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Enhancement of Ornithine Production in Proline-Supplemented Corynebacterium Glutamicum by Ornithine Cyclodeaminase

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Enhancement of Ornithine Production in Proline-Supplemented Corynebacterium Glutamicum by Ornithine Cyclodeaminase J. Microbiol. Biotechnol. (2010), 20(1), 127–131 doi: 10.4014/jmb.0907.07034 First published online 10 November 2009 Enhancement of Ornithine Production in Proline-Supplemented Corynebacterium glutamicum by Ornithine Cyclodeaminase Lee, Soo Youn1, Jae-Yong Cho2, Hyun Jeong Lee3, Yang-Hoon Kim4*, and Jiho Min1,3,5* 1Department of Bioprocess Engineering, Chonbuk National University, Jeonju 561-756, Korea 2Division of Animal Science and Biotechnology, Sangji University, Wonju-si 220-702, Korea 3Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, Jeonju 561-756, Korea 4School of Life Science, Chungbuk National University, Cheongju 361-763, Korea 5Division of Chemical Engineering, Chonbuk National University, Jeonju 561-756, Korea Received: July 20, 2009 / Revised: August 17, 2009 / Accepted: August 18, 2009 In this study, Corynebacterium glutamicum and its derived to produce certain amino acids results from several mutants were used to demonstrate the relationship between improvements made through repeated random mutations proline, glutamate, and ornithine. The maximum ornithine and selection [9, 10]. production was shown in the culture medium (3,295.0 mg/l) The intermediate metabolite in arginine biosynthesis, when the cells were cultured with 20 mM proline, and was ornithine, is effective in the treatment of liver diseases and 15.5 times higher than in the presence of 1 mM proline. helps strengthen the heart [21]. In bacteria, ornithine is However, glutamate, which is known as an intermediate in synthesized by converting glutamate to ornithine in five the process of converting proline to ornithine, did not have steps through a series of acetylated intermediates [16]. any positive effect on ornithine production. This suggests Subsequently, ornithine is converted to citrulline by that the conversion of proline to ornithine through carbamoyltransferase (argF), and its biosynthesis is controlled glutamate, is not possible in C. glutamicum. Comparative by the repressor (argR) on the arginine operon [5, 17]. In analysis between the wild-type strain, SJC 8043 (argF-, addition, glutamate, the primary metabolite of ornithine and argR-), and SJC 8064 (argF-, argR-, and ocd-), showed arginine biosynthesis, is converted to glutamyl phosphate that C. glutamicum could regulate ornithine production by by γ-glutamyl kinase [4, 22, 23]. ornithine cyclodeaminase (Ocd) under proline-supplemented Several studies have reported an increase in ornithine conditions. Therefore, proline directly caused an increase production by fermentation using auxotrophic mutants, in the endogenous level of ornithine by Ocd, which would which are citrulline- and arginine-required mutants of be a primary metabolite in the ornithine biosynthesis Acinetobacter lwoffi and Brevibacterium ketoglutamicum, pathway. respectively [2, 14]. However, these methods have several Keywords: Corynebacterium glutamicum, ornithine, proline, problems, such as the production of by-products or the ornithine cyclodeaminase induction of undesirable metabolic pathways caused by the overexpression of specific genes or a reversion of the auxotrophic mutants [13, 15]. On the other hand, an ornithine production-controlling reaction such as feeding Corynebacterium glutamicum is a Gram-positive soil of feedback effectors and culture condition control using bacterium that is widely used for the production of a variety supplements is able to simplify and minimize the process of amino acids [9, 20]. The ability of C. glutamicum strains of amino acid production desired [8, 10]. Therefore, this study examined the production of ornithine *Corresponding author J.M. through proline and glutamate supplementation to increase Phone: +82-63-270-2436; Fax: +82-63-270-2306; the primary metabolite in the ornithine biosynthesis pathway E-mail: [email protected] in C. glutamicum. This is the first report of ornithine Y.-H.K. Phone: +82-43-261-3575; Fax: +82-43-264-9600; production via ornithine cyclodeaminase (Ocd) in only E-mail: [email protected] proline-supplemented C. glutamicum. 