Skip to main content

β-Carotene Biosynthesis in Probiotic Bacteria

Abstract

Susceptibility to deadly diarrheal diseases is partly due to widespread pediatric vitamin A deficiency. To increase vitamin A coverage in malnourished children, we propose to engineer a probiotic bacterium that will produce β-carotene in the intestine, which will be metabolized to vitamin A. Such a therapy has the potential to broadly stimulate mucosal immunity and simultaneously reduce the incidence and duration of diarrheal disease. To that end, a β-carotene-producing variant of the probiotic Escherichia coli strain Nissle 1917 (EcN-BETA) was generated. Notably, the strain produces β-carotene under anaerobic conditions, reflective of the gut environment. EcN-BETA also retains β-carotene production capability after lyophilization, suggesting that it may be amenable to dry formulation. Moreover, EcN-BETA activates murine dendritic cells in vitro, suggesting that the presence of β-carotene may not diminish the immunostimulatory capacity of EcN. Finally, we present a framework through which further improvements may enable approaches such as the one described in this report to yield innovative life-saving therapies for the developing world.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Black RE, Morris SS, Bryce J (2003) Where and why are 10 million children dying every year? Lancet 361(9376):2226–2234. doi:10.1016/S0140-6736(03)13779-8

    Article  Google Scholar 

  2. Thapar N, Sanderson IR (2004) Diarrhoea in children: an interface between developing and developed countries. Lancet 363(9409):641–653. doi:10.1016/S0140-6736(04)15599-2

    Article  Google Scholar 

  3. Mora JR, Iwata M, von Andrian UH (2008) Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol 8(9):685–698. doi:10.1038/nri2378

    Article  CAS  Google Scholar 

  4. Boy E, Mannar V, Pandav C, de Benoist B, Viteri F, Fontaine O, Hotz C (2009) Achievements, challenges, and promising new approaches in vitamin and mineral deficiency control. Nutr Rev 67(Suppl 1):S24–S30. doi:10.1111/j.1753-4887.2009.00155.x

    Article  Google Scholar 

  5. Saurer L, McCullough KC, Summerfield A (2007) In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid. J Immunol 179:3504–3514

    CAS  Google Scholar 

  6. Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, Hall J, Sun CM, Belkaid Y, Powrie F (2007) A functionally specialized population of mucosal CD103 + DCs induces Foxp3 + regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med 204(8):1757–1764. doi:10.1084/jem.20070590

    Article  CAS  Google Scholar 

  7. Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, Cheroutre H (2007) Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 317(5835):256–260. doi:10.1126/science.1145697

    Article  CAS  Google Scholar 

  8. Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, Song SY (2004) Retinoic acid imprints gut-homing specificity on T cells. Immunity 21:527–538

    Article  CAS  Google Scholar 

  9. Mora JR, Iwata M, Eksteen B, Song SY, Junt T, Senman B, Otipoby KL, Yokota A, Takeuchi H, Ricciardi-Castagnoli P, Rajewsky K, Adams DH, von Andrian UH (2006) Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 314(5802):1157–1160. doi:10.1126/science.1132742

    Article  CAS  Google Scholar 

  10. Watson DS, Huang Z, Szoka FC Jr (2009) All-trans retinoic acid potentiates the antibody response in mice to a lipopeptide antigen adjuvanted with liposomal lipid A. Immunol Cell Biol 87(8):630–633. doi:10.1038/icb.2009.48

    Article  CAS  Google Scholar 

  11. Ross A, Chen Q, Ma Y (2009) Augmentation of antibody responses by retinoic acid and costimulatory molecules. Semin Immunol 21:42–50

    Article  CAS  Google Scholar 

  12. Ma Y, Ross AC (2005) The anti-tetanus immune response of neonatal mice is augmented by retinoic acid combined with polyriboinosinic:polyribocytidylic acid. Proc Natl Acad Sci 102:13556–13561

    Article  CAS  Google Scholar 

  13. DePaolo RW, Abadie V, Tang F, Fehlner-Peach H, Hall JA, Wang W, Marietta EV, Kasarda DD, Waldmann TA, Murray JA, Semrad C, Kupfer SS, Belkaid Y, Guandalini S, Jabri B (2011) Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens. Nature 471(7337):220–224. doi:10.1038/nature09849

