Abstract
The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the l-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved l-ornithine production by Corynebacterium glutamicum. Production of l-ornithine requires 2 moles of NADPH per mole of l-ornithine. Thus, the effect of NADPH availability on l-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of l-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum ΔAPRE::rocG, produced 14.84 g l−1 of l-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.
Similar content being viewed by others
References
Belitsky BR, Sonenshein AL (1998) Role and regulation of Bacillus subtilis glutamate dehydrogenase genes. J Bacteriol 180:6298–6305
Chinard FP (1952) Photometric estimation of proline and ornithine. J Biol Chem 199:91–95
Choi DK, Ryu WS, Choi CY, Park YH (1996) Production of l-ornithine by arginine auxotrophic mutants of Brevibacterium ketoglutamicum in dual substrate limited continuous culture. J Ferment Bioeng 81:216–219
Eikmanns BJ, Thum-Schmitz N, Eggeling L, Ludtke KU, Sahm H (1994) Nucleotide sequence, expression, and transcription analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology 140:1817–1828
Huang MT, Wang Y, Liu JZ, Mao ZW (2011) Multiple strategies for metabolic engineering of Escherichia coli for efficient production of coenzyme Q10. Chin J Chem Eng 19:316–326
Hwang G-H, Cho J-Y (2010) Identification of a suppressor gene for the arginine-auxotrophic argJ mutation in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 37:1131–1136
Hwang GH, Cho JY (2012) Implication of gluconate kinase activity in l-ornithine biosynthesis in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 39:1869–1874
Hwang JH, Hwang GH, Cho JY (2008) Effect of increased glutamate availability on l-ornithine production in Corynebacterium glutamicum. J Microbiol Biotechnol 18:704–710
Inui M, Kawaguchi H, Murakami S, Vertès AA, Yukawa H (2004) Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254
Kabus A, Georgi T, Wendisch VF, Michael B (2007) Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation. Appl Microbiol Biotechnol 75:47–53
Kholy ER, Eikmanns BJ, Gutmann M, Sahm H (1993) Glutamate dehydrogenase is not essential for glutamate formation by Corynebacterium glutamicum. Appl Environ Microbiol 59:2329–2331
Kinoshita S, Nakayama K, Udaka S (1957) The fermentative production of l-ornithine. J Gen Appl Microbiol 3:276–277
Kirchner O, Tauch A (2003) Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J Biotechnol 104:287–299
Lee HC, Kim JS, Jang W, Kim SY (2010) High NADPH/NADP ratio improves thymidine production by a metabolically engineered Escherichia coli strain. J Biotechnol 149:24–32
Lee HW, Yoon SJ, Jang HW, Kim CS, Kim TH, Ryu WS, Jung JK, Park YH (2000) Effects of mixing on fed-batch fermentation of l-ornithine. J Biosci Bioeng 89:539–544
Lee SY, Cho JY, Lee HJ, Kim YH, Min J (2010) Enhancement of ornithine production in proline-supplemented Corynebacterium glutamicum by ornithine cyclodeaminase. J Microbiol Biotechnol 20:127–131
Lee SY, Kim YH, Min J (2009) The effect of ArgR-DNA binding affinity on ornithine production in Corynebacterium glutamicum. Curr Microbiol 59:483–488
Lee YJ, Cho JY (2006) Genetic manipulation of a primary metabolic pathway for l-ornithine production in Escherichia coli. Biotechnol Lett 28:1849–1856
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408
Lu DM, Jiang L-Y, Chen L-A, Liu J-Z, Mao Z-W (2011) Optimization of fermentation conditions of the engineered Corynebacterium glutamicum to enhance l-ornithine production by response surface methodology. J Biotechnol Biomater 1:116. doi:10.4172/2155-952X.1000116,15
Lu DM, Liu JZ, Mao ZW (2012) Engineering of Corynebacterium glutamicum to enhance l-ornithine production by gene knockout and comparative proteomic analysis. Chin J Chem Eng 20:731–739
Martínez A, Zhu J, Lin H, Bennett GN, San KY (2008) Replacing Escherichia coli NAD-dependent glyceraldehydes 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. Metab Eng 10:352–359
Meiswinkel TM, Gopinath V, Lindner SN, Nampoothiri M, Wendisch VF (2012) Accelerated pentose utilization by Corynebacterium glutamicum for accelerated production lysine, glutamate, ornithine and putrescine. Microb Biotechnol 6:131–140
Salvatore F, Cimino F, Maria C, Cittadini D (1964) Mechanism of the protection by l-ornithine-l-aspartate mixture and by l-arginine in ammonia intoxication. Arch Biochem Biophys 107:499–503
Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73
Schneider J, Niermann K, Wendisch VF (2011) Production of the amino acids l-glutamate, l-lysine, l-ornithine and l-arginine from arabinose by recombinant Corynebacterium glutamicum. J Biotechnol 154:191–198
Shi A, Zhu X, Lu J, Zhang X, Ma Y (2013) Activating transhydrogenase and NAD kinase in combination for improving isobutanol production. Metab Eng 16:1–10
Shi HP, Fishel RS, Efron DT, Williams JZ, Fishel MH, Barbul A (2002) Effect of supplemental ornithine on wound healing. J Surg Res 106:299–302
Shirai T, Fujimura K, Furusawa C, Nagahisa K, Shioya S, Shimizu H (2007) Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis. Microb Cell Fact 6:19
Takeno S, Murata R, Kobayashi R, Mitsuhashi S, Ikeda M (2010) Engineering of Corynebacterium glutamicum with an NADPH-generating glycolytic pathway for l-lysine production. Appl Environ Microbiol 76:7154–7160
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
Xu D, Tan Y, Shi F, Wang X (2010) An improved shuttle vector constructed for metabolic engineering research in Corynebacterium glutamicum. Plasmid 64:85–91
Zajac A, Poprzecki S, Zebrowska A, Chalimoniuk M, Langfort J (2010) Arginine and ornithine supplementation increases growth hormone and insulin-like growth factor-1 serum levels after heavy-resistance exercise in strength-trained athletes. J Strength Cond Res 24:1082–1090
Zhang JF, Wang JB, Huang JM, Zhang J (2009) Breeding of high-yield l-ornithine-producing strain by protoplast fusion. Amino acids Biotic Resour 31:53–57
Acknowledgments
We are grateful to the National Natural Science Foundation of China (grant nos. 30970089, 20876181, 21276289) and the Natural Science Foundation of Guangdong Province (nos. 9351027501000003, S2011010001396) for their financial support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Jiang, LY., Zhang, YY., Li, Z. et al. Metabolic engineering of Corynebacterium glutamicum for increasing the production of l-ornithine by increasing NADPH availability. J Ind Microbiol Biotechnol 40, 1143–1151 (2013). https://doi.org/10.1007/s10295-013-1306-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10295-013-1306-2