Detection of D-Ornithine Extracellularly Produced by Corynebacterium Glutamicum ATCC 13032::Argf

Biosci. Biotechnol. Biochem., 74 (12), 2507–2510, 2010 Note Detection of D-Ornithine Extracellularly Produced by Corynebacterium glutamicum ATCC 13032::argF

y Daisuke MATSUI and Tadao OIKAWA

Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka 564-8680, Japan

Received July 21, 2010; Accepted September 7, 2010; Online Publication, December 23, 2010 [doi:10.1271/bbb.100523]

We found that Corynebacterium glutamicum ATCC the method of Hwang et al. using Escherichia coli 13032::argF extracellularly produced a large amount of JM110 instead of E. coli DH5 for non-methylation of a D-ornithine when cultivated in a CGXII medium vector transformed into C. glutamicum ATCC 13032.2) containing 1 mML-arginine. This is the first report that C. glutamicum ATCC 13032::argF was pre-cultivated C. glutamicum ATCC 13032 or its mutant produces a on a brain-heart infusion (BHI) agar medium (Difco, D-amino acid extracellularly. C. glutamicum ATCC BD, France), and then a single colony on the medium 13032::argF produced 13 mMD-ornithine in 45 h of was inoculated into 50 ml of a BHI medium in a 500-ml cultivation. Erlenmeyer flask with a baffle and cultivated at 30 C for 24 h with shaking (150 rpm) until the growth of the Key words: Corynebacterium glutamicum ATCC microorganism reached stationary phase. After centrifu- 13032; D-ornithine; ornithine carbamoyl- gation at 5;500 g for 15 min at 4 C, the collected cells transferase; ornithine racemase were washed with an ice-chilled CGXII medium plus 1mML-arginine,7) and inoculated into 50 ml of the same Corynebacterium glutamicum ATCC 13032 is a facul- medium at an A600 of 1.0. After 0, 3, 6, 9, 21, 24, 27, 30, tative anaerobe that is useful in the industrial production 45, 48, 54, 69, and 93 h, 0.5 ml of the culture broth was of various amino acids,1) including L-glutamic acid, taken from the flask and centrifuged at 16;100 g for L-lysine, L-threonine, and L-valine. In addition, C. 15 min at 4 C. The supernatant was diluted with a 0.1 M glutamicum ATCC 13032::argF, whose structural gene sodium acetate buffer (pH 7.2) 1 or 10 times, and encodes the ornithine carbamoyltransferase (argF, delivertized with o-phthalaldehyde after filtration with accession no. AAC24816) when the chromosome is a GL chromatodisc (0.2 mm, GL Science, Tokyo). The disrupted, produces L-ornithine as reported by Hwang sample was subjected to HPLC analysis. The course of et al.2) Hence various researchers have studied the D- and L-ornithine production by C. glutamicum ATCC enzymes in various L-amino acids biosynthetic pathways 13032 was also studied by the method described above. of C. glutamicum ATCC 13032 and their genes related to C. glutamicum ATCC 13032::argF was cultivated the regulation of various forms of L-amino acid produc- under the conditions described above to determine the tion.3–6) However, D-amino acids production of C. course of D- and L-ornithine production by C. glutami- glutamicum ATCC 13032 has not been reported so cum ATCC 13032::argF. After cultivation at 30 C far. During the course of biochemical studies of for 30 h, the cells were collected by centrifugation at C. glutamicum ATCC 13032::argF, we found that it 5;500 g for 15 min at 4 C. A portion of the collected extracellularly produces D-ornithine in addition to cells was suspended in a 10 mM Tris–HCl buffer L-ornithine. Here we describe the D-ornithine production (pH 8.0) plus 10% TritonX-100, and disrupted at 4 C of C. glutamicum ATCC 13032::argF as compared with twice by French pressure cell press FA-078 (SLM that of the wild-type strain. Aminco, Urbana, IL). After centrifugation at 5;500 g D-Ornithine was purchased from Watanabe Chemical for 15 min at 4 C, the supernatant solution obtained was Industries (Hiroshima, Japan). L-Ornithine was from used as a cell-free extract. Separately, a portion of the Wako Chemical (Osaka, Japan). All other chemicals collected cells was suspended in a 10 mM Tris–HCl used were from Kanto Kagaku (Tokyo), Kishida Chemi- buffer (pH 8.0) and disrupted by the French press. After cal (Osaka, Japan), Sigma-Aldrich (St. Louis, MO), or ultracentrifugation at 27;500 g for 30 min at 30 C Tokyo Kasei Kogyo (Tokyo), unless otherwise stated, (P70AT Rotor; Himac CP70MX Preparative Ultracen- and were of the best grade commercially available. trifuge, Hitachi Koki, Tokyo), the insoluble fraction Corynebacterium glutamicum ATCC 13032 was obtained was washed 4 times with deionised water and obtained as an NBRC 12168, from the type-culture used as a peptidoglycan fraction. A cell-free extract and collection of the Japanese National Institute of Tech- a peptidoglycan fraction of C. glutamicum ATCC 13032 nology and Evaluation (Ciba, Japan). C. glutamicum were prepared by the method described above using a ATCC 13032::argF, the ornithine carbamoyltransferase BHI and a CGXII medium without 1 mML-arginine. deletion mutant (accession no. AAC24816) of C. gluta- After 24 h of cultivation, the absorbance at 600 nm of the micum ATCC 13032, was prepared by a modification of culture broth of C. glutamicum ATCC 13032::argF and

