinal tract microbiota revisited. Environ Microbiol 9: 2125–2136.
[5]
Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, et al. (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464: 59–65.
[6]
Kleerebezem M (2010) Metagenomic approaches to unravel the composition and function of the human small intestine microbiota. In:Heidt PJ, Snel J, Midtvedt T, Rusch V editors. Intestinal microbiomics: novel indicators of health and disease. Old Herborn University. 27–42.
[7]
Zoetendal EG, Raes J, van den Bogert B, Arumugam M, Booijink C, et al. (2012) The human small intestinal microbiota is driven by rapid uptake and conversion of simple carbohydrates. ISME J 6: 1415–1426.
[8]
Lepage P, Leclerc MC, Joossens M, Mondot S, Blottière HM, et al. (2013) A metagenomic insight into our gut's microbiome. Gut 62: 146–158.
[9]
Ottman N, Smidt H, deVos WM, Belzer C (2012) The function of our microbiota: who is out there and what do they do? Front Cell Infect Microbiol 2, DOI 10.3389/fcimb.2012.00104.
[10]
Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, et al. (2006) Metagenomic analysis of the human distal gut microbiome. Science 312: 1355–1359.
[11]
Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA (2008) Polysaccharide utilization by gut bacteria: Potential for new insights from genomic analysis. Nat Rev Microbiol 6: 121–131.
[12]
Cummings JH, Macfarlane GT (1991) The control and consequences of bacterial fermentation in the human colon. J Appl Bacteriol 70: 443–459.
[13]
Grabitske HA, Slavin JL (2008) Low-digestible carbohydrates in practice. J Am Diet Assoc 108: 1677–1681.
[14]
Roberfroid M (2007) Prebiotics: the concept revisited. J Nutr 137: 830S–837S.
[15]
Figueroa-González I, Cruz-Guerrero A, Quijano G (2011) The benefits of probiotics on human health. J Microbial Biochem Technol S1: 003.
[16]
Bird AR, Conlon MA, Christophersen CT, Topping DL (2010) Resistant starch, large bowel fermentation and a broader perspective of prebiotics and probiotics. Benef Microbes 1: 423–431.
[17]
Jaskari J, Kontula P, Siitonen A, Jousimies-Somer H, Mattila-Sandholm T, et al. (1998) Oat beta-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains. Appl Microbiol Biotechnol 49: 175–181.
[18]
Djouzi Z, Andrieux C, Pelenc V, Somarriba S, Popot F, et al. (1995) Degradation and fermentation of alpha-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats. J Appl Bacteriol 79: 117–127.
[19]
Sarbini SR, Kolida S, Naeye T, Einerhand A, Brison Y, et al. (2011) In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota. Appl Environ Microbiol 77: 5307–5315.
[20]
Herfel TM, Jacobi SK, Lin X, Fellner V, Walker DC, et al. (2011) Polydextrose enrichment of infant formula demonstrates prebiotic characteristics by altering intestinal microbiota, organic acid concentrations, and cytokine expression in suckling piglets. J Nutr 141: 2139–2145.
[21]
Ohkusa T, Ozaki Y, Sato C, Mikuni K, Ikeda H (1995) Long-term ingestion of lactosucrose increases Bifidobacterium sp. in human fecal flora. Digestion 56: 415–420.
[22]
Gullón B, Gullón P, Sanz Y, Alonso JL, Parajó JC (2011) Prebiotic potential of a refined product containing pectic oligosaccharides. LWT-Food Sci Technol 44: 1687–1696.
[23]
Hopkins MJ, Cummings JH, Macfarlane GT (1998) Inter-species differences in maximum specific growth rates and cell yields of bifidobacteria cultured on oligosaccharides and other simple carbohydrate sources. J Appl Bacteriol 85: 381–386.
[24]
Rask Licht T, Ebersbach T, Fr？ki？r H (2012) Prebiotics for prevention of gut infections. Trends in Food Science & Technology 23: 70–82.
[25]
Macfarlane S, Macfarlane GT, Cummings JH (2006) Review article: prebiotics in the gastrointestinal tract. Aliment Pharmacol Ther 24: 701–714.
[26]
Goulas T, Goulas A, Tzortzis G, Gibson GR (2009) Expression of four β-galactosidases from Bifidobacterium bifidum NCIMB41171 and their contribution on the hydrolysis and synthesis of galactooligosaccharides. Appl Microbiol Biotechnol. 84 (5): 899–907.