128 Lee et al. MATERIALS AND METHODS Bacterial Strains and Growth Conditions C. glutamicum ATCC 13032 was used in this study. The deletion mutants of C. glutamicum, SJC 8043 (argF-, argR-) and SJC 8064 (argF-, argR-, ocd-), were kindly provided by Sangji University, Korea [11]. For the ornithine production experiments, a seed culture was prepared by inoculating the LB medium, and growing the inoculated cells to an optical density (OD) of 18.0-20.0, with further growth for 20 h. The cells were inoculated in 100 ml of MMY medium [0.8 g KH2PO4, 10 g (NH4)2SO4, 1 g MgSO4·7H2O, 1.2 g Na2HPO4, 20 mg MnSO4·H2O, 20 mg FeSO4·7H2O, 10 mg ZnSO4·7H2O, 10 g yeast extract, 20 g CaCO3, and 60 g glucose/l] for the main culture. All cultures were grown at 30oC and 150 rpm on a rotary Fig. 1. The time profiles of ornithine, cell growth, and residual glucose concentration of the Corynebacterium glutamicum wild- shaker, and the samples were withdrawn at regular intervals so that type strain. the ornithine and biomass concentrations could be determined. Cell density [▲]; residual glucose concentration [□]; ornithine concentration [●]. Analytical Methods The level of cell growth was determined by measuring the OD at 600 nm. Ornithine analysis was performed according to the method 1 mM arginine, and 1 mM proline. The final cell density of ± reported by Chinard [3]. The reaction mixture contained 3 N HCl C. glutamicum was 24.9 1.7 at 48 h (Fig. 1), and the and 1% ninhydrin in a total volume of 1.0 ml. After incubation for concentration of residual glucose was observed to be between 40 min and 1 h at 100oC, 4 ml of 85% phosphoric acid was approximately 4% (w/v) at the stationary phase. The level added and the mixture was measured at 526 nm. The residual glucose of ornithine production by C. glutamicum in the stationary in the MMY broth was measured using a glucose (GO) assay kit phase (at 24 h) was 290.1 mg/l. In this study, we found that (Sigma-Aldrich, Inc., U.S.A.). the ornithine was produced by the consumption of glucose at high yield in MMY, and it consistent with the effect of Statistical Analysis organic acid production through glucose consumption [12, All the results were obtained from three independent samples from 19]. In addition, the concentrations of consumed glucose triplicate cultures carried out simultaneously for error analysis, and were similar in two other strains used in this study (data are shown along with the standard deviation represented by the error bars. The standard deviations, as well as the correlation between the not shown), but the amounts of ornithine produced by the concentration of proline and ornithine production, were otained two mutant strains were remarkably higher than the wild- using SigmaPlot software (SPSS, Chicago, IL. U.S.A.). The statistical type strain (Table 1). significance was determined using a Student’s t-test for two points. P values <0.05 were considered significant. Ornithine Production Depending on Proline and Glutamate Concentrations It has been reported that proline is converted to glutamate, RESULTS which is the primary metabolite in the biosynthesis pathway of ornithine by pyrroline-5-carboxylate reductase (ProC) Growth Rate and Ornithine Production in C. glutamicum and proline dehydrogenase (PutA) in the metabolic pathway The wild-type strain C. glutamicum ATCC 13032 was of proline [6, 18, 23]. Therefore, an attempt was made to cultivated in a MMY medium containing 6% (w/v) glucose, regulate the concentration of glutamate, known as an Table 1. Ornithine production by C. glutamicum wild type, SJC 8043 (argF-, argR-), and SJC 8064(argF -, argR-, ocd-) during growth in proline-supplemented MMY medium in the presence of 1 mM arginine. Ornithine concentration Strain MMYa MMY+5 mM Prolineb mg/l mg/g (dcw)c mg/l mg/g (dcw)c Relative C. glutamicum wild type 101.1±30.2 76.6±22.9 514.0±65.4 389.4±49.5 5.1 C. glutamicum SJC 8043 130.4±58.7 111.4±50.2 598.3±15.44 511.4±13.2 4.6 C. glutamicum SJC 8064 249.1±107 220.4±37.0 415.9±20.9 368.1±18.5 1.7 aThe cells were grown on MMY medium with 1 mM arginine for 15 h. bThe cells were grown on MMY medium with 1 mM arginine and 5 mM proline for 15 h. cOrnithine concentration was calculated from the free amount of ornithine production in 15 h by a known quantity of cells (dry cell weight). The data represent the mean of three independent cultures±standard deviation. ORNITHINE PRODUCTION IN PROLINE-SUPPLEMENTED C. GLUTAMICUM 129 Fig. 3. Suggested pathway of ornithine biosynthesis by ornithine cyclodeaminase in C. glutamicum, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database search. type and two mutant strains, were used and the level of Fig. 2. Ornithine production in C. glutamicum. ornithine production from supplemented proline 5 mM The cultures were grown on MMY medium in the presence of 0 to 30 mM after 15 h incubation was determined. All the strains tested proline and glutamate, respectively. Black bars, proline supplement; White showed different levels of ornithine production under ± bars, glutamate supplement. The results are reported as the mean SD and normal conditions because of their mutant type. In SJC are statistically significant at *p<0.05 and **p<0.01 (n=3). 8064, the basal level of ornithine was about 2-fold higher than the wild type and the SJC 8043. However under intermediate metabolite from proline to ornithine in MMY proline (5 mM)-supplemented conditions, there was no medium containing 6% glucose (w/v), 1 mM arginine, and significant increase in ornithine production from SJC 8064, 1 mM proline. As shown in Fig. 2, there was no increase considering the basal level of ornithine production. Table 1 in ornithine production by glutamate in C.
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