    Article  CAS  Google Scholar 

  14. de Vrese M, Marteau PR (2007) Probiotics and prebiotics: effects on diarrhea. J Nutr 137(3 Suppl 2):803S–811S

    Google Scholar 

  15. Van Niel CW, Feudtner C, Garrison MM, Christakis DA (2002) Lactobacillus therapy for acute infectious diarrhea in children: a meta-analysis. Pediatrics 109(4):678–684

    Article  Google Scholar 

  16. Trebichavsky I, Splichal I, Rada V, Splichalova A (2010) Modulation of natural immunity in the gut by Escherichia coli strain Nissle 1917. Nutr Rev 68(8):459–464. doi:10.1111/j.1753-4887.2010.00305.x

    Article  Google Scholar 

  17. Schultz M (2008) Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm Bowel Dis 14(7):1012–1018. doi:10.1002/ibd.20377

    Article  Google Scholar 

  18. Christensen HR, Frokiaer H, Pestka JJ (2002) Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. J Immunol 168(1):171–178

    CAS  Google Scholar 

  19. Panigrahi P, Parida S, Pradhan L, Mohapatra SS, Misra PR, Johnson JA, Chaudhry R, Taylor S, Hansen NI, Gewolb IH (2008) Long-term colonization of a Lactobacillus plantarum synbiotic preparation in the neonatal gut. J Pediatr Gastroenterol Nutr 47(1):45–53. doi:10.1097/MPG.0b013e31815a5f2c

    Article  Google Scholar 

  20. Barth S, Duncker S, Hempe J, Breves G, Baljer G, Bauerfeind R (2009) Escherichia coli Nissle 1917 for probiotic use in piglets: evidence for intestinal colonization. J Appl Microbiol 107(5):1697–1710. doi:10.1111/j.1365-2672.2009.04361.x

    Article  CAS  Google Scholar 

  21. Allen LH, Haskell M (2002) Estimating the potential for vitamin A toxicity in women and young children. J Nutr 132(9 Suppl):2907S–2919S

    CAS  Google Scholar 

  22. Redmond TM, Gentleman S, Duncan T, Yu S, Wiggert B, Gantt E, Cunningham FX Jr (2001) Identification, expression, and substrate specificity of a mammalian beta-carotene 15,15′-dioxygenase. J Biol Chem 276(9):6560–6565. doi:10.1074/jbc.M009030200

    Article  CAS  Google Scholar 

  23. Yan W, Jang GF, Haeseleer F, Esumi N, Chang J, Kerrigan M, Campochiaro M, Campochiaro P, Palczewski K, Zack DJ (2001) Cloning and characterization of a human beta, beta-carotene-15,15′-dioxygenase that is highly expressed in the retinal pigment epithelium. Genomics 72(2):193–202. doi:10.1006/geno.2000.6476

    Article  CAS  Google Scholar 

  24. Mezger M, Wozniok I, Blockhaus C, Kurzai O, Hebart H, Einsele H, Loeffler J (2008) Impact of mycophenolic acid on the functionality of human polymorphonuclear neutrophils and dendritic cells during interaction with Aspergillus fumigatus. Antimicrob Agents Chemother 52(7):2644–2646. doi:10.1128/AAC.01618-07

    Article  CAS  Google Scholar 

  25. Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Adams JL, Silverstone AL, Drake R (2005) Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nat Biotechnol 23(4):482–487. doi:10.1038/nbt1082

    Article  CAS  Google Scholar 

  26. Farre G, Bai C, Twyman RM, Capell T, Christou P, Zhu C (2011) Nutritious crops producing multiple carotenoids—a metabolic balancing act. Trends Plant Sci 16(10):532–540. doi:10.1016/j.tplants.2011.08.001

    Article  CAS  Google Scholar 

  27. Cunningham FX Jr, Gantt E (2007) A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynth Res 92(2):245–259. doi:10.1007/s11120-007-9210-0

    Article  CAS  Google Scholar 

  28. Misawa N, Shimada H (1997) Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 59(3):169–181

    Article  CAS  Google Scholar 

  29. Alper H, Miyaoku K, Stephanopoulos G (2005) Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat Biotechnol 23(5):612–616. doi:10.1038/nbt1083

    Article  CAS  Google Scholar 

  30. Sun CM, Hall JA, Blank RB, Bouladoux N, Oukka M, Mora JR, Belkaid Y (2007) Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J Exp Med 204(8):1775–1785. doi:10.1084/jem.20070602

