Skip to main content
SpringerLink
Log in

Cloning and knockout of phytoene desaturase gene in Sphingomonas elodea ATCC 31461 for economic recovery of gellan gum

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.

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

  1. Armstrong G, Alberti M, Leach F, Hearst J (1989) Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet 216:254–268

    Article  PubMed  CAS  Google Scholar 

  2. Armstrong GA, Alberti M, Hearst JE (1990) Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes. Proc Natl Acad Sci USA 87:9975–9979

    Article  PubMed  CAS  Google Scholar 

  3. Bajaj IB, Survase SA, Saudagar PS, Singhal RS (2007) Gellan gum: fermentative production, downstream processing and applications. Food Technol Biotechnol 45:341–354

    CAS  Google Scholar 

  4. Banik R, Kanari B, Upadhyay S (2000) Exopolysaccharide of the gellan family: prospects and potential. World J Microbiol Biotechnol 16:407–414

    Article  CAS  Google Scholar 

  5. Banik RM, Santhiagu A, Upadhyay SN (2007) Optimization of nutrients for gellan gum production by Sphingomonas paucimobilis ATCC 31461 in molasses-based medium using response surface methodology. Bioresour Technol 98:792–797

    Article  PubMed  CAS  Google Scholar 

  6. Bartley GE, Schmidhauser TJ, Yanofsky C, Scolnik PA (1990) Carotenoid desaturases from Rhodobacter capsulatus and Neurospora crassa are structurally and functionally conserved and contain domains homologous to flavoprotein disulfide oxidoreductases. J Biol Chem 265:16020–16024

    PubMed  CAS  Google Scholar 

  7. Ehrenshaft M, Daub M (1994) Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene. Appl Environ Microbiol 60:2766–2771

    PubMed  CAS  Google Scholar 

  8. Fialho AM, Monteiro GA, Sa-Correia I (1991) Conjugal transfer of recombinant plasmids into gellan gum-producing and non-producing variants of Pseudomonas elodea ATCC 31461. Lett Appl Microbiol 12:85–87

    Article  PubMed  CAS  Google Scholar 

  9. Fialho AM, Moreira LM, Granja AT, Popescu AO, Hoffmann K, Sa-Correia I (2008) Occurrence, production, and applications of gellan: current state and perspectives. Appl Microbiol Biotechnol 79:889–900

    Article  PubMed  CAS  Google Scholar 

  10. Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652

    Article  PubMed  CAS  Google Scholar 

  11. Garrido-Fernández J, Maldonado-Barragán A, Caballero-Guerrero B, Hornero-Méndez D, Ruiz-Barba J (2010) Carotenoid production in Lactobacillus plantarum. Int J Food Microbiol 140:34–39

    Article  PubMed  Google Scholar 

  12. Gay P, Le Coq D, Steinmetz M, Berkelman T, Kado C (1985) Positive selection procedure for entrapment of insertion sequence elements in Gram-negative bacteria. J Bacteriol 164:918–921

    PubMed  CAS  Google Scholar 

  13. Giavasis I, Harvey L, McNeil B (2000) Gellan gum. Crit Rev Biotechnol 20:177–211

    Article  PubMed  CAS  Google Scholar 

  14. Grasdalen H, Smidsrød O (1987) Gelation of gellan gum. Carbohydr Polym 7:371–393

    Article  CAS  Google Scholar 

  15. Harding N, Patel Y, Coleman R (2006) Targeted gene deletions for polysaccharide slime formers. US Patent 0199201

  16. Jansson P-E, Lindberg B, Sandford PA (1983) Structural studies of gellan gum, an extracellular polysaccharide elaborated by Pseudomonas elodea. Carbohydr Res 124:135–139

    Article  CAS  Google Scholar 

  17. Jenkins C, Andrewes A, McQuade T, Starr M (1979) The pigment of Pseudomonas paucimobilis is a carotenoid (nostoxanthin), rather than a brominated aryl-polyene (xanthomonadin). Curr Microbiol 3:1–4

    Article  CAS  Google Scholar 

  18. Kanari B, Banik R, Upadhyay S (2002) Effect of environmental factors and carbohydrate on gellan gum production. Appl Biochem Biotechnol 102:129–140

    Article  PubMed  Google Scholar 

  19. Kang K, Veeder G (1982) Polysaccharide S-60 and bacterial fermentation process for its preparation. US Patent 4326053

  20. Kang K, Veeder G, Mirrasoul P, Kaneko T, Cottrell I (1982) Agar-like polysaccharide produced by a Pseudomonas species: production and basic properties. Appl Environ Microbiol 43:1086–1091

