Cloning and sequence determination of the aspartase-encoding gene from Brevibacterium flavum MJ233

Gene, 158 (1995) 87-90 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

87

GENE 08835

Cloning and sequence determination of the aspartase-encoding gene from Brevibacterium flavum M J233 (Recombinant DNA; aspA; Brevibacterium flavum)

Y o k o Asai, M a s a y u k i l n u i , A l a i n V e r t r s , M i k i K o b a y a s h i a n d H i d e a k i Y u k a w a Tsukuba Research Center,MitsubishiChemicalCorporation, lnashiki, lbaraki 300-03, Japan Receivedby A.M.Campbell: 1 September 1994;Revised/Accepted:21 October/24October 1994;Receivedat publishers:6 February 1995

SUMMARY A 2.5-kb EcoRI fragment containing the aspartase-encoding gene (aspA) of Brevibacteriumflavum MJ233 was cloned into plasmid pUC18 using Southern hybridization with the Escherichia coli aspA gene as a probe. The complete nucleotide (nt) sequence of the cloned DNA indicated that the deduced gene product of the Br.flavum aspA is composed of 526 amino acids (aa). Comparison of the aa sequence to the corresponding sequences from E. coli, Bacillus subtilis and Pseudomonas fluorescens revealed 63, 47 and 57% homology, respectively. The aspA product was determined to have a size of approx. 57 kDa by SDS-PAGE.

INTRODUCTION L-Aspartic acid is used in parenteral nutrition as a food additive. Particularly, it is used for the chemical synthesis of the artificial sweetener aspartame (N-L-~-aspartyl-Lphenylalanine-l-methyl ester) (Homier et al., 1991). We previously reported the use of the coryneform bacterium Brevibacterium fla~')um MJ233 for the industrial production of L-aspartic acid from fumaric acid (Terasawa et al., 1985). Genetic engineering techniques should be used to improw,~ the process. Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1, Aspa) catalyzes the reversfble conversion of fumarate and ammonia to L-aspartate. It has been found in various Correspondence to: Dr. H. Yukawa, Tsukuba Research Center, Mitsubishi Chemical Corporation, Ami, Inashiki 300-03, Japan. Tel. (81-298) 87-1011; Fax (81-298) 87-3259. Abbreviations: aa, amino acid(s); Ap, ampicillin; Aspa, aspartase(s); aspA, gene encoding aspartasels); B., Bacillus;bp, base pair(s); Br., Brevibacterium; E., Escherichia;kb, kilobase(s) or 1000bp; Km, Kanamycin;nt, nucleotide(s);ORF, open reading frame;PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; R, resistance/resistant; SDS, sodium dodecyl sulfate; [], denotes plasmid-carrier state. SSDI 0378-1119(95)00117-4

bacteria, plants and in some animal tissues. Comparison of Aspa should provide useful information on the structure-function relationship. Recently, several studies including chemical modification and site-directed mutagenesis have been undertaken to elucidate functionally essential aa and functional conformation of Aspa (Ida and Tokushige, 1985; Murase et al., 1991; Zhang et al., 1993). However, nt sequence information about the enzymes is still needed. In this paper, we report the cloning and nt sequencing of the aspA gene from Br. flavum which is a coryneform bacteria that has a long history of use in the industrial production of a variety of compounds.

EXPERIMENTALAND DISCUSSION

(a) Cloning of aspA A pool of 2.5-kb EcoRI-digested chromosomal DNA fragments was used to construct a library in plasmid pUC18. 500 clones were subsequently screened by colony hybridization performed under the same conditions as genomic Southern hybridization (Fig. 1). Two positive

88 12345

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Fig. 1. Southern hybridization of the E. coli aspA DNA to genomic DNA of Br. flavum MJ233. Chromosomal DNA from Br. flavum was digested with EcoRI, BamHI, PstI or HindIII, separated on 0.7% agarose gel, and transferred to a nylon membrane (Amersham) by electroblotting. E. coil aspA 1.5-kb fragment amplified by PCR as a probe was purified from agarose gels and radiolabeled with [et-32P] dCTP by random primer labeling (Takara). Hybridization performed overnight at 50°C in a solution containing 5 x SSC (75 mM NaCI/7.5mM Na3'citrate pH 7.6), 5 x Denhardt's solution (0.04% Ficoll/0.04% polyvinylpyrrolidone/0.04% bovine serum albumin)/0.5% SDS/20 mg per ml of denaturated sonicated salmon sperm DNA. Following hybridization, filters were washed twice at 50°C for 20 min in 2 x SSC/0.1% SDS and in 1 x SSC/0.1%SDS. It revealed a single 2.5-kb EcoRI fragment (lane 1). Lanes 1-4, Br.flavum MJ233 chromosomal DNA digested with, respectively, EcoRI, BamHI, PstI and HindIII. Lane 5, ~, HindlII molecular weight markers.

