Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts
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.
KeywordViolacein Deoxyviolacein Biosynthesis Duganella sp. B2 Heterologous expression
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).
- Antonio RV, Creczynski-Pasa TB (2004) Genetic analysis of violacein biosynthesis by Chromobacterium violaceum. Genet Mol Res 3(1):85–91Google 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):513–519Google Scholar
- Creczynski-Pasa TB, Antonio RV (2004) Energetic metabolism of Chromobacterium violaceum. Genet Mol Res 3(1):162–166Google 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):4392–4401Google 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):11660–11665CrossRefGoogle Scholar
- Demoss RD, Evans NR (1960) Incorporation of C14-labeled substrates into violacein. J Bacteriol 79:729–733Google Scholar
- Durán N, Erazo S, Campos V (1983) Bacterial Chemistry-II: antimicrobial photoproduct from pigment of Chromobacterium violaceum. An Acad Bras Ciênc 55:231–234Google Scholar
- Moss MO, Ryall C, Logan NA (1978) The classification and characterization of chromobacteria from a lowland river. J Gen Microbiol 105:11–21Google 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):992–999Google 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):4140–4142Google Scholar
- 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):131–140Google 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 59:630–635Google Scholar