    Article  CAS  Google Scholar 

  31. Rohdich F, Kis K, Bacher A, Eisenreich W (2001) The non-mevalonate pathway of isoprenoids: genes, enzymes and intermediates. Curr Opin Chem Biol 5(5):535–540

    Article  CAS  Google Scholar 

  32. Cunningham FX, Gantt E (1998) Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 49:557–583. doi:10.1146/annurev.arplant.49.1.557

    Article  CAS  Google Scholar 

  33. Cunningham FX Jr, Pogson B, Sun Z, McDonald KA, DellaPenna D, Gantt E (1996) Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8(9):1613–1626. doi:10.1105/tpc.8.9.1613

    CAS  Google Scholar 

  34. Kajiwara S, Fraser PD, Kondo K, Misawa N (1997) Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochem J 324(Pt 2):421–426

    CAS  Google Scholar 

  35. Stokes JL (1949) Fermentation of glucose by suspensions of Escherichia coli. J Bacteriol 57(2):147–158

    CAS  Google Scholar 

  36. Geissmann F, Revy P, Brousse N, Lepelletier Y, Folli C, Durandy A, Chambon P, Dy M (2003) Retinoids regulate survival and antigen presentation by immature dendritic cells. J Exp Med 198(4):623–634

    Article  CAS  Google Scholar 

  37. Abdel-Fatth G, Watzl B, Huang D, Watson RR (1993) Beta-carotene in vitro stimulates tumor necrosis factor alpha and interleukin 1 alpha secretion by human peripheral blood mononuclear cells. Nutr Res 13(8):863–871

    Article  CAS  Google Scholar 

  38. Uhlig HH, McKenzie BS, Hue S, Thompson C, Joyce-Shaikh B, Stepankova R, Robinson N, Buonocore S, Tlaskalova-Hogenova H, Cua DJ, Powrie F (2006) Differential activity of IL-12 and IL-23 in mucosal and systemic innate immune pathology. Immunity 25(2):309–318. doi:10.1016/j.immuni.2006.05.017

    Article  CAS  Google Scholar 

  39. Aujla SJ, Chan YR, Zheng M, Fei M, Askew DJ, Pociask DA, Reinhart TA, McAllister F, Edeal J, Gaus K, Husain S, Kreindler JL, Dubin PJ, Pilewski JM, Myerburg MM, Mason CA, Iwakura Y, Kolls JK (2008) IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nat Med 14(3):275–281. doi:10.1038/nm1710

    Article  CAS  Google Scholar 

  40. Mayer MP, Beyer P, Kleinig H (1990) Quinone compounds are able to replace molecular oxygen as terminal electron acceptor in phytoene desaturation in chromoplasts of Narcissus pseudonarcissus L. Eur J Biochem 191(2):359–363

    Article  CAS  Google Scholar 

  41. Armstrong GA, Hearst JE (1996) Carotenoids 2: genetics and molecular biology of carotenoid pigment biosynthesis. FASEB J 10(2):228–237

    CAS  Google Scholar 

  42. Dailey TA, Dailey HA (1998) Identification of an FAD superfamily containing protoporphyrinogen oxidases, monoamine oxidases, and phytoene desaturase. Expression and characterization of phytoene desaturase of Myxococcus xanthus. J Biol Chem 273(22):13658–13662

    Article  CAS  Google Scholar 

  43. West CE, Eilander A, van Lieshout M (2002) Consequences of revised estimates of carotenoid bioefficacy for dietary control of vitamin A deficiency in developing countries. J Nutr 132(9 Suppl):2920S–2926S

    CAS  Google Scholar 

  44. Kim SW, Keasling JD (2001) Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnol Bioeng 72(4):408–415. doi:10.1002/1097-0290(20000220)72:4<408:AID-BIT1003>3.0.CO;2-H

    Article  CAS  Google Scholar 

  45. Wilding EI, Brown JR, Bryant AP, Chalker AF, Holmes DJ, Ingraham KA, Iordanescu S, So CY, Rosenberg M, Gwynn MN (2000) Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci. J Bacteriol 182(15):4319–4327

    Article  CAS  Google Scholar 

  46. Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21(7):796–802. doi:10.1038/nbt833

