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Putrescine production by engineered Corynebacterium glutamicum

  • Biotechnological Products and Process Engineering
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Abstract

Here, we report the engineering of the industrially relevant Corynebacterium glutamicum for putrescine production. C. glutamicum grew well in the presence of up to 500 mM of putrescine. A reduction of the growth rate by 34% and of biomass formation by 39% was observed at 750 mM of putrescine. C. glutamicum was enabled to produce putrescine by heterologous expression of genes encoding enzymes of the arginine- and ornithine decarboxylase pathways from Escherichia coli. The results showed that the putrescine yield by recombinant C. glutamicum strains provided with the arginine-decarboxylase pathway was 40 times lower than the yield by strains provided with the ornithine decarboxylase pathway. The highest production efficiency was reached by overexpression of speC, encoding the ornithine decarboxylase from E. coli, in combination with chromosomal deletion of genes encoding the arginine repressor ArgR and the ornithine carbamoyltransferase ArgF. In shake-flask batch cultures this strain produced putrescine up to 6 g/L with a space time yield of 0.1 g/L/h. The overall product yield was about 24 mol% (0.12 g/g of glucose).

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References

  • Abe S, Takayarna K, Kinoshita S (1967) Taxonomical studies on glutamic acid producing bacteria. J Gen Appl Microbiol 13:279–301

    Article  Google Scholar 

  • Altenburger P, Kämpfer P, Akimov VN, Lubitz W, Busse H-J (1997) Polyamine distribution in actinomycetes with group B peptidoglycan and species of the genera Brevibacterium, Corynebacterium, and Tsukamurella. Int J Syst Bacteriol 47:270–277

    Article  CAS  Google Scholar 

  • Barrett E, Stanton C, Zelder O, Fitzgerald G, Ross RP (2004) Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey. Appl Environ Microbiol 70:2861–2866

    Article  CAS  Google Scholar 

  • Bellmann A, Vrljic M, Patek M, Sahm H, Kramer R, Eggeling L (2001) Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum. Microbiology 147:1765–1774

    CAS  Google Scholar 

  • Bethesda Research Laboratories (1986) BRL pUC host: E. coli DH5a competent cells. Focus 8:9

    Google Scholar 

  • Blethen SL, Boeker EA, Snell EE (1968) Arginine decarboxylase from Escherichia coli. I. Purification and specificity for substrates and coenzyme. J Biol Chem 243:1671–1677

    CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Carvajal N, Orellana MS, Salas M, Enriquez P, Alarcon R, Uribe E, Lopez V (2004) Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group. Arch Biochem Biophys 430:185–190

    Article  CAS  Google Scholar 

  • Cunin R, Glansdorff N, Piérard A, Stalon V (1986) Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 50:314–352

    CAS  Google Scholar 

  • Eikmanns BJ, Kleinertz E, Liebl W, Sahm H (1991) A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene 102:93–98

    Article  CAS  Google Scholar 

  • Einarsson S, Josefsson B, Lagerkvist S (1983) Determination of amino acids with 9-fluorenylmethyl chloroformate and reversed-phase liquid chromatography. J Chromatogr A 282:609–618

    Article  CAS  Google Scholar 

  • Entian KD, Kötter P (2007) Twenty five yeast genetic strain and plasmid collections. Methods Microbiol 36:629–666

    Article  CAS  Google Scholar 

  • Eppelmann K, Nossin PMM, Kremer SM, Wubbolts MG (2006) Biochemical synthesis of 1,4-Butanediamine. WO/2006/005604

  • Georgi T, Rittmann D, Wendisch VF (2005) Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1, 6-bisphosphatase. Metab Eng 7:291–301

    Article  CAS  Google Scholar 

  • Guyer MS, Reed RR, Steitz JA, Low KB (1981) Identification of a sex-factor affinity site in E. coli gamma delta. Cold Spring Harb Symp Quant Biol 1:135–140

