10.1128/AEM.68.1.93-101.2002.

2002, 68(1):93. DOI:Appl. Environ. Microbiol. SteinbüchelTran Hai, Fred Bernd Oppermann-Sanio and Alexander Polyamides

RelatedVitro Synthesis of Cyanophycin and sp. Strain MA19 and InSynechococcus

from the Thermophilic Cyanobacterium Thermostable Cyanophycin Synthetase Molecular Characterization of a

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/02/$04.00�0 DOI: 10.1128/AEM.68.1.93–101.2002

Jan. 2002, p. 93–101 Vol. 68, No. 1

Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of a Thermostable CyanophycinSynthetase from the Thermophilic Cyanobacterium

Synechococcus sp. Strain MA19 and In Vitro Synthesis ofCyanophycin and Related Polyamides

Tran Hai, Fred Bernd Oppermann-Sanio, and Alexander Steinbuchel*Institut fur Mikrobiologie, Westfalische Wilhelms-Universitat, D-48149 Munster, Germany

Received 29 June 2001/Accepted 26 September 2001

The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyano-phycin synthetase (cphA) and cyanophycinase (cphB), which were identified, cloned, and sequenced in thisstudy. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteinsof other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coliharboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the drycell matter in the presence of isopropyl-�-D-thiogalactopyranoside (IPTG). The cyanophycin synthetase wasenriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyano-phycin synthetase maintained the parental thermophilic character and was active even after prolongedincubation at 50°C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h ofincubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, L-arginine andL-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycincontaining less arginine, �-arginyl aspartic acid dipeptide, and poly-�,�-DL-aspartic acid as primers and alsoincorporated �-hydroxyaspartic acid instead of L-aspartic acid or L-canavanine instead of L-arginine at asignificant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, thethermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production ofcyanophycin make it an interesting candidate for biotechnological processes.

Cyanophycin is a reserve material for nitrogen and energyfound only in cyanobacteria and was first observed by Borzi in1887 (5). Accumulation of cyanophycin normally occurs whenthe cells enter the stationary growth phase (2, 17, 19, 28), incells grown under stress conditions (14, 15), or in some specificcell forms, such as akinetes and heterocysts (10). Synthesis ofcyanophycin occurs via a nonribosomal pathway catalyzed bycyanophycin synthetase and can be stimulated by adding anti-biotics, such as chloramphenicol, to growing cultures (29) or byreducing the photon flow and cultivation temperature (38).Cyanophycin granules are not the primary nitrogen source forde novo synthesis of protein. However, they are mobilizedduring akinete germination (35). Cyanophycin may also serveas an energy source. Under strictly anaerobic conditions ATPis formed during ornithine formation from arginine (33). Cya-nophycin contents depend on the species and range from 0.1 to16% (wt/wt) of the dry cell matter. Cyanophycin is a branchedpolypeptide that normally consists of L-aspartic acid and L-arginine at a molar ratio of approximately 1:1 and is thereforealso referred to as multi-L-arginine-poly-L-aspartic acid (30, 32,34). The polymer is of biotechnological interest since if thearginine content is reduced (11), the oligo-arginyl polyaspartic

acid obtained may be used as a biodegradable substitute invarious technical products and processes (27).

Cyanophycin synthetases have been enriched 94-fold fromAnabaena cylindrica (31) and 144-fold from the thermostablecyanobacterium Synechococcus sp. strain MA19 (8), and a cya-nophycin synthetase was also purified to electrophoretic ho-mogeneity from Anabaena variabilis (39). In Synechocystis sp.strain PCC6803 the enzyme is encoded by cphA (39), which isrepresented by slr2002 in the genomic library of this strain (12).The cphA genes from A