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Bio-indigo Production using Wild-type Acinetobacter sp. and Indole-3-acetate Monooxygenase (iacA) Expressed in Escherichia coli

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Abstract

This study reports the identification of a novel bio-indigo-producing bacterium from soil sources. Blue-colored colonies were first screened and isolated from a screening plate containing M9 minimal medium and 1.0 mM indole. The blue colony was selected among various colonies and identified as an Acinetobacter species. The purified blue dye exhibited a distinct spectral feature of λmax at 490 nm. The structure of the dye was then analyzed. Thin-layer chromatography separation and liquid chromatography/mass spectrometry analysis confirmed that the blue dye was indigo (with Rf and m/z values of 0.8 and 263.4, respectively). Wild-type Acinetobacter sp. could produce bio-indigo up to 1.018 ± 0.013 mg/L in an M9 minimal medium supplemented with 1.0 mM indole. Next, the genes involved in the production of indigo were investigated using sequence analysis and by comparing them with those in related Acinetobacter species. The indole-3-acetate monooxygenase-encoding gene iacA was found to be responsible for indigo synthesis from indole. The iacA gene was then amplified and expressed in Escherichia coli BL21(DE3), and the recombinant E. coli strain could produce bio-indigo at levels up to 0.291 ± 0.027 g/L over 24 h. Indigo production was highly dependent on indole substrate feeding. These findings may facilitate the industrial bioprocess of bio-indigo production.

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References

  1. Choi, K.-Y. (2020) A review of recent progress in the synthesis of bio-indigoids and their biologically assisted end-use applications. Dyes Pigm. 181: 108570.

    Article  CAS  Google Scholar 

  2. Grieve, M. C., T. W. Biermann, and K. Schaub (2006) The use of indigo derivatives to dye denim material. Sci. Justice 46: 15–24.

    Article  CAS  PubMed  Google Scholar 

  3. Candlish, E., J. La Croix, and A. M. Unrau (1969) The biosynthesis of 3-nitropropionic acid in creeping indigo (Indigofera spicata). Biochemistry 8: 182–186.

    Article  CAS  PubMed  Google Scholar 

  4. Dutta, S., S. Roychoudhary, and B. K. Sarangi (2017) Effect of different physico-chemical parameters for natural indigo production during fermentation of Indigofera plant biomass. 3 Biotech 7: 322.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Sarangi, B. K., Y. Minami, and S. T. Thul (2015) RNA-Seq analysis for indigo biosynthesis pathway genes in Indigofera tinctoria and Polygonum tinctorium. Genom. Data 6: 212–213.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Park, H., G. Park, W. Jeon, J.-O. Ahn, Y.-H. Yang, and K.-Y. Choi (2020) Whole-cell biocatalysis using cytochrome P450 monooxygenases for biotransformation of sustainable bioresources (fatty acids, fatty alkanes, and aromatic amino acids). Biotechnol. Adv. 40: 107504.

    Article  PubMed  Google Scholar 

  7. Zhang, X., Y. Qu, Q. Ma, C. Kong, H. Zhou, X. Cao, W. Shen, E. Shen, and J. Zhou (2014) Production of indirubin from tryptophan by recombinant Escherichia coli containing naphthalene dioxygenase genes from Comamonas sp. MQ. Appl. Biochem. Biotechnol. 172: 3194–3206.

    Article  CAS  PubMed  Google Scholar 

  8. Ameria, S. P., H. S. Jung, H. S. Kim, S. S. Han, H. S. Kim, and J. H. Lee (2015) Characterization of a flavin-containing monooxygenase from Corynebacterium glutamicum and its application to production of indigo and indirubin. Biotechnol. Lett. 37: 1637–1644.

    Article  CAS  PubMed  Google Scholar 

  9. Choi, H. S., J. K. Kim, E. H. Cho, Y. C. Kim, J. I. Kim, and S. W. Kim (2003) A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli. Biochem. Biophys. Res. Commun. 306: 930–936.

    Article  CAS  PubMed  Google Scholar 

  10. Mercadal, J. P., P. Isaac, F. Siñeriz, and M. A. Ferrero (2010) Indigo production by Pseudomonas sp. J26, a marine naphthalene-degrading strain. J. Basic Microbiol. 50: 290–293.