    Google Scholar 

  • Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172

    Article  CAS  Google Scholar 

  • Huser AT, Chassagnole C, Lindley ND, Merkamm M, Guyonvarch A, Elisakova V, Patek M, Kalinowski J, Brune I, Puhler A, Tauch A (2005) Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling. Appl Environ Microbiol 71:3255–3268

    Article  CAS  Google Scholar 

  • Hwang J-H, Hwang G-H, Cho J-Y (2008) Effect of increased glutamate availability on L-ornithine production in Corynebacterium glutamicum. J Microbiol Biotechnol 18:704–710

    CAS  Google Scholar 

  • Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol 42:39–51

    Article  CAS  Google Scholar 

  • Ikeda M, Mitsuhashi S, Tanaka K, Hayashi M (2009) Re-engineering of an L-Arginine and L-citrulline producer of Corynebacterium glutamicum. Appl Environ Microbiol 75:1635–1641

    Article  CAS  Google Scholar 

  • Inui M, Kawaguchi H, Murakami S, Vertes AA, Yukawa H (2004) Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254

    Article  CAS  Google Scholar 

  • Jo SJ, Maeda M, Ooi T, Taguchi S (2006) Production system for biodegradable polyester polyhydroxybutyrate by Corynebacterium glutamicum. J Biosci Bioeng 102:233–236

    Article  CAS  Google Scholar 

  • Kashiwagi K, Shibuya S, Tomitori H, Kuraishi A, Igarashi K (1997) Excretion and uptake of putrescine by the PotE protein in Escherichia coli. J Biol Chem 272:6318–6323

    Article  CAS  Google Scholar 

  • Kawaguchi H, Vertès AA, Okino S, Inui M, Yukawa H (2006) Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. Appl Environ Microbiol 72:3418–3428

    Article  CAS  Google Scholar 

  • Kawaguchi H, Sasaki M, Vertès AA, Inui M, Yukawa H (2008) Engineering of an L-arabinose metabolic pathway in Corynebacterium glutamicum. Appl Microbiol Biotechnol 77:1053–1062

    Article  CAS  Google Scholar 

  • Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175:5595–5603

    CAS  Google Scholar 

  • Kikuchi Y, Itaya H, Date M, Matsui K, Wu LF (2008) Production of Chryseobacterium proteolyticum protein-glutaminase using the twin-arginine translocation pathway in Corynebacterium glutamicum. Appl Microbiol Biotechnol 78:67–74

    Article  CAS  Google Scholar 

  • Kurihara S, Oda S, Tsuboi Y, Kim HG, Oshida M, Kumagai H, Suzuki H (2008) gamma-Glutamylputrescine synthetase in the putrescine utilization pathway of Escherichia coli K-12. J Biol Chem 283:19981–19990

    Article  CAS  Google Scholar 

  • Lee H, Yoon S, Jang H, Kim C, Kim T, Ryu W, Jung J, Park Y (2000) Effects of mixing on fed-batch fermentation of L-ornithine. J Biosci Bioeng 89:539–544

    Article  CAS  Google Scholar 

  • Michaels R, Kim KH (1966) Comparative studies of putrescine degradation by microorganisms. Biochim Biophys Acta 115:59–64

    CAS  Google Scholar 

  • Mimitsuka T, Sawai H, Hatsu M, Yamada K (2007) Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation. Biosci Biotechnol Biochem 71:2130–2135

    Article  CAS  Google Scholar 

  • Miyamoto S, Kashiwagi K, Ito K, Watanabe S, Igarashi K (1993) Estimation of polyamine distribution and polyamine stimulation of protein synthesis in Escherichia coli. Arch Biochem Biophys 300:63–68

    Article  CAS  Google Scholar 

  • Okino S, Inui M, Yukawa H (2005) Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 68:475–480

    Article  CAS  Google Scholar 

  • Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Möckel B, Sahm H, Eikmanns BJ (2001) Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol 3:295–300

    CAS  Google Scholar 

  • Qian Z-G, Xia X-X, Lee SY (2009) Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine. Biotechnol Bioeng 104:651–662

