Abstract
In this study, Corynebacterium glutamicum ATCC 13032 was engineered to produce l-citrulline through a metabolic engineering strategy. To prevent the flux away from l-citrulline and to increase the expression levels of genes involved in the citrulline biosynthesis pathway, the argininosuccinate synthase gene (argG) and the repressor gene (argR) were inactivated. The engineered C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2) produced higher amounts of l-citrulline (5.43 g/L) compared to the wildtype strain (0.15 g/L). To determine new strategies for further enhancement of l-citrulline production, the effect of l-citrulline on ornithine acetyltransferase (EC 2.3.1.35; OATase; ArgJ) was first investigated. Citrulline was determined to inhibit Ornithine acetyltransferase; for 50 % inhibition, citrulline concentration was 30 mM. The argJ gene from C. glutamicum ATCC 13032 was cloned, and the recombinant shuttle plasmid pXMJ19-argJ was constructed and expressed in C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2). Overexpression of the argJ gene exhibited increased OAT activity and resulted in a positive effect on citrulline production (8.51 g/L). These results indicate that OAT plays a vital role during l-citrulline production in C. glutamicum.
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References
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
Chun JY, Lee EJ, Lee HS et al (1998) Molecular cloning and analysis of the argC gene from Corynebacterium glutamicum. Biochem Mol Biol Int 46:437–447
Cunin R, Glansdorff N, Pierard A et al (1986) Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 50:314–352
Friedrich B, Friedrich CG, Magasanik B (1978) Catabolic N-2-acetylornithine 5-aminotransferase of Klebsiella aerogenes: control of synthesis by induction, catabolite repression, and activation by glutamine synthetase. J Bacteriol 133:686–691
Hayashi M, Ohnishi J, Mitsuhashi S et al (2006) Transcriptome analysis reveals global expression changes in an industrial l-lysine producer of Corynebacterium glutamicum. Biosci Biotechnol Biochem 70:546–550
Hecker M, Sessa WC, Harris HJ et al (1990) The metabolism of l-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle l-citrulline to l-arginine. Proc Natl Acad Sci USA 87:8612–8616
Hwang GH, Cho JY (2010) Identification of a suppressor gene for the arginine-auxotrophic argJ mutation in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 37:1131–1136
Ikeda M, Mitsuhashi S, Tanaka K et al (2009) Reengineering of a Corynebacterium glutamicum l-arginine and l-citrulline producer. Appl Environ Microbiol 75:1635–1641
Jakoby M, Ngouoto-Nkili CE, Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors. Biotechnol Tech 13:437–441
Kabus A, Niebisch A, Bott M (2007) Role of cytochrome bd oxidase from Corynebacterium glutamicum in growth and lysine production. Appl Environ Microbiol 73:861–868
Kumar A, Vij N, Randhawa GS (2003) Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti. Indian J Exp Biol 41:1198–1204
Kwon NS, Nathan CF, Gilker C et al (1990) l-citrulline production from l-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen. J Biol Chem 265:13442–13445
Liu Y, Robyn Van Heeswijck R, Høj P et al (1995) Purification and characterization of ornithine acetyltransferase from Saccharomyces cerevisiae. Eur J Biochem 228:291–296
Lu CD (2006) Pathways and regulation of bacterial arginine metabolism and perspectives for obtaining arginine overproducing strains. Appl Microbiol Biotechnol 70:261–272
Moinard C, Cynober L (2007) Citrulline: a new player in the control of nitrogen homeostasis. J Nutr 137:1621S–1625S
Park SD, Lee JY, Sim SY et al (2007) Characteristics of methionine production by an engineered Corynebacterium glutamicum strain. Metab Eng 9:327–336
Petri K, Walter F, Persicke M et al (2013) A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum. BMC Genomics 14:713
Rimando AM, Perkins-Veazie PM (2005) Determination of citrulline in watermelon rind. J Chromatogr A 1078:196–200
Sakanyan V, Petrosyan P, Lecocq M et al (1996) Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway. Microbiology 142:99–108
Schäfer A, Tauch A, Jäger W et al (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(1):69–73
Tang J, Hao N, Xu S et al (2013) Construction of Corynebacterium glutamicum mutant with knockout of argG gene. J Nanjing Univ Technol (Nat Sci Ed) 35:86–90
Udaka S (1966) Pathway-specific pattern of control of arginine biosynthesis in bacteria. J Bacteriol 91:617–621
Udaka S, Kinoshita S (1958) Studies on l-ornithine fermentation. I. The biosynthetic pathway of l-ornithine in Micrococcus glutamicum. J Gen Appl Microbiol 4:272–282
Xu Y, Labedan B, Glansdorff N (2007) Surprising arginine biosynthesis: a reappraisal of the enzymology and evolution of the pathway in microorganisms. Microbiol Mol Biol Rev 71:36–47
Yim SH, Jung S, Lee SK et al (2011) Purification and characterization of an arginine regulatory protein, ArgR, in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 38:1911–1920
Acknowledgments
This study was supported by the National Basic Research Program of China (973 Program) (No. 2011CBA00807), the National High Technology Research and Development Program of China (863 Program) (No. 2012AA022101), the National Natural Science Foundation of China (No. 31270162), the Natural Science Foundation of Jiangsu Province (No. BK20140932), the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (No. 13KJB530008), and the PAPD Project of Jiangsu Province, a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Hao, N., Mu, J., Hu, N. et al. Improvement of l-citrulline production in Corynebacterium glutamicum by ornithine acetyltransferase. J Ind Microbiol Biotechnol 42, 307–313 (2015). https://doi.org/10.1007/s10295-014-1561-x
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DOI: https://doi.org/10.1007/s10295-014-1561-x