y To whom correspondence should be addressed. Tel: +81-6-6368-0812; Fax: +81-6-6388-8609; E-mail: [email protected] Abbreviations: AA, amino acid residue; PLP, pyridoxal 50-phosphate 2508 D. MATSUI and T. OIKAWA

A 300,000 Time 21.307 Inten. 149,142 250,000 L-Orn D-Orn

200,000

uV 150,000 Standard 100,000 Sample A 50,000 Sample B 0

14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 Retention time (min)

B (x10,000) Max Intensity : 41.988 Time 34.272 Scan 2.914 Inten. -8.701 7.5 D-Orn (m/z: 216)

5.0 L-Orn (m/z: 216) MIC

2.5 Standard Sample A Sample B 0.0

31.031.5 32.0 32.5 33.0 33.5 34.0 34.5 Retention time (min)

Fig. 1. Detection and Identification of D-Ornithine in the Culture Broth of Corynebacterium glutamicum ATCC 13032::argF. A, HPLC analysis. Sample A, D- and L-ornithine extracellularly produced by C. glutamicum ATCC 13032::argF after cultivation for 21 h. Sample B, D- and L-ornithine extracellularly produced by C. glutamicum ATCC 13032 after cultivation for 21 h. Standards, 10 mMD- and L-ornithine in deionized water. B, GC-MS analysis. Sample A, D- and L-ornithine extracellularly produced by C. glutamicum ATCC 13032::argF after cultivation for 21 h. Sample B, D- and L-ornithine extracellularly produced by C. glutamicum ATCC 13032 after cultivation for 21 h. Standards, 20 mMD- and L-ornithine in deionized water. of C. glutamicum ATCC 13032 reached 54:20 0:5 and solvent B for 5.5 min. The sample for HPLC analysis was 54:17 0:2 respectively. delivertized with o-phthalaldehyde and N-tert-butyloxy- A peptidoglycan fraction was hydrolyzed with hydro- carbonyl-L-cysteine (BocC) by the method of Buck gen chloride with a Pico-Tag Work Station (Waters, et al.12) A sample (10 ml) was mixed with methanol Tokyo) at 110 C for 20 h following the protocol of the (0.1 ml) containing 7.5 mg of o-phthalaldehyde and manufacturer. The hydrolyzed samples were dried under 7.5 mg of N-tert-butyloxycarbonyl-L-cysteine (BocC), reduced pressure and resuspened in a mixture (95%, and then 5.0 ml of a 0.4 M sodium borate buffer (pH 10.4) v/v) containing 5 mM disodium phosphate in deionized containing 15 ml of Brij 30 was added to the mixture. water and adjusted to pH 7.4 with 1 N NaOH and The growth of the microorganism in the culture broth acetonitrile (5%, v/v). An aliquot of this solution was was measured photometrically at 600 nm with a UV-VIS subjected to GC-MS analysis. photometer V-550 (Jasco, Tokyo). GC-MS analysis was carried out with a gas chroma- We found that C. glutamicum ATCC 13032::argF