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Re-evaluation of cyanophycin synthesis in Corynebacterium glutamicum and incorporation of glutamic acid and lysine into the polymer

  • Lars Wiefel
  • Karen Wohlers
  • Alexander SteinbüchelEmail author
Biotechnological products and process engineering
  • 72 Downloads

Abstract

Corynebacterium glutamicum was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-l-arginyl-poly-[l-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, C. glutamicum was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of C. glutamicum to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains C. glutamicum pVWEx1::cphAΔ1 and C. glutamicum pVWEx1::cphABP1 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40 mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in C. glutamicum in previous studies. Additionally, an incorporation of up to 6 mol% of glutamic acid into the backbone of CGP synthesized by C. glutamicum pVWEx1::cphADh was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.

Keywords

Cyanophycin Corynebacterium glutamicum Glutamic acid Lysine 

Notes

Acknowledgments

We thank Cysal GmbH (Münster, Germany) for providing purified CphE.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain studies with human participants or animals performed by any of the authors.

References

  1. Aboulmagd E, Oppermann-Sanio FB, Steinbüchel A (2000) Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC 6308. Arch Microbiol 174:297–306CrossRefGoogle Scholar
  2. Aboulmagd E, Voss I, Oppermann-Sanio FB, Steinbüchel A (2001) Heterologous expression of cyanophycin synthetase and cyanophycin synthesis in the industrial relevant bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida. Biomacromolecules 2:1338–1342CrossRefGoogle Scholar
  3. Arai T, Kino K (2008) A cyanophycin synthetase from Thermosynechococcus elongatus BP-1 catalyzes primer-independent cyanophycin synthesis. Appl Microbiol Biotechnol 81:69–78CrossRefGoogle Scholar
  4. Becker J, Wittmann C (2012) Systems and synthetic metabolic engineering for amino acid production – the heartbeat of industrial strain development. Curr Opin Biotechnol 23:718–726CrossRefGoogle Scholar
  5. Eberhardt D, Jensen JV, Wendisch VF (2014) L-Citrulline production by metabolically engineered Corynebacterium glutamicum from glucose and alternative carbon sources. AMB Express 4:85CrossRefGoogle Scholar
  6. Eikmanns BJ, Kleinertz E, Liebl W, Sahm H (1991) A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, con-trolled gene expression, and promoter probing. Gene 102:93–98CrossRefGoogle Scholar
  7. Frey KM, Oppermann-Sanio FB, Schmidt H, Steinbüchel A (2002) Technical scale production of cyanophycin with recombinant strains of Escherichia coli. Appl Environ Microbiol 68:3377–3384CrossRefGoogle Scholar
  8. Frommeyer M, Steinbüchel A (2013) Increased lysine content is the main characteristic of the soluble form of the polyamide cyanophycin synthesized by recombinant Escherichia coli. Appl Environ Microbiol 79:4474–4483CrossRefGoogle Scholar
  9. Frommeyer M, Wiefel L, Steinbüchel A (2016) Features of the biotechnologically relevant polyamide family “cyanophycins” and their biosynthesis in prokaryotes and eukaryotes. Crit Rev Biotechnol 36:153–164CrossRefGoogle Scholar
  10. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefGoogle Scholar
  11. Hühns M, Neumann K, Hausmann T, Ziegler K, Klemke F, Kahmann U, Staiger D, Lockau W, Pistorius EK, Broer I (2008) Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum. Plant Biotechnol J 6:321–336CrossRefGoogle Scholar
  12. Hühns M, Neumann K, Hausmann T, Klemke F, Lockau W, Kahmann U, Kopertekh L, Staiger D, Pistorius EK, Reuther J, Waldvogel E, Wohlleben W, Effmert M, Junghans H, Neubauer K, Kragl U, Schmidt K, Schmidtke J, Broer I (2009) Tuber-specific cphA expression to enhance cyanophycin production in potatoes. Plant Biotechnol J 7:883–898CrossRefGoogle Scholar
  13. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28CrossRefGoogle Scholar