    Article  CAS  Google Scholar 

  47. Bloch K, Papismedov E, Yavriyants K, Vorobeychik M, Beer S, Vardi P (2006) Photosynthetic oxygen generator for bioartificial pancreas. Tissue Eng 12(2):337–344. doi:10.1089/ten.2006.12.337

    Article  CAS  Google Scholar 

  48. Ribaya-Mercado JD, Maramag CC, Tengco LW, Blumberg JB, Solon FS (2008) Relationships of body mass index with serum carotenoids, tocopherols and retinol at steady-state and in response to a carotenoid-rich vegetable diet intervention in Filipino schoolchildren. Biosci Rep 28(2):97–106. doi:10.1042/BSR20070045

    Article  CAS  Google Scholar 

  49. Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38(6):e92. doi:10.1093/nar/gkp1193

    Article  Google Scholar 

  50. Steidler L, Neirynck S, Huyghebaert N, Snoeck V, Vermeire A, Goddeeris B, Cox E, Remon JP, Remaut E (2003) Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 21(7):785–789. doi:10.1038/nbt840

    Article  CAS  Google Scholar 

  51. Saif LJ, Ward LA, Yuan L, Rosen BI, To TL (1996) The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses. Arch Virol Suppl 12:153–161

    CAS  Google Scholar 

  52. Sankaranarayanan S, Ma Y, Bryson MC, Li NQ, Ross AC (2007) Neonatal-age treatment with vitamin A delays postweaning vitamin A deficiency and increases the antibody response to T-cell dependent antigens in young adult rats fed a vitamin A-deficient diet. J Nutr 137(5):1229–1235

    CAS  Google Scholar 

  53. Autieri SM, Lins JJ, Leatham MP, Laux DC, Conway T, Cohen PS (2007) L-fucose stimulates utilization of D-ribose by Escherichia coli MG1655 DeltafucAO and E. coli Nissle 1917 DeltafucAO mutants in the mouse intestine and in M9 minimal medium. Infect Immun 75(11):5465–5475. doi:10.1128/IAI.00822-07

    Article  CAS  Google Scholar 

  54. Blum-Oehler G, Oswald S, Eiteljorge K, Sonnenborn U, Schulze J, Kruis W, Hacker J (2003) Development of strain-specific PCR reactions for the detection of the probiotic Escherichia coli strain Nissle 1917 in fecal samples. Res Microbiol 154(1):59–66

    Article  CAS  Google Scholar 

  55. Craft NE, Soares JH Jr (1992) Relative solubility, stability, and absorptivity of lutein and beta-carotene in organic solvents. J Agric Food Chem 40:431–434

    Article  CAS  Google Scholar 

Download references

Acknowledgments

E. coli Nissle 1917 was a generous gift from Dr. Paul Cohen (University of Rhode Island). Several plasmids containing the requisite genes of the β-carotene biosynthesis pathway, including pSTBlue1-BETAipi and pAC-BETA, were generously provided by Dr. Francis Cunningham (University of Maryland). A plasmid containing the human BCDO gene was graciously provided by Prof. Don Zack (Johns Hopkins University). A plasmid containing the mouse BCMO (pBAD/BCD) and a doubly transformed E. coli clone (pBAD/BCD + pAC-BETA) were generously provided by Dr. Michael Redmond (National Eye Institute). Thanks are extended to Dr. Walter Moos (Vice President, SRI Biosciences) and Dr. Amit Galande (Director, SRI Biosciences) for encouragement and support, to Dr. Eugenia Poliankov (National Eye Institute) and Dr. Kevin Ramkissoon (SRI International) for helpful discussions, and to Than-Thuy Tran for technical assistance with the BMDC experiments. This work was supported by a grant from the Bill & Melinda Gates Foundation through the Grand Challenges Explorations Initiative. SRI International’s Center for Advanced Drug Research was established through funding support from the Commonwealth of Virginia.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Douglas S. Watson.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 30 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Miller, J.K., Harrison, M.T., D’Andrea, A. et al. β-Carotene Biosynthesis in Probiotic Bacteria. Probiotics & Antimicro. Prot. 5, 69–80 (2013). https://doi.org/10.1007/s12602-013-9133-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12602-013-9133-3

Keywords

  • Vitamin A deficiency
  • Probiotic
  • β-Carotene
  • Pediatric malnutrition
  • Mucosal immunity
  • Diarrheal disease