Applied Microbiology and Biotechnology

, Volume 94, Issue 6, pp 1521–1532 | Cite as

Pathway redesign for deoxyviolacein biosynthesis in Citrobacter freundii and characterization of this pigment

  • Pei-xia Jiang
  • Hai-sheng Wang
  • Su Xiao
  • Ming-yue Fang
  • Rui-ping Zhang
  • Shu-ying He
  • Kai Lou
  • Xin-Hui Xing
Biotechnological products and process engineering


Violacein (Vio) is an important purple pigment with many potential bioactivities. Deoxyviolacein, a structural analog of Vio, is always synthesized in low concentrations with Vio in wild-type bacteria. Due to deoxyviolacein’s low production and difficulties in isolation and purification, little has been learned regarding its function and potential applications. This study was the first effort in developing a stable and efficient biosynthetic system for producing pure deoxyviolacein. A recombinant plasmid with vioabce genes was constructed by splicing using an overlapping extension-polymerase chain reaction, based on the Vio-synthesizing gene cluster of vioabcde, originating from Duganella sp. B2, and was introduced into Citrobacter freundii. With the viod gene disrupted in the Vio synthetic pathway, Vio production was completely abolished and the recombinant C. freundii synthesized only deoxyviolacein. Interestingly, vioe gene expression was strongly stimulated in the viod-deleted recombinant strain, indicating that viod disruptions could potentially induce polar effects upon the downstream vioe gene within this small operon. Deoxyviolacein production by this strain reached 1.9 g/L in shaker flasks. The product exhibited significant acid/alkali and UV resistance as well as significant inhibition of hepatocellular carcinoma cell proliferation at low concentrations of 0.1–1 μM. These physical characteristics and antitumor activities of deoxyviolacein contribute to illuminating its potential applications.


Violacein Deoxyviolacein Biosynthesis Heterologous expression Pathway redesign 



This work was supported in part by the National Science Fund of China (Grant no. 21006058), a China Postdoctoral Science Foundation funded project (Grant no. 20080430367), a Basic Research Fund of CAAS (0042009001), and a Xinjiang-supporting project by Science and Technology (Grant no. 200991132).

