Skip to main content
SpringerLink
Log in

Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts

  • Biotechnological Products and Process Engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Violacein is a bacteria-originated indolocarbazole pigment with potential applications due to its various bioactivities such as anti-tumor, antiviral, and antifungal activities. However, stable mass production of this pigment is difficult due to its low productivities and the instability of wild-type violacein-producing strains. In order to establish a stable and efficient production system for violacein, the violacein synthesis pathway from a new species of Duganella sp. B2 was reconstructed in different bacterial strains including Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes by using different vectors. The gene cluster that encodes five enzymes involved in the violacein biosynthetic pathway was first isolated from Duganella sp. B2, and three recombinant expression vectors were constructed using the T7 promoter or the alkane-responsive promoter PalkB. Our results showed that violacein could be stably synthesized in E. coli, C. freundii, and E. aerogenes. Interestingly, we found that there were great differences between the different recombinant strains, not only in the protein expression profiles pertaining to violacein biosynthesis but also in the productivity and composition of crude violacein. Among the host strains tested, the crude violacein production by the recombinant C. freundii strain reached 1.68 g L−1 in shake flask cultures, which was 4-fold higher than the highest production previously reported in flask culture by other groups. To the best of our knowledge, this is the first report on the efficient production of violacein by genetically engineered strains.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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. Int J Antimicrob Agents 32(3):256–261

    Article  CAS  Google Scholar 

  • Antonio RV, Creczynski-Pasa TB (2004) Genetic analysis of violacein biosynthesis by Chromobacterium violaceum. Genet Mol Res 3(1):85–91

    CAS  Google Scholar 

  • August PR, Grossman TH, Minor C, Draper MP, MacNeil IA, Pemberton JM, Call KM, Holt D, Osburne MS (2000) Sequence analysis and functional characterization of the violacein biosynthetic pathway from Chromobacterium violaceum. J Mol Microbiol Biotechnol 2(4):513–519

    CAS  Google Scholar 

  • Brady SF, Chao CJ, Handelsman J, Clardy J (2001) Cloning and heterologous expression of a natural product biosynthetic gene cluster from eDNA. Org Lett 3(13):1981–1984

    Article  CAS  Google Scholar 

  • Bromberg Natalia DN (2001) Violacein transformation by peroxidases and oxidases: implications on its biological properties. J Mol Catal B Enzym 11:463–467

    Article  Google Scholar 

  • Conrado RJ, Varner JD, DeLisa MP (2008) Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr Opin Biotechnol 19(5):492–499

    Article  CAS  Google Scholar 

  • Creczynski-Pasa TB, Antonio RV (2004) Energetic metabolism of Chromobacterium violaceum. Genet Mol Res 3(1):162–166

    CAS  Google Scholar 

  • Daniel R, Stuertz K, Gottschalk G (1995) Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii. J Bacteriol 177(15):4392–4401

    CAS  Google Scholar 

  • De Almeida DF, Hungria M, Guimaraes CT, Antonio RV, Almeida FC, de Almeida LGP, de Almeida R, Alves-Gomes JA, Andrade EM, Araripe J (2003) The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc Natl Acad Sci USA 100(20):11660–11665

    Article  Google Scholar 

  • Demoss RD, Evans NR (1960) Incorporation of C14-labeled substrates into violacein. J Bacteriol 79:729–733

    CAS  Google Scholar 

  • Durán N, Antonio RV, Haun M, Pilli RA (1994) Biosynthesis of a trypanocide by Chromobacterium violaceum. World J Microbiol Biotechnol V10(6):686–690

    Article  Google Scholar 

  • Durán N, Erazo S, Campos V (1983) Bacterial Chemistry-II: antimicrobial photoproduct from pigment of Chromobacterium violaceum. An Acad Bras Ciênc 55:231–234

    Google Scholar 

  • Duran N, Justo GZ, Ferreira CV, Melo PS, Cordi L, Martins D (2007) Violacein: properties and biological activities. Biotechnol Appl Biochem 48:127–133

    Article  CAS  Google Scholar 

  • Duran N, Menck CF (2001) Chromobacterium violaceum: a review of pharmacological and industiral perspectives. Crit Rev Microbiol 27(3):201–222

    Article  CAS  Google Scholar 

  • Gillis M, Ley J (2006) The Genera Chromobacterium and Janthinobacterium. The Prokaryotes 5:737–746

    Article  Google Scholar 

  • Hirano S, Asamizu S, Onaka H, Shiro Y, Nagano S (2008) Crystal structure of VioE, a key player in the construction of the molecular skeleton of violacein. J Biol Chem 283(10):6459–6466

    Article  CAS  Google Scholar 

  • John M, Pemberton KMV, Penfold RJ (1991) Cloning and heterologous expression of the violacein biosynthesis gene cluster from Chromobacterium violaceum. Curr Microbiol 22(6):355–358

    Article  Google Scholar 

  • Leon LL, Miranda CC, De Souza AO, Duran N (2001) Antileishmanial activity of the violacein extracted from Chromobacterium violaceum. J Antimicrob Chemother 48(3):449–450

