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Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for l-lysine production from methanol at 50°C

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Abstract

We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial l-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the l-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by l-threonine and l-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l l-lysine and 59 g/l l-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l l-lysine and 69 g/l of l-glutamate, while a sixfold higher l-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced l-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric l-lysine yield and the l-lysine yield on methanol, and caused significantly reduced volumetric l-glutamate yield and l-glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving l-lysine overproduction, PC activity plays an important role in controlling l-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for l-lysine overproduction by B. methanolicus.

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References

  • Anthony C (1991) Assimilation of carbon by methylotrophs. In: Biology of methylotrophs, Goldberg I, Rokem JS eds. Butterworth-Heinemann, Boston, USA pp. 70–109

  • Brautaset T, Williams MD, Dillingham RD, Kaufmann C, Bennaars A, Crabbe E, Flickinger MC (2003) The role of Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of l-glutamate in l-lysine secreting mutants. Appl Environ Microbiol 69:3986–3995

    Article  CAS  Google Scholar 

  • Brautaset T, Jakobsen ØM, Flickinger MC, Valla S, Ellingsen TE (2004) Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus. J Bacteriol 186:1229–1238

    Article  CAS  Google Scholar 

  • Brautaset T, Jakobsen ØM, Flickinger MC, Josefsen KD, Ellingsen TE (2007) Bacillus methanolicus: a candidate for industrial production of amino acids from methanol at 50 °C. Appl Microbiol Biotechnol 74:22–34

    Article  CAS  Google Scholar 

  • Brooke AG, Watling EM, Attwood MM, Tempest DW (1989) Environmental control of metabolic fluxes in thermotolerant methylotrophic Bacillus strains. Arch Microbiol 151:268–273

    Article  CAS  Google Scholar 

  • Cahyanto MN, Kawasaki H, Nagashio M, Fujiyama K, Seki T (2006) Regulation of aspartokinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase in Lactobacillus plantarum. Microbiology 152:105–112

    Article  CAS  Google Scholar 

  • Cue D, Lam H, Dillingham RL, Hanson RS, Flickinger MC (1997) Genetic manipulation of Bacillus methanolicus, a Gram-positive thermotolerant methylotroph. Appl Environ Microbiol 63:1406–1420

    CAS  Google Scholar 

  • Dunn MF, Encarnación S, Araíza G, Vargas C, Dávalos A, Peralta H, Mora Y, Mora J (1996) Pyruvate carboxylase from Rhizobium etli: mutant characterization, nucleotide sequence, and physiological role. J Bacteriol 178:5960–5970

    CAS  Google Scholar 

  • 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 

  • Haima P, Bron S, Venema G (1989) The effect of restriction and shotgun cloning and plasmid stability in Bacillus subtilis Marburg. Mol Gen Genet 209:335–342

    Article  Google Scholar 

  • Hamer G, Pal HS, Hamdan IY (1979) Yield depression in methanol-utilizing batch cultures. Biotechnol Lett 1:9–14

    Article  CAS  Google Scholar 

  • Hanson RS, Dillingham RL, Olson P, Lee GH, Cue D, Schendel FJ, Bremmon C, Flickinger MC (1996) Production of l-lysine and some other amino acids by mutants of Bacillus methanolicus. In: Lidstrom ME, Tabita FR (eds) Microbial growth on C1 compounds. Kluwer Academic Publishers, Dordrecht, the Netherlands, pp 227–234

    Google Scholar 

  • Hasegawa T, Hashimoto K, Kawasaki H, Nakamutsu T (2008) Changes in enzyme activities at the pyruvate node in glutamate-producing Corynebacterium glutamicum. J Biosci Bioeng 105:12–19

    Article  CAS  Google Scholar 

  • Ikeda M, Nakagawa S (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109

    Article  CAS  Google Scholar 

  • Ikeda M, Ohnishi J, Hayashi M, Mitsuhashi S (2006) A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient l-lysine production. J Ind Microbiol Biotechnol 33:610–615

    Article  CAS  Google Scholar 

  • Jakobsen ØM, Benichou A, Flickinger MC, Valla S, Ellingsen TE, Brautaset T (2006) Upregulated transcription of plasmid and chromosomal ribulose monophosphate pathway genes is critical for methanol assimilation and methanol tolerance in the methylotrophic bacterium Bacillus methanolicus. J Bacteriol 188:3063–3072

    Article  CAS  Google Scholar 

  • Jakobsen ØM, Brautaset T, Degnes KF, Heggeset TMB, Balzer S, Flickinger MC, Valla S, Ellingsen TE (2009) Overexpression of wild-type aspartokinase increases l-lysine production in thermotolerant methylotrophic Bacillus methanolicus. Appl Environ Microbiol 75:652–661

    Article  CAS  Google Scholar 

  • Koffas MAG, Ramamoorthi R, Pine WA, Sinskey AJ, Stephanopoulos G (1998) Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene. Appl Microbiol Biotechnol 50:346–352

    Article  CAS  Google Scholar 

  • Koffas MA, Jung GY, Aon JC, Stephanopoulos G (2002) Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum. Appl Environ Microbiol 68:5422–5428

    Article  CAS  Google Scholar 

  • Koffas MA, Jung GY, Stephanopoulos G (2003) Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression. Metab Eng 5:32–41

    Article  CAS  Google Scholar 

  • Kondo H, Kazuta Y, Saito A, Fuji K (1997) Cloning and nucleotide sequence of the Bacillus stearothermophilus pyruvate carboxylase. Gene 191:47–50