  14. Joentgen W, Groth T, Steinbüchel A. 2001. Polyasparaginic acid homopolymers and copolymers, biotechnical production and use thereof. US Patent 6,180,752 B1Google Scholar
  15. Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175:5595–5603CrossRefGoogle Scholar
  16. Kimura E (2005) L-Glutamate production. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CDC Press, Boca Raton, pp 439–464CrossRefGoogle Scholar
  17. Kroll J, Klinter S, Steinbüchel A (2011) A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium. Appl Microbiol Biotechnol 89:593–604CrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  19. Merritt MV, Sid SS, Mesh L, Allen MM (1994) Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions. Arch Microbiol 162:158–166CrossRefGoogle Scholar
  20. Persicke M, Plassmeier J, Neuweger H, Rückert C, Pühler A, Kalinowski J (2011) Size exclusion chromatography - an improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites. J Biotechnol 154:171–178CrossRefGoogle Scholar
  21. Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Mockel B, Sahm H, Eikmanns BJ (2001) Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol 3:295–300Google Scholar
  22. Rittmann D, Lindner SN, Wendisch VF (2009) Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl Environ Microbiol 74:6216–6222CrossRefGoogle Scholar
  23. Sallam A, Kast A, Przybilla S, Meiswinkel T, Stenbüchel A (2009) Biotechnological process for production of b-dipeptides from cyanophycin on a technical scale and its optimization. Appl Environ Microbiol 75:29–38CrossRefGoogle Scholar
  24. Sallam A, Steinbüchel A (2010) Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production. Appl Microbiol Biotechnol 87:815–828CrossRefGoogle Scholar
  25. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USAGoogle Scholar
  26. Siebert D, Wendisch VF (2015) Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum. Biotechnol Biofuels 8:91–104CrossRefGoogle Scholar
  27. Simon RD (1976) The biosynthesis of multi-L-arginyl-poly(L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. Biochim Biophys Acta 422:407–418CrossRefGoogle Scholar
  28. Steinle A, Bergander K, Steinbüchel A (2009) Metabolic engineering of Saccharomyces cerevisiae for production of novel cyanophycins with an extended range of constituent amino acids. Appl Environ Microbiol 75:3437–3446CrossRefGoogle Scholar
  29. Steinle A, Witthoff S, Krause JP, Steinbüchel A (2010) Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases. Appl Environ Microbiol 76:1062–1070CrossRefGoogle Scholar
  30. Tauch A, Kirchner O, Löffler B, Götker S, Pühler A, Kalinowski J (2002) Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr Microbiol 45:362–367CrossRefGoogle Scholar
  31. van der Rest ME, Lange C, Molenaar D (1999) A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl Microbiol Biotechnol 52:541–545CrossRefGoogle Scholar
  32. von der Osten CH, Gioannetti C, Sinskey AJ (1989) Design of a defined medium for growth of Corynebacterium glutamicum in which citrate facilitates iron uptake. Biotechnol Lett 11:11–16CrossRefGoogle Scholar
  33. Voss I, Steinbüchel A (2006) Application of a KDPG-aldolase gene dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16. Metab Eng 8:66–78CrossRefGoogle Scholar
  34. Wiefel L, Bröker A, Steinbüchel A (2011) Synthesis of a citrulline-rich cyanophycin by use of Pseudomonas putida ATCC 4359. Appl Microbiol Biotechnol 90:1755–1762CrossRefGoogle Scholar
  35. Wiefel L, Steinbüchel A (2014) Solubility behavior of cyanophycin depending on lysine content. Appl Environ Microbiol 80:1091–1096CrossRefGoogle Scholar
  36. Ziegler K, Deutzmann R, Lockau W (2002) Cyanophyc in synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense. Z Naturforsch 57:522–529CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institut für Molekulare Mikrobiologie und BiotechnologieWestfälische Wilhelms-UniversitätMünsterGermany
  2. 2.Environmental Science DepartmentKing Abdulaziz UniversityJeddahSaudi Arabia

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