clones were isolated. The plasmids present in these two clones were shown by restriction analysis to contain the same insert. One of these plasmids, named pYA1, was selected for further analysis.

coli. However, the N terminus of the Br. flavum Aspa was approx. 50-aa longer than that of E. coli. The E. coli Aspa has a tetrameric structure (Suzuki et al., 1973) and consists of two pairs of dimers, in which one subunit interacts with the other subunits at at least two or more different sites (Watanabe et al., 1981). The regions surrounding C y s 339 and C y s 4°° may be involved in subunit recognition, and the region surrounding Cys88 may participate in interactions between subunits (Yumoto et al., 1992). These aa (Cys339, Cys4°° and Cys88) of the E. coli enzyme, were also recognised in the Br. flavum Aspa, as approx. 50 aa downstream, C y s 390, Cys 451 and C y s 139, respectively. On the other hand, Cys 14° of E. coli Aspa, which had been described as functionally essential (Ida and Tokushige, 1985), was not recognised either in the surrounding region of B. subtilis, P. fluorescens enzyme or the corresponding region of Br. flavum.

(c) Identification of the aspA product SDS PAGE analysis of the total protein contents of the Br. flavum transformants is shown in Fig. 4. In order to transform to Br. flavum, we constructed plasmid pYA2 based on pCRY40 (legend to Fig. 4) carrying the aspA gene. pYA2 enhanced the production of polypeptide of approx. 57 kDa, above that seen with the vector alone. The molecular mass of the native enzyme of Br. flavum was determined to be about 230 kDa by native PAGE analysis (data not shown). These results indicated that the native enzyme of Br. flavum is also composed of four subunits.

(b) Sequencing of the Br.flavum MJ233C aspA gene The coding region corresponding to the Br. flavum aspA gene is shown in Fig. 2. It includes an ORF compris-

(d) Conclusions (I) The aspA gene from Br. flavum was isolated using the E. coli aspA gene as a probe.

ing 1578 nt (526 aa). A sequence favouring hairpin-loop structures formation was identified downstream from its stop codon (nt 1918-1963) and designated T1 (cf., Fig. 2). T1 showed a high structural similarity to Rho-independent E. coli transcription terminators (d'Aubenton et al., 1990). In B. subtilis, the aspA gene and the gene encoding L-asparaginase are located in the same operon (Sun and Setlow, 1991). However, this organization was not conserved in Br. flavum as no sequence coresponding to a L-asparaginase-encoding gene was found either upstream or downstream. Comparison of the aa sequence with that of the E. coli (Takagi et al., 1985), B. subtilis (Sun and Setlow, 1991) and P. fluorescens (Takagi et al., 1986) Aspa revealed 63, 47 and 57% homology, respectively (Fig. 3). The Br. flavum Aspa appears relatively homologous to that of E.

(2) The ORF consists of 1578 nt. The deduced polypeptide is 526-aa long. (3) Comparison of this Aspa aa sequence with the corresponding sequences from E. coli, B. subtilis and P.fluorescens revealed 63, 47 and 57% homology, respectively. (4) A sequence favouring hairpin-loop formation was identified downstream from its stop codon (nt 1918-1963). This structure showed a high structural similarity to Rho-independent E. coli transcription terminators. (5) The N terminus of the Br. flavum Aspa was approx. 50-aa longer than that of the E. coli, B. subtilis or P. fluorescens aspA gene product. (6) The aspA product was determined to have a molecular mass of about 57 kDa by SDS-PAGE.