[27]
Rycroft CE, Jones MR, Gibson GR, Rastall RA (2001) A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. J Appl Microbiol 91: 878–887.
[28]
Van der Meulen R, Makras L, Verbrugghe K, Adriany T, De Vuyst L (2006) In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms. Appl Environ Microbiol 72: 1006–1012.
[29]
Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, et al. (2009) Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii. Brit J Nutr 101: 541–550.
[30]
Kleessen B, Hartmann L, Blaut M (2001) Oligofructose and long-chain inulin: influence on the gut microbial ecology of rats associated with a human faecal flora. Brit J Nutr 86: 291–300.
[31]
Langlands SJ, Hopkins MJ, Coleman N, Cummings JH (2004) Prebiotic carbohydrates modify the mucosa-associated microflora of the human large bowel. Gut 53: 1610–1616.
[32]
Duncan SH, Hold GL, Harmsen HJM, Stewart CS, Flint HJ (2002) Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov. Int J Syst Evol Microbiol 52: 2141–2146.
[33]
Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B, et al. (2002) The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. PNAS 99: 14422–14427.
[34]
Barrangou R, Altermann E, Hutkins R, Cano R, Klaenhammer RT (2003) Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus. PNAS 100: 8957–8962.
[35]
Goh JY, Zhang C, Benson KA, Schlegel V, Lee JH, et al. (2006) Identification of a putative operon involved in fructooligosaccharide utilization by Lactobacillus paracasei. Appl Environ Microbiol 72: 7518–7530.
[36]
Ryan MS, Fitzgerald FG, van Sinderen D (2005) Transcriptional regulation and characterization of a novel beta-fructofuranosidase-encoding gene from Bifidobacterium breve UCC2003. Appl Environ Microbiol 71: 3475–3482.
[37]
Scott KP, Martina JC, Chassarda C, Clergeta M, Potrykusa J, et al. (2011) Substrate-driven gene expression in Roseburia inulinivorans: Importance of inducible enzymes in the utilization of inulin and starch. PNAS 108: 4672–4679.
[38]
Majumder A, Sultan A, Jersie-Christensen RR, Ejby M, Schmidt BG, et al. (2011) Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol. Proteomics 11: 3470–3481.
[39]
Imamura L, Hisamitsu K, Kobashi K (1994) Purification and characterization of beta-fructofuranosidase from Bifidobacterium infantis. Biol Pharm Bull 17: 596–602.
[40]
Van Laere KMJ, Hartemink R, Beldman G, Pitson S, Dijkema C, et al. (1999) Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase. Appl Microbiol Biotechnol 52: 681–688.
[41]
Sonnenburg ED, Zheng H, Joglekar P, Higginbottom SK, Firbank SJ, et al. (2010) Secificity of polysaccharide use in intestinal Bacteroides species determines diet-induced microbiota alterations. Cell 141: 1241–1252.
[42]
Lagaert S, Pollet A, Delcour JA, Lavigne R, Courtin CM, et al. (2011) Characterization of two β-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides. Appl Microbiol Biotechnol 92: 1179–1185.
[43]
Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, et al. (2012) Human gut microbiome viewed across age and geography. Nature 486: 222–228.
[44]
Tasse L, Bercovici J, Pizzut-Serin S, Robe P, Tap J, et al. (2010) Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes. Genome Res 20: 1605–1612.
[45]
Aider M, de Halleux D (2007) Isomerization of lactose and lactulose production: review. Trends Food Sci Tech 18: 356–364.
[46]
Kunz C, Rudloff S, Baier W, Klein N, Strobel S (2000) Oligosaccharides in human milk: structural, functional, and metabolic aspects. Annu Rev Nutr 20: 699–722.
[47]
Urashima T, Saito T, Nakamura T, Messer M (2001) Oligosaccharides of milk and colostrum in non-human mammals. Glycoconjugate J 18: 357–371.
[48]
Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, et al. (2007) Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes. DNA Res 14: 169–181.
[49]
Turnbaugh PJ, Gordon JI (2009) The core gut microbiome, energy balance and obesity. J Physiol 587: 4153–4158.
[50]
Kullin B, Abratt VR, Reid SJ (2006) A functional analysis of the Bifidobacterium longum cscA and scrP genes in sucrose utilization. Appl Microbiol Biotechnol 72: 975–981.
[51]
Bujacz A, Jedrzejczak-Krzepkowska M, Bielecki S, Redzynia I, Bujacz G (2011) Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose. FEBS J 278: 1728–1744.