tography GC-2010 system equipped with a Model extracellularly produced large amount of an unknown QP2010 mass spectrometer (GC-MS, Shimadzu, Kyoto, amino acid when cultivated in a CGXII medium Japan). A fused silica capillary column, Chirasil-L-Val containing 1 mML-arginine. The unknown amino acid (Varian, Darmstadt, Germany) was used to separate and produced was identified by GC-MS analysis and HPLC quantify D- and L-amino acids by a method previously analysis. It was eluted at a retention time of 15.85 min reported.8–10) on HPLC analysis, and this retention time coincided HPLC analysis was carried out with a high-performance well with that of an authentic D-ornithine (Fig. 1A). liquid chromatography LC-10AD system (Shimadzu). GC-MS analysis indicated that the unknown amino acid A Hypersil ODS column (particle size, 5 mm, 4 250 was eluted at a retention time of 32.25 min, and it mm; Agilent, Santa Clara, CA) was used to separate showed an m=z value of 216 (Fig. 1B). This m=z value D- and L-ornithine method of Erbe et al.11) The column agreed well with the theoretical m=z value of D-ornithine temperature was kept at 40 C, and the adsorbed sample (216). Accordingly, the unknown amino acid produced was eluted with a linear gradient of a 0.1 M acetate buffer, by C. glutamicum ATCC 13032::argF was identified as pH 7.2 (solvent A) and methanol (solvent B). The D-ornithine. This is the first report that C. glutamicum column was equilibrated with solvent A containing ATCC 13032 or its mutant produces a D-amino acid 62% of solvent B at a flow rate of 0.8 ml min1, and then extracellularly. Since Hwang et al. measured the amino the concentration of solvent B was increased to 70% acids extracellularly produced by C. glutamicum ATCC from 0 to 13 min. After elution of D- and L-ornithine, the 13032::argF spectrophotometrically using a nynhydrin concentration of solvent B was increased to 85% from 13 reaction, they probably did not notice D-ornithine to 17 min. Before analysis of the next sample, the column production by this organism.2) Our finding also suggests was re-equilibrated with solvent A containing 62% of that the D-ornithine intracellularly produced can be D-Ornithine Extracellularly Produced by C. glutamicum 2509 AB128.0 30.0 128.0 0.5

64.0 25.0 64.0 0.4 )

32.0 32.0 M

20.0 ) M 16.0 16.0 0.3 600 15.0 600 A A A A 8.0 8.0 0.2 10.0 4.0 4.0 Ornithine conc. (m Ornithine 0.1 5.0 2.0 conc. (m Ornithine 2.0

1.0 0.0 1.0 0.0 0 24 48 72 96 0 24 48 72 96 Cultivation time (h) Cultivation time (h)