    CAS  Google Scholar 

  • Rittmann D, Lindner SN, Wendisch VF (2008) Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl Environ Microbiol 74:6216–6222

    Article  CAS  Google Scholar 

  • Sambrook J (2001) Molecular cloning: a laboratory manual/Joseph Sambrook, David W. Russell. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sanders J, Scott E, Weusthuis R, Mooibroek H (2007) Bio-refinery as the bio-inspired process to bulk chemicals. Macromol Biosci 7:105–117

    Article  CAS  Google Scholar 

  • Sasaki M, Jojima T, Inui M, Yukawa H (2010) Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 86:1057–1066

    Article  CAS  Google Scholar 

  • Schneider J, Niermann K, Wendisch VF (2010) Production of the amino acids L-glutamate, L-lysine, L-ornithine and L-arginine from arabinose by recombinant Corynebacterium glutamicum: accepted. doi:10.1016/j.jbiotec.2010.07.009

  • Scott E, Peter F, Sanders J (2007) Biomass in the manufacture of industrial products—the use of proteins and amino acids. Appl Microbiol Biotechnol 75:751–762

    Article  CAS  Google Scholar 

  • Seibold G, Auchter M, Berens S, Kalinowski J, Eikmanns BJ (2006) Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production. J Biotechnol 124:381–391

    Article  CAS  Google Scholar 

  • Tabor CW, Tabor H (1976) 1, 4-Diaminobutane (putrescine), spermidine, and spermine. Annu Rev Biochem 45:285–306

    Article  CAS  Google Scholar 

  • Tabor CW, Tabor H (1985) Polyamines in microorganisms. Microbiol Rev 49:81–99

    CAS  Google Scholar 

  • Tateno T, Fukuda H, Kondo A (2007) Production of L-Lysine from starch by Corynebacterium glutamicum displaying alpha-amylase on its cell surface. Appl Microbiol Biotechnol 74:1213–1220

    Article  CAS  Google Scholar 

  • Tateno T, Okada Y, Tsuchidate T, Tanaka T, Fukuda H, Kondo A (2009) Direct production of cadaverine from soluble starch using Corynebacterium glutamicum coexpressing alpha-amylase and lysine decarboxylase. Appl Microbiol Biotechnol 82:115–121

    Article  CAS  Google Scholar 

  • 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 52:541–545

    Article  Google Scholar 

  • Vrljic M, Sahm H, Eggeling L (1996) A new type of transporter with a new type of cellular function: L-lysine export from Corynebacterium glutamicum. Mol Microbiol 22:815–826

    Article  CAS  Google Scholar 

  • Wendisch VF (ed) (2007) Amino acid biosynthesis—pathways, regulation and metabolic engineering. Springer-Verlag, Berlin

    Google Scholar 

  • Wendisch VF, Bott M, Eikmanns BJ (2006) Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids. Curr Opin Microbiol 9:268–274

    Article  CAS  Google Scholar 

  • Yamanobe T, Kurihara Y, Uehara H, Komoto T (2007) Structure and characterization of nylon 46. J Mol Struct 829:80–87

    Article  CAS  Google Scholar 

  • Zelder O, Jeong WK, Klopprogge C, Herold A, Schröder H (2007) Process for the production of cadaverine. WO2007/113127

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  1. Genetics of Prokaryotes, Faculty of Biology & CeBiTec, University of Bielefeld, P.O. Box 100131, 33501, Bielefeld, Germany

    Jens Schneider & Volker F. Wendisch

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  1. Jens Schneider
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  2. Volker F. Wendisch
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Correspondence to Volker F. Wendisch.

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Schneider, J., Wendisch, V.F. Putrescine production by engineered Corynebacterium glutamicum . Appl Microbiol Biotechnol 88, 859–868 (2010). https://doi.org/10.1007/s00253-010-2778-x

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  • DOI: https://doi.org/10.1007/s00253-010-2778-x

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