[52]
Jedrzejczak-Krzepkowska M, Tkaczuk KL, Bielecki S (2011) Biosynthesis, purification and characterization of β-fructofuranosidase from Bifidobacterium longum KN29.1. Process Biochem 46: 1963–1972.
[53]
Lagaert S, Van Campenhout S, Pollet A, Bourgois TM, Delcour JA, et al. (2007) Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis. Appl Environ Microbiol 73: 5374–5377.
[54]
Lagaert S, Pollet A, Delcour JA, Lavigne R, Courtin CM, et al. (2010) Substrate specificity of three recombinant α-L-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides. Biochem Bioph Res Co 402: 644–650.
[55]
González R, Klaassens ES, Malinen E, de Vos WM, Vaughan EE (2008) Differential transcriptional response of Bifidobacterium longum to human milk, formula milk, and galactooligosaccharide. Appl Environ Microbiol 74: 4686–4694.
[56]
Belenguer A, Duncan SH, Calder AG, Holtrop G, Louis P, et al. (2006) Two routes of metabolic cross-feeding between Bifidobacterium adolescentis and butyrate-producing anaerobes from the human gut. Appl Environ Microbiol 72: 3593–3599.
[57]
Rossi M, Corradini C, Amaretti A, Nicolini M, Pompei A, et al. (2005) Fermentation of fructooligosaccharides and inulin by Bifidobacteria: A comparative study of pure and fecal cultures. Appl Environ Microbiol 71: 6150–6158.
[58]
Conte MP, Schippa S, Zamboni I, Penta M, Chiarini F, et al. (2006) Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease. Gut 55: 1760–1767.
Rhimi M, Boisson A, Dejob M, Boudebouze S, Maguin E, et al. (2010) Efficient bioconversion of lactose in milk and whey: immobilization and biochemical characterization of a beta-galactosidase from the dairy Streptococcus thermophilus LMD9 strain. Res Microbiol 161: 515–525.
[61]
Stabler SP (1999) B12 and nutrition. In: Barnejee R editor. Chemistry and biochemistry of B12. Wiley: New York. 343–365.
[62]
Barnerjee R (2006) B12 trafficking in mammals: A for coenzyme escort service. ACS Chem Biol 1: 149–159.
[63]
Santos F, Vera JL, van der Heijden R, Valdez G, de Vos WM, et al. (2008) The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098. Microbiology 154: 81–93.
[64]
Van Hylckama Vlieg JE, Veiga P, Zhang C, Derrien M, Zhao L (2011) Impact of microbial transformation of food on health – from fermented foods to fermentation in the gastro-intestinal tract. Curr Opin Biotechnol 22: 211–219.
[65]
Temperton B, Field D, Oliver A, Tiwari B, Muhling M, et al. (2009) Bias in assessments of marine microbial biodiversity in fosmid libraries as evaluated by pyrosequencing. Isme J 3: 792–796.
[66]
Gabor EM, Alkema WB, Janssen DB (2004) Quantifying the accessibility of the metagenome by random expression cloning techniques. Environ Microbiol 6: 879–886.
[67]
Taras D, Simmering R, Collins MD, Lawson PA, Blaut M (2002) Reclassification of Eubacterium formicigenerans Holdeman and Moore 1974 as Dorea formicigenerans gen. nov., comb. nov., and description of Dorea longicatena sp. nov., isolated from human faeces. Int J Syst and Evol Microbiol 52: 423–428.
[68]
Smillie CS, Smith MB, Friedman J, Cordero OX, David LA, et al. (2011) Ecology drives a global network of gene exchange connecting the human microbiome. Nature 480: 241–244.
[69]
Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, et al. (2011) Enterotypes of the human gut microbiome. Nature 473: 174–180.
[70]
Peterson J, Garges S, Giovanni M, McInnes P, Wang L, et al. (2009) The NIH Human Microbiome Project. Genome Res. 19: 2317–2323.
[71]
Gloux K, Berteau O, El Oumami H, Beguet F, Leclerc M, et al. (2011) A metagenomic β-glucuronidase uncovers a core adaptive function of the human intestinal microbiome. PNAS 108 Suppl 14539–4546.
[72]
Huang X, Madan A (1999) CAP3: A DNA sequence assembly program. Genome Res 9: 868–877.
[73]
Noguchi H, Park J, Takagi T (2006) MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic Acids Res 34: 5623–5630.
[74]
Huson DH, Auch AF, Qi J, Schuster SC (2007) MEGAN analysis of metagenomic data. Genome Res 17: 377–386.