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Fig. 2. The nt sequence of the Br.flavum MJ233 aspA gene. The nt sequence of the 2.5-kb EcoRI fragment was determined on both strands by dideoxy chain termination, as described by Sanger et al. (1977) using a 373A DNA sequencer (Applied Biosystems, Foster City, CA, USA). The putative ribosome-binding site is indicated in bold characters. The putative Rho-independent terminator is indicated by arrows below the sequence. The nt sequence presented in this paper has been deposited in GenBank/EMBL under accession No. D25316.

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Fig. 3. Comparison or the deduced aa sequence of the Br.fla~um Aspa with that of E. ¢oli (accession No. X04066), B. sub~ilis (accession No. M63264) and P. fluorescens (accession N o D0010). Identical aa in more than three proteins are shown in shaded areas. Dashes indicate gaps inserted in order to optimize the protein alignments.

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42.4 30.0 20.1 Fig. 4. SDS-PAGE analysis of cell extracts prepared from Br. flavum harboring various plasmids. Cell extracts were analyzed by 4-20% gradient PAGE (Laemmli, 1970) and stained with Coomassie brilliant blue R-250. Lane 1, marker proteins (97.4 kDa, phosphorylase b; 66.2 kDa, bovine serum albumin; 42.4 kDa, ovalbumin; 30.0 kDa, carbonic anhydrase; 20.1 kDa, trypsin inhibitor); lane 2, Br. flavum[pCRY40]; lane 3, Br. flavum[pYA2], pCRY40, Coryneform origin of replication, 8.6-kb Km R. pYA2, Plasmid pCRY40 containing a 2.5-kb EcoRI DNA fragment encoding the aspA gene of Br. flavum MJ233, 11.1 kb, Km R.

ACKNOWLEDGEMENTS

We thank members of the Biochemical Laboratory in the Tsukuba Research Center, Mitsubishi Chemical Co. Ltd., for useful discussions.

REFERENCES d'Aubenton, C.Y., Brody, E. and Thermes, C.: Prediction of rhoindependent Escherichia coli transcription terminators. A statistical analysis of their RNA stem-loop structures. J. Mol. Biol. 216 (1990) 835-853. Homler, B.E., Deis, R.C. and Shazer, W.H.: Aspartame. Alternative Sweeteners, 2nd ed. Marcel Dekker, New York, NY, 1991, pp. 39-69. Ida, N. and Tokushige, M.: Assignment of catalytically essential cysteine residues in aspartase by selective chemical modification with N-(7-dimethylamino-4-methylcoumarynyl) maleimide. J. Biochem. 98 (1985) 793-797. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Murase, S., Takagi, J.S., Higashi, Y., Imaishi, H., Yumoto, N. and Tokushige, M.: Activation of aspartase by site-directed mutagenesis. Biochem. Biophys. Res. Commun. 177 (1991)414-419. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Sun, D. and Setlow, P.: Cloning, nt sequence and expression of the Bacillus subtilis arts operon, which codes for L-asparaginase and L-aspartase. J. Bacteriol. 173 (1991) 3831 3845. Suzuki, S., Yamaguchi, J. and Tokushige, M.: Purification and molecular properties of aspartase from Escherichia coli. Biochim. Biophys. Acta 321 (1973) 369-381. Takagi, J.S., Ida, N., Tokushige, M., Sakamoto, H. and Shimura, Y.: Cloning and nucleotide sequence of the aspartase gene of Escherichia coli W. Nucleic Acids Res. 13 (1985) 2063 2074. Takagi, J.S., Tokushige, M. and Shimura, Y.: Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens. J. Biochem. 100 (1986) 697-705. Terasawa, M., Yukawa, H. and Takayama, Y.: Production of L-aspartic acid from Brevibacterium by the cell re-using process. Process Biochem. 1 (1985) 124-128. Watanabe, Y., Iwakura, M., Tokushige, M. and Eguchi, G.: Subunit arrangement of Escherichia coli aspartase. Biochim. Biophys. Acta 661 (1981) 261-266. Yumoto, N., Murase, S., Imaishi, H. and Tokushige, M.: Determination of the subunit contact region of aspartase. Biochem. Int. 28 (1992) 413 422. Zhang, H.Y., Zhang, J., Lin, L., Du, W.Y. and Lu, J.: Enhancement of the stability and activity of aspartase by random and site-directed mutagenesis. Biochem. Biophys. Res. Commun. 192 (1993) 15-21.