    Article  CAS  PubMed  Google Scholar 

  11. Wongsaroj, L., R. Sallabhan, J. M. Dubbs, S. Mongkolsuk, and S. Loprasert (2015) Cloning of toluene 4-monooxygenase genes and application of two-phase system to the production of the anticancer agent, indirubin. Mol. Biotechnol. 57: 720–726.

    Article  CAS  PubMed  Google Scholar 

  12. Yen, K. M., M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt, and K. K. Chen (1991) Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase. J. Bacteriol. 173: 5315–5327.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kim, H.-J., S. Jang, J. Kim, Y.-H. Yang, Y.-G. Kim, B.-G. Kim, and K.-Y. Choi (2017) Biosynthesis of indigo in Escherichia coli expressing self-sufficient CYP102A from Streptomyces cattleya. Dyes Pigm. 140: 29–35.

    Article  CAS  Google Scholar 

  14. Lee, J., J. Kim, J. E. Song, W. S. Song, E. J. Kim, Y. G. Kim, H. J. Jeong, H. R. Kim, K. Y. Choi, and B. G. Kim (2021) Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat. Chem. Biol. 17: 104–112.

    Article  CAS  PubMed  Google Scholar 

  15. Namgung, S., H. A. Park, J. Kim, P.-G. Lee, B.-G. Kim, Y.-H. Yang, and K.-Y. Choi (2019) Ecofriendly one-pot biosynthesis of indigo derivative dyes using CYP102G4 and PrnA halogenase. Dyes Pigm. 162: 80–88.

    Article  CAS  Google Scholar 

  16. Li, Y., J. Zhu, J. Wang, H. Xia, and S. Wu (2016) [Biosynthesis of indigo and indirubin by whole-cell catalyst designed by combination of protein engineering and metabolic engineering]. Sheng Wu Gong Cheng Xue Bao 32: 41–50.

    CAS  PubMed  Google Scholar 

  17. Dai, C., Q. Ma, Y. Li, D. Zhou, B. Yang, and Y. Qu (2019) Application of an efficient indole oxygenase system from Cupriavidus sp. SHE for indigo production. Bioprocess Biosyst. Eng. 42: 1963–1971.

    Article  CAS  PubMed  Google Scholar 

  18. Han, G. H., G. H. Gim, W. Kim, S. I. Seo, and S. W. Kim (2012) Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation. J. Biotechnol. 164: 179–187.

    Article  CAS  PubMed  Google Scholar 

  19. O’Connor, K. E., A. D. Dobson, and S. Hartmans (1997) Indigo formation by microorganisms expressing styrene monooxygenase activity. Appl. Environ. Microbiol. 63: 4287–4291.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Pasdaran, A., N. Azarpira, R. Heidari, S. Nourinejad, M. Zare, and A. Hamedi (2022) Effects of some cosmetic dyes and pigments on the proliferation of human foreskin fibroblasts and cellular oxidative stress; potential cytotoxicity of chlorophyllin and indigo carmine on fibroblasts. J. Cosmet. Dermatol. 21: 3979–3985.

    Article  PubMed  Google Scholar 

  21. Traglia, G., K. Chiem, B. Quinn, J. S. Fernandez, S. Montaña, M. Almuzara, M. A. Mussi, M. E. Tolmasky, A. Iriarte, D. Centrón, and M. S. Ramírez (2018) Genome sequence analysis of an extensively drug-resistant Acinetobacter baumannii indigo-pigmented strain depicts evidence of increase genome plasticity. Sci. Rep. 8: 16961.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Traglia, G., E. Vilacoba, M. Almuzara, L. Diana, A. Iriarte, D. Centrón, and M. S. Ramírez (2014) Draft genome sequence of an extensively drug-resistant Acinetobacter baumannii indigo-pigmented strain. Genome Announc. 2: e01146–14.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Vilacoba, E., M. Almuzara, L. Gulone, R. Rodriguez, E. Pallone, R. Bakai, D. Centrón, and M. S. Ramírez (2013) Outbreak of extensively drug-resistant Acinetobacter baumannii indigo-pigmented strains. J. Clin. Microbiol. 51: 3726–3730.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lin, G. H., H. P. Chen, J. H. Huang, T. T. Liu, T. K. Lin, S. J. Wang, C. H. Tseng, and H. Y. Shu (2012) Identification and characterization of an indigo-producing oxygenase involved in indole 3-acetic acid utilization by Acinetobacter baumannii. Antonie Van Leeuwenhoek 101: 881–890.