    Article  CAS  Google Scholar 

  • Lu Y, Wang L, Xue Y, Zhang C, Xing X-H, Lou K, Zhang Z, Li Y, Zhang G, Bi J (2009) Production of violet pigment by a newly isolated psychrotrophic bacterium from a glacier in Xinjiang, China. Biochem Eng J 43(2):135–141

    Article  CAS  Google Scholar 

  • Manukhov IV, Mamaeva DV, Morozova EA, Rastorguev SM, Faleev NG, Demidkina TV, Zavilgelsky GB (2006) l-methionine gamma-lyase from Citrobacter freundii: cloning of the gene and kinetic parameters of the enzyme. Biochemistry (Moscow) 71(4):361–369

    Article  CAS  Google Scholar 

  • Matz C, Deines P, Boenigk J, Arndt H, Eberl L, Kjelleberg S, Jurgens K (2004) Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates. Appl Environ Microbiol 70(3):1593–1599

    Article  CAS  Google Scholar 

  • 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):e2744

    Article  Google Scholar 

  • Mendes AS, de Carvalho JE, Duarte MCT, Duran N, Bruns RE (2001) Factorial design and response surface optimization of crude violacein for Chromobacterium violaceum production. Biotechnol Lett 23:1963–1969

    Article  CAS  Google Scholar 

  • Moss MO, Ryall C, Logan NA (1978) The classification and characterization of chromobacteria from a lowland river. J Gen Microbiol 105:11–21

    CAS  Google Scholar 

  • Nakamura ST, 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. Biochem Eng J 12(1):79–86

    Article  CAS  Google Scholar 

  • Pantanella F, Berlutti F, Passariello C, Sarli S, Morea C, Schippa S (2007) Violacein and biofilm production in Janthinobacterium lividum. J Appl Microbiol 102(4):992–999

    CAS  Google Scholar 

  • Rettori D, Durán N (1998) Production, extraction and purificationof violacein: an antibiotic pigment producedby Chromobacterium violaceum. World J Microbiol Biotechnol V14(5):685–688

    Article  Google Scholar 

  • Sanchez C, Brana AF, Mendez C, Salas JA (2006) Reevaluation of the violacein biosynthetic pathway and its relationship to indolocarbazole biosynthesis. ChemBioChem 7(8):1231–1240

    Article  CAS  Google Scholar 

  • 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):4140–4142

  • Shirata ATT, Yasui H, Hata T, Hayasaka S, Kojima A, Kato H (2000) Isolation of bacteria producing bluish-purple pigment and use for dyeing. JARQ. Jpn Agric Res Q 34(2):131–140

    Google Scholar 

  • Smits TH, Seeger MA, Witholt B, van Beilen JB (2001) New alkane-responsive expression vectors for Escherichia coli and pseudomonas. Plasmid 46(1):16–24

    Article  CAS  Google Scholar 

  • 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. Microb Ecol 55(2):184–193

    Article  CAS  Google Scholar 

  • 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. Biochem Eng J 44(2-3):119–124

    Article  CAS  Google Scholar 

  • Wang HS, Lu Y, Xue Y, Ruan ZY, Jiang RB, Xing XH, Lou K, Wei D (2008) Separation, purification and structure identification of purple pigments from Duganella sp. B2. J Chem Ind Eng 59:630–635

    CAS  Google Scholar 

  • Wilkinson B, Micklefield J (2007) Mining and engineering natural-product biosynthetic pathways. Nat Chem Biol 3(7):379–386

    Article  CAS  Google Scholar 

  • Yada S, Wang Y, Zou Y, Nagasaki K, Hosokawa K, Osaka I, Arakawa R, Enomoto K (2008) Isolation and characterization of two groups of novel marine bacteria producing violacein. Mar Biotechnol (NY) 10(2):128–132

    Article  CAS  Google Scholar 

  • Yang LH, Xiong H, Lee OO, Qi SH, Qian PY (2007) Effect of agitation on violacein production in Pseudoalteromonas luteoviolacea isolated from a marine sponge. Lett Appl Microbiol 44(6):625–630

    Article  CAS  Google Scholar 

  • Yoshitoshi Nakamura CA, Tatsuro S (2003) Production of antibacterial violet pigment by psychrotropic bacterium RT102 strain. Biotechnol Bioprocess Eng 8:37–40

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank Prof. B. Witholt of ETH, Swiss for kindly donating the plasmid pCom10. This work was supported in part by the National Science Fund of China (Grant No. 20676071 and 20836004), China Postdoctoral Science Foundation funded project (Grant No. 023206061) and Xinjiang-supporting project by Science and Technology (Grant No. 200991132).

Author information

Authors and Affiliations

  1. Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People’s Republic of China

    Pei-xia Jiang, Chong Zhang & Xin-Hui Xing

  2. Graduate School of Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China

    Hai-sheng Wang

  3. Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, 830091, People’s Republic of China

    Kai Lou

Authors
  1. Pei-xia Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hai-sheng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kai Lou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xin-Hui Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xin-Hui Xing.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jiang, Px., Wang, Hs., Zhang, C. et al. Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts. Appl Microbiol Biotechnol 86, 1077–1088 (2010). https://doi.org/10.1007/s00253-009-2375-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-009-2375-z

Keyword

Search

Navigation