Fig. 2. Course of D- and L-Ornithine Production by Corynebacterium glutamicum ATCC 13032::argF and Corynebacterium glutamicum ATCC 13032. , Absorbance at 600 nm; , L-Ornithine concentration; , D-Ornithine concentration (n ¼ 3). A, Corynebacterium glutamicum ATCC 13032::argF. B, Corynebacterium glutamicum ATCC 13032. exported to a medium by C. glutamicum ATCC 13032 Bacillus licheniformis and B. subtilis.14–16) D-Ornithine cells. produced in C. sticklandii DSM 519 was catalyzed by After 45 h of cultivation of C. glutamicum ATCC an ornithine racemase.13) In the case of B. cereus ATCC 13032::argF, the D-ornithine concentration reached 10876, the putative ornithine racemase gene was found 13 mM, and L-ornithine, 21 mM (Fig. 2A). In contrast, in the genomic DNA of B. cereus ATCC 10876 C. glutamicum ATCC 13032 produced only 0.07 mM (accession no. ZP 04321077), but the function of its D-ornithine and 0.17 mML-ornithine at the same culti- gene product remains unknown. The transamination vation time (Fig. 2B). activity of D-ornithine was also detected in B. lichen- L-Proline and L-glutamic acid are known as two of the iformis,15) but no enzyme related to D-ornithine catab- precursors in the L-ornithine biosynthetic pathway of olism or anabolism in C. glutamicum ATCC 13032 has C. glutamicum ATCC 13032. We examined the effects been reported. Two amino acid racemase genes were of L-proline and L-glutamic acid addition to the medium found in the genomic DNA of C. glutamicum ATCC on the production of D-ornithine by C. glutamicum 13032: the alanine racemase gene (alr, accession no. ATCC 13032::argF and C. glutamicum ATCC 13032. YP 224879), and the glutamate racemase gene (murI, As compared with L-ornithine production at 24 h of culti- accession no. YP 226751).17,18) As we have reported, vation in a CGXII medium containing 1 mML-arginine these amino acid racemases of C. glutamicum ATCC (15.1 mM), L-ornithine production increased to 17.3 and 13032 showed high substrate specificity, and probably to 17.8 mM when 5 mML-proline and 5 mML-glutamic do not act on ornithine. D-Amino acid dehydrogenase acid was added to a CGXII medium containing 1 mM (dadA, accession no. YP 227262) and D-amino acid L-arginine respectively. But in the case of D-ornithine, oxidase (thiO, accession no. YP 226279) genes also only the addition of 5 mML-glutamic acid to a CGXII exist in the genomic DNA of C. glutamicum ATCC medium containing 1 mML-arginine increased D- 13032, but the functions of their gene products are still ornithine production, from 14.3 to 17.3 mM, and the unknown. addition of 5 mML-proline to a CGXII medium contain- We detected almost the same amount of D-ornithine ing 1 mML-arginine decreased D-ornithine production, (about 0.2 mM) in the cell-free extract of both C. glu- from 14.3 to 9.2 mM. The addition of D-orL-ornithine to tamicum ATCC 13032::argF and C. glutamicum ATCC the medium had no effect on D-ornithine production by 13032, but only C. glutamicum ATCC 13032::argF C. glutamicum ATCC 13032::argF or C. glutamicum extracellularly produced D-ornithine (13 mM after 45 h ATCC 13032. The cell-free extract of C. glutamicum cultivation). This suggests that disruption of the orni- ATCC 13032::argF and of C. glutamicum ATCC 13032 thine carbamoyltransferase gene (argF) increases the also contained D-ornithine at concentrations, of 0:20 L-ornithine production of C. glutamicum ATCC 13032. 