Supplementary material

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  1. Ahmed AM, Shimamoto T (2008) Emergence of a cefepime- and cefpirome-resistant Citrobacter freundii clinical isolate harbouring a novel chromosomally encoded AmpC beta-lactamase, CMY-37. International Journal of Antimicrobial Agents 32(3):256–261CrossRefGoogle Scholar
  2. Andrighetti-Frohner CR, Antonio RV, Creczynski-Pasa TB, Barardi CR, Simoes CM (2003) Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum. Mem Inst Oswaldo Cruz 98(6):843–848CrossRefGoogle Scholar
  3. Antonio RV, Creczynski-Pasa TB (2004) Genetic analysis of violacein biosynthesis by Chromobacterium violaceum. Genet Mol Res 3(1):85–91Google Scholar
  4. Antonisamy P, Ignacimuthu S (2010) Immunomodulatory, analgesic and antipyretic effects of violacein isolated from Chromobacterium violaceum. Phytomedicine 17(3–4):300–304CrossRefGoogle Scholar
  5. Antonisamy P, Kannan P, Ignacimuthu S (2009) Anti-diarrhoeal and ulcer-protective effects of violacein isolated from Chromobacterium violaceum in Wistar rats. Fundam Clin Pharmacol 23(4):483–490CrossRefGoogle Scholar
  6. Asamizu S, Igarashi Y, Onaka H (2007) VioE, a prodeoxyviolacein synthase involved in violacein biosynthesis, is responsible for intramolecular indole rearrangement. Tetrahedron Letters 48:2923–2926CrossRefGoogle Scholar
  7. Balibar CJ, Walsh CT (2006) In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum. Biochemistry 45(51):15444–15457CrossRefGoogle Scholar
  8. Becker MH, Brucker RM, Schwantes CR, Harris RN, Minbiole KP (2009) The bacterially produced metabolite violacein is associated with survival of amphibians infected with a lethal fungus. Appl Environ Microbiol 75(21):6635–6638CrossRefGoogle Scholar
  9. Bromberg N, Justo GZ, Haun M, Duran N, Ferreira CV (2005) Violacein cytotoxicity on human blood lymphocytes and effect on phosphatases. J Enzyme Inhib Med Chem 20(5):449–454CrossRefGoogle Scholar
  10. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity. Cancer Res 47(4):943–946Google Scholar
  11. Cazoto LL, Martins D, Ribeiro MG, Duran N, Nakazato G (2011) Antibacterial activity of violacein against Staphylococcus aureus isolated from Bovine mastitis. J Antibiot (Tokyo) 64(5):395–397CrossRefGoogle Scholar
  12. Durán N, Antonio RV, Haun M, Pilli RA (1994) Biosynthesis of a trypanocide by Chromobacterium violaceum. World J Microbiol Biotechnol V10(6):686–690CrossRefGoogle Scholar
  13. Duran N, Justo GZ, Ferreira CV, Melo PS, Cordi L, Martins D (2007) Violacein: properties and biological activities. Biotechnol Appl Biochem 48(Pt 3):127–133Google Scholar
  14. Ferreira CV, Bos CL, Versteeg HH, Justo GZ, Duran N, Peppelenbosch MP (2004) Molecular mechanism of violacein-mediated human leukemia cell death. Blood 104(5):1459–1464CrossRefGoogle Scholar
  15. Jiang PX, Wang HS, Zhang C, Lou K, Xing XH (2010) Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts. Appl Microbiol Biotechnol 86(4):1077–1088CrossRefGoogle Scholar
  16. Konzen M, De Marco D, Cordova CA, Vieira TO, Antonio RV, Creczynski-Pasa TB (2006) Antioxidant properties of violacein: possible relation on its biological function. Bioorg Med Chem 14(24):8307–8313CrossRefGoogle Scholar
  17. Kuwayama H, Obara S, Morio T, Katoh M, Urushihara H, Tanaka Y (2002) PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. Nucleic Acids Res 30(2):E2CrossRefGoogle Scholar
  18. Matz C, Webb JS, Schupp PJ, Phang SY, Penesyan A, Egan S, Steinberg P, Kjelleberg S (2008) Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense. PLoS ONE 3(7):e2744CrossRefGoogle Scholar
  19. Momen AZ, Hoshino T (2000) Biosynthesis of violacein: intact incorporation of the tryptophan molecule on the oxindole side, with intramolecular rearrangement of the indole ring on the 5-hydroxyindole side. Biosci Biotechnol Biochem 64(3):539–549CrossRefGoogle Scholar
  20. Morohoshi T, Fukamachi K, Kato M, Kato N, Ikeda T (2010) Regulation of the violacein biosynthetic gene cluster by acylhomoserine lactone-mediated quorum sensing in Chromobacterium violaceum ATCC 12472. Biosci Biotechnol Biochem 74(10):2116–2119CrossRefGoogle Scholar
  21. Nakamura Y, Sawada T (2003) Production of antibacterial violet pigment by psychrotropic bacterium RT102 strain. Biotechnology and Bioprocess Engineering 8:37–40CrossRefGoogle Scholar
  22. Nakamura Y, Morita Y, Tamiya E (2002) Isolation of a psychrotrophic bacterium from the organic residue of a water tank keeping rainbow trout and antibacterial effect of violet pigment produced from the strain. Biochemical Engineering Journal 12:79–86CrossRefGoogle Scholar
  23. Ryan KS, Drennan CL (2009) Divergent pathways in the biosynthesis of bisindole natural products. Chem Biol 16(4):351–364CrossRefGoogle Scholar
  24. Ryan KS, Balibar CJ, Turo KE, Walsh CT, Drennan CL (2008) The violacein biosynthetic enzyme VioE shares a fold with lipoprotein transporter proteins. J Biol Chem 283(10):6467–6475CrossRefGoogle Scholar
  25. Sanchez C, Brana AF, Mendez C, Salas JA (2006) Reevaluation of the violacein biosynthetic pathway and its relationship to indolocarbazole biosynthesis. Chembiochem 7(8):1231–1240CrossRefGoogle Scholar
  26. Shinoda K, Hasegawa T, Sato H, Shinozaki M, Kuramoto H, Takamiya Y, Sato T, Nikaidou N, Watanabe T, Hoshino T (2007) Biosynthesis of violacein: a genuine intermediate, protoviolaceinic acid, produced by VioABDE, and insight into VioC function. Chem Commun (Camb)(40):4140–4142Google Scholar
  27. Smits TH, Seeger MA, Witholt B, van Beilen JB (2001) New alkane-responsive expression vectors for Escherichia coli and pseudomonas. Plasmid 46(1):16–24CrossRefGoogle Scholar
  28. Srinivasan V, Nam HM, Sawant AA, Headrick SI, Nguyen LT, Oliver SP (2008) Distribution of tetracycline and streptomycin resistance genes and class 1 integrons in Enterobacteriaceae isolated from dairy and nondairy farm soils. Microbial Ecology 55(2):184–193CrossRefGoogle Scholar
  29. Tsutomu H, Takeo U, Nagahiro O (1987) Biosynthesis of violacein: a novel rearrangement in tryptophan metabolism with a 1, 2-shift of the indole ring. Agricultural and Biological Chemistry 51:965–996CrossRefGoogle Scholar
  30. Wang HS, Jiang PX, Lu Y, Ruan ZY, Jiang RB, Xing XH, Lou K, Wei D (2009) Optimization of culture conditions for violacein production by a new strain of Duganella sp B2. Biochemical Engineering Journal 44(2–3):119–124CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Pei-xia Jiang
    • 1
    • 2
  • Hai-sheng Wang
    • 3
  • Su Xiao
    • 2
  • Ming-yue Fang
    • 2
  • Rui-ping Zhang
    • 2
  • Shu-ying He
    • 4
  • Kai Lou
    • 5
  • Xin-Hui Xing
    • 2
  1. 1.Department of Industrial Microbiology and Biotechnology, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Institute of Biochemical Engineering, Department of Chemical EngineeringTsinghua UniversityBeijingPeople’s Republic of China
  3. 3.Graduate School of Chinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  4. 4.School of Life Science and TechnologyChina Pharmaceutical UniversityNanjingPeople’s Republic of China
  5. 5.Institute of MicrobiologyXinjiang Academy of Agricultural SciencesÜrümqiPeople’s Republic of China

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