    Article  CAS  Google Scholar 

  • Lee GH, Hur W, Bremmon CE, Flickinger MC (1996) l-lysine production from methanol at 50 °C with Bacillus methanolicus: modelling volume control, l-lysine concentration, and productivity with a three-phase continuous simulation. Biotechnol Bioeng 49:639–653

    Article  CAS  Google Scholar 

  • Leuchtenberger W, Huthmacher K, Drauz K (2005) Biotechnological production of amino acids and derivatives: current status and prospects. Appl Microbiol Biotechnol 69:1–8

    Article  CAS  Google Scholar 

  • Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K, Ikeda M (2002) A novel methodology employing Corynebacterium glutamicum genome information to generate a new l-lysine producing mutant. Appl Microbiol Biotechnol 58:217–223

    Article  CAS  Google Scholar 

  • Parsot C, Cohen GN (1988) Cloning and nucleotide sequence of the Bacillus subtilis hom gene coding for homoserine dehydrogenase. Structural and evolutionary relationships with Escherichia coli aspartokinases-homoserine dehydrogenases I and II. J Biol Chem 263:14654–14660

    CAS  Google Scholar 

  • Peterson S, de Graaf AA, Eggeling L, Möllney M, Viechert W, Sahm H (2000) In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum. J Biol Chem 275:35932–35941

    Article  Google Scholar 

  • Peters-Wendisch PG, Wendisch VF, Paul S, Eikmanns BJ, Sahm H (1997) Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum. Microbiology 143:1095–1103

    Article  CAS  Google Scholar 

  • Peters-Wendisch PG, Kreutzer C, Kaliniwski J, Patek M, Sahm H, Eikmanns BJ (1998) Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene. Microbiology 144:915–927

    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 

  • Pfefferle W, Möckel B, Bathe B, Marx A (2003) Microbial production of l-amino acids: biotechnological manufacture of lysine. Adv Biochem Eng Biotechnol 79:59–112

    CAS  Google Scholar 

  • Sahm H, Eggeling L, Eikmanns B, Kramer R (1996) Construction of l-lysine-, l-threonine-, or l-isoleucine-overproducing strains of Corynebacterium glutamicum. Ann N Y Acad Sci 782:25–39

    Article  CAS  Google Scholar 

  • Sahm H, Eggeling L, de Graaf AA (2000) Pathway analysis and metabolic engineering in Corynebacterium glutamicum. Biol Chem 381:899–910

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Sanchez S, Demain AL (2008) Metabolic regulation and overproduction of primary metabolites. Microbial Biotechnol 1:283–319

    Article  CAS  Google Scholar 

  • Schendel FJ, Bremmon CE, Flickinger MC, Guettler M, Hanson RS (1990) l-lysine production at 50 °C by mutants of a newly isolated and characterized Bacillus sp. Appl Environ Microbiol 56:963–970

    CAS  Google Scholar 

  • Schrader J, Schilling M, Holtmann D, Sell D, Filho MV, Marx A, Vorholt JA (2009) Methanol-based industrial biotechnology: current status and future perspectives on methylotrophic bacteria. Trends Biotechnol 27:107–115

    Article  CAS  Google Scholar 

  • Shirai T, Fujimura K, Furuzawa C, Nagahisa K, Shioya S, Shimizu H (2007) Study of the roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis. Microb Cell Fact 6:19–30

    Article  Google Scholar 

  • Thèze J, Margarita D, Cohen GN, Borne F, Patte JC (1974) Mapping of the structural genes of the three aspartokinases and of the two homoserine dehydrogenases of Escherichia coli K-12. J Bacteriol 117:133–143

    Google Scholar 

  • Vrljic M, Kronemeyer W, Sahm H, Eggeling L (1995) Unbalance of l-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-deficient mutants. J Bacteriol 177:4021–4027

    CAS  Google Scholar 

  • Yao W, Deng X, Zhong H, Liu M, Zheng P, Sun Z, Zhang Y (2009) Double deletion of dtsR1 and pyc induce efficient l-glutamate overproduction in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 36:911–922

    Article  CAS  Google Scholar 

  • Yong-Biao J, Islam MN, Sueda S, Kondo H (2004) Identification of the catalytic residues involved in the carboxyl transfer of pyruvate carboxylase. Biochemistry 43:5912–5920

    Article  CAS  Google Scholar 

  • Zhang M, Lidstrom ME (2003) Promoters and transcripts of genes involved in methanol oxidation in Methylobacterium extorquens AM1. Microbiology 149:1033–1040

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the Research Council of Norway. We thank Halvor L. Holen and Trine Aakvik for their valuable contribution to cloning the pyc gene.

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Authors and Affiliations

  1. Department of Biotechnology, SINTEF Materials and Chemistry, 7465, Trondheim, Norway

    Trygve Brautaset, Øyvind M. Jakobsen, Kristin F. Degnes, Roman Netzer, Ingemar Nærdal & Trond E. Ellingsen

  2. Department of Biotechnology, Norwegian University of Science and Technology, 7491, Trondheim, Norway

    Anne Krog

  3. BioTechnology Institute, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN, 55108, USA

    Rick Dillingham

  4. Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695-7928, USA

    Michael C. Flickinger

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  1. Trygve Brautaset
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  2. Øyvind M. Jakobsen
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Correspondence to Trygve Brautaset.

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Brautaset, T., Jakobsen, Ø.M., Degnes, K.F. et al. Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for l-lysine production from methanol at 50°C. Appl Microbiol Biotechnol 87, 951–964 (2010). https://doi.org/10.1007/s00253-010-2559-6

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