    PubMed  CAS  Google Scholar 

  21. Kang K, Veeder G, Colegrove G (1983) Deacetylated polysaccharide S-60. US Patent 4385123

  22. Lenz O, Friedrich B (1998) A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus. Proc Natl Acad Sci USA 95:12474–12479

    Article  PubMed  CAS  Google Scholar 

  23. Manna B, Gambhir A, Ghosh P (1996) Production and rheological characteristics of the microbial polysaccharide gellan. Lett Appl Microbiol 23:141–145

    Article  CAS  Google Scholar 

  24. Miller J (1973) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  25. Nampoothiri K, Singhania R, Sabarinath C, Pandey A (2003) Fermentative production of gellan using Sphingomonas paucimobilis. Process Biochem 38:1513–1519

    Article  CAS  Google Scholar 

  26. O’ Neill MA, Selvendran RR, Morris VJ (1983) Structure of the acidic extracellular gelling polysaccharide produced by Pseudomonas elodea. Carbohydr Res 124:123–133

    Article  Google Scholar 

  27. Pasamontes L, Hug D, Tessier M, Hohmann HP, Schierle J, van Loon AP (1997) Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534. Gene 185:35–41

    Article  PubMed  CAS  Google Scholar 

  28. Paul F, Morin A, Monsan P (1986) Microbial polysaccharides with actual potential industrial applications. Biotechnol Adv 4:245–259

    Article  PubMed  CAS  Google Scholar 

  29. Sambrook J, Russell D (1986) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, 999 pp

  30. Sandmann G (1994) Carotenoid biosynthesis in microorganisms and plants. Eur J Biochem 223:7–24

    Article  PubMed  CAS  Google Scholar 

  31. Sutherland I (2001) Microbial polysaccharides from Gram-negative bacteria. Int Dairy J 11:663–674

    Article  CAS  Google Scholar 

  32. Takaichi S, Shimada K (1992) Characterization of carotenoids in photosynthetic bacteria. Method Enzymol 213:374–385

    Article  CAS  Google Scholar 

  33. Tan G, Gao Y, Shi M, Zhang X, He S, Chen Z et al (2005) SiteFinding-PCR: a simple and efficient PCR method for chromosome walking. Nucleic Acids Res 33:e122

    Article  PubMed  Google Scholar 

  34. Tonhosolo R, D’Alexandri F, De Rosso V, Gazarini M, Matsumura M, Peres V et al (2009) Carotenoid biosynthesis in intraerythrocytic stages of plasmodium falciparum. J Biol Chem 284:9974–9985

    Article  PubMed  CAS  Google Scholar 

  35. Videira PA, Cortes LL, Fialho AM, Sa-Correia I (2000) Identification of the pgmG gene, encoding a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461. Appl Environ Microbiol 66:2252–2258

    Article  PubMed  CAS  Google Scholar 

  36. Wang X, Xu P, Yuan Y, Liu C, Zhang D, Yang Z et al (2006) Modeling for gellan gum production by Sphingomonas paucimobilis ATCC 31461 in a simplified medium. Appl Environ Microbiol 72:3367–3374

    Article  PubMed  CAS  Google Scholar 

  37. West T (2002) Isolation of a mutant strain of Pseudomonas sp. ATCC 31461 exhibiting elevated polysaccharide production. J Ind Microbiol Biotechnol 29:185–188

    Article  PubMed  CAS  Google Scholar 

  38. Yuan C, Morrison N, Clark R (2005): Calcium stable high acyl gellan gum for enhanced colloidal stability in beverages. US Patent 0266138

Download references

Acknowledgments

This work was supported by Zhejiang DSM Zhongken Biotechnology Co., Ltd. We thank Dr. Figurski for providing pRK2013, Dr. Lenz for supplying pLO3, and Xinhang Jiang of Zhejiang University for assisting in the HPLC analysis.

Author information

Authors and Affiliations

  1. Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, People’s Republic of China

    Liang Zhu, Xuechang Wu, Ou Li, Yamin Chen, Chaodong Qian, Yi Teng, Xianglin Tao & Haichun Gao

Authors
  1. Liang Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xuechang Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ou Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yamin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chaodong Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yi Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xianglin Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Haichun Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xuechang Wu.

Additional information

Liang Zhu and Xuechang Wu contributed equally to this article.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhu, L., Wu, X., Li, O. et al. Cloning and knockout of phytoene desaturase gene in Sphingomonas elodea ATCC 31461 for economic recovery of gellan gum. J Ind Microbiol Biotechnol 38, 1507–1513 (2011). https://doi.org/10.1007/s10295-010-0937-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-010-0937-9

Keywords

Search

Navigation