0:04 or 0:19 0:05 mM respectively. The genomic DNA of C. glutamicum ATCC 13032 GC-MS analysis of D-amino acids in the peptidogly- encodes one protein partially similar to the ornithine can fraction indicated that peptidoglycan fractions, of racemase from C. sticklandii DSM 519 (accession no. both C. glutamicum ATCC 13032::argF and C. gluta- CAQ42981) as found by a Psi- or Phi-BLAST (http: micum ATCC 13032 contained only D-alanine (retention //blast.ncbi.nlm.nih.gov/Blast.cgi) as an accession num- time, 8.25 min; m=z, 190) and D-glutamic acid (retention ber of BA000036. This gene product is a candidate for time, 31.75 min; m=z, 202), but not D-ornithine or other the enzyme that catalyzes the conversion of L-ornithine D-amino acids. to D-ornithine in C. glutamicum ATCC 13032. Using D-Ornithine has been reported previously to be a C. glutamicum ATCC 13032::argF, it is possible to biochemically important molecule in several micro- produce D-ornithine easily at high concentration. organisms. It was detected as the intermediate of the The peptidoglycan of animal and human pathogenic L-ornithine catabolic pathway in Clostridium sticklandii corynebacteria such as Corynebacterium diphteriae and DSM 519.13) It was also detected as an essential C. glutamicum ATCC 13032 contains a directly cross- component of a bacteriocin and the cell walls of linked meso-diaminopimelic acid (variation A1)asan 2510 D. MATSUI and T. OIKAWA essential component, but in the phytopathogenic cor- 3) de Graaf AA, Eggeling L, and Sahm H, Adv. Biochem. Eng. ynebacteria, such as Corynebacterium poinsettiae NCPP Biotechnol., 73, 9–29 (2001). 177, Corynebacterium flaccumfaciens ATCC 6887, and 4) Leyval D, Uy D, Delaunay S, Georgen JL, and Engasser JM, J. Biotechnol., 104, 241–252 (2003). Corynebacterium betae ATCC 13437, the peptidoglycan 5) Peters-Wendisch P, Stilz M, Etterich H, Kennerknecht N, Sahm contains D-ornithine instead of meso-diamino pimelic H, and Eggeling L, Appl. Environ. Microbiol., 71, 7139–7144 19) acid (variation B2). We measured the D-amino acids (2005). contents in a peptidoglycan fraction of C. glutamicum 6) Ru¨hler C, Pu¨hler A, and Kalinowski J, J. Biotechnol., 104, 213– ATCC 13032::argF and C. glutamicum ATCC 13032, 228 (2003). J. Bacteriol. 175 but did not detect D-ornithine in either fraction. 7) Keilhauer C, Eggeling L, and Sahm H, , , 5595– 5603 (1993). Accordingly, D-ornithine is not an essential component 8) Ali H, Pa¨tzold R, and Bru¨ckner H, Food Chem., 99, 803–812 of the peptidoglycan in C. glutamicum ATCC 13032, (2006). but may function as an intermediate or a precursor of 9) Pa¨tzold R and Bru¨ckner H, Amino Acids, 31, 63–71 (2006). a biochemically important compound in C. glutamicum 10) Pa¨tzold R and Bru¨ckner H, Eur. Food Res. Technol., 223, 347– ATCC 13032. 354 (2006). We are currently studying the D-ornithine production 11) Erbe T and Bru¨ckner H, J. Chromatogr. A, 881, 81–91 (2000). system of C. glutamicum ATCC 13032 with various 12) Buck RH and Krummen K, J. Chromatogr. A, 315, 279–285 biochemical systems. (1984). 13) Fonknechten N, Perret A, Perchat N, Tricot S, Lechaplais C, Vallenet D, Vergne C, Zaparucha A, Paslier DL, Weissenbach J, Acknowledgment and Salanoubat M, J. Bacteriol., 191, 3162–3167 (2009). 14) Kuromitsu HK and Snoke JE, Biochim. Biophys. Acta, 62, 114– This work was supported in part by a Research Grant 121 (1962). from the Strategic Project to Support the Formation of 15) Bernlohr RW and Novelli GD, Arch. Biochem. Biophys., 103, Research Bases at Private Universities in Japan. 94–104 (1963). 16) Duc-Nguyen H and Weed LL, J. Biol. Chem., 239, 3372–3376 (1964). References 17) Oikawa T, Tauch A, Schaffer S, and Fujioka T, J. Biotechnol., 125, 503–512 (2006). 1) Hermann T, J. Biotechnol., 104, 155–172 (2003). 18) Kouguchi S, Krumbach K, Eggeling L, Sahm H, and Oikawa T, 2) Hwang J-H, Hwang G-H, and Cho J-Y, J. Microbiol. Bio- JBS, 75, 1080 (2003). technol., 18, 704–710 (2008). 19) Elizabeth W, Int. J. Syst. Bacteriol., 20, 425–433 (1970).