    Article  CAS  PubMed  Google Scholar 

  25. Qu, Y., W. Pi, F. Ma, J. Zhou, and X. Zhang (2010) Influence and optimization of growth substrates on indigo formation by a novel isolate Acinetobacter sp. PP-2. Bioresour. Technol. 101: 4527–4532.

    Article  CAS  PubMed  Google Scholar 

  26. Doukyu, N., K. Toyoda, and R. Aono (2003) Indigo production by Escherichia coli carrying the phenol hydroxylase gene from Acinetobacter sp strain ST-550 in a water-organic solvent two-phase system. Appl. Microbiol. Biotechnol. 60: 720–725.

    Article  CAS  PubMed  Google Scholar 

  27. Doukyu, N., T. Nakano, Y. Okuyama, and R. Aono (2002) Isolation of an Acinetobacter sp. ST-550 which produces a high level of indigo in a water-organic solvent two-phase system containing high levels of indole. Appl. Microbiol. Biotechnol. 58: 543–546.

    Article  CAS  PubMed  Google Scholar 

  28. Lin, H. R., H. Y. Shu, and G. H. Lin (2018) Biological roles of indole-3-acetic acid in Acinetobacter baumannii. Microbiol. Res. 216: 30–39.

    Article  CAS  PubMed  Google Scholar 

  29. Seo, D. and K.-Y. Choi (2020) Heterologous production of pyomelanin biopolymer using 4-hydroxyphenylpyruvate dioxygenase isolated from Ralstonia pickettii in Escherichia coli. Biochem. Eng. J. 157: 107548.

    Article  CAS  Google Scholar 

  30. Jung, H.-R., T.-R. Choi, Y. H. Han, Y.-L. Park, J. Y. Park, H.-S. Song, S.-Y. Yang, S. K. Bhatia, R. Gurav, H. Park, S. Namgung, K.-Y. Choi, and Y.-H. Yang (2020) Production of blue-colored polyhydroxybutyrate (PHB) by one-pot production and coextraction of indigo and PHB from recombinant Escherichia coli. Dyes Pigm. 173: 107889.

    Article  CAS  Google Scholar 

  31. Ahn, S.-Y., M. Choi, D. Jeong, S. Park, H. Park, K.-S. Jang, and K.-Y. Choi (2019) Synthesis and chemical composition analysis of protocatechualdehyde-based novel melanin dye by 15T FT-ICR: high dyeing performance on soft contact lens. Dyes Pigm. 160: 546–554.

    Article  CAS  Google Scholar 

  32. Jang, S., H. Gang, B.-G. Kim, and K.-Y. Choi (2018) FCS and ECH dependent production of phenolic aldehyde and melanin pigment from l-tyrosine in Escherichia coli. Enzyme Microb. Technol. 112: 59–64.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2021R1 A2C1007519). This study was also supported by the R&D Program of MOTIE/KEIT (grant number: 20014350).

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Author notes

  1. Sooyeon Ahn and SeoA Park contributed equally to this work.

Authors and Affiliations

  1. Institute of Environmental Engineering, Ajou University, Suwon, 16499, Korea

    Sooyeon Ahn, Pradeep Kumar & Kwon-Young Choi

  2. Graduate School of Environmental Engineering, Ajou University, Suwon, 16499, Korea

    SeoA Park & Kwon-Young Choi

  3. Environmental Biotechnology Laboratory, Department of Environmental and Safety Engineering, College of Engineering, Ajou University, Suwon, 16499, Korea

    Kwon-Young Choi

  4. Department of Energy Systems Research, Ajou University, Suwon, 16499, Korea

    Kwon-Young Choi

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  1. Sooyeon Ahn
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  2. SeoA Park
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Correspondence to Kwon-Young Choi.

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Ahn, S., Park, S., Kumar, P. et al. Bio-indigo Production using Wild-type Acinetobacter sp. and Indole-3-acetate Monooxygenase (iacA) Expressed in Escherichia coli. Biotechnol Bioproc E 28, 281–288 (2023). https://doi.org/10.1007/s12257-022-0163-0

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  • DOI: https://doi.org/10.1007/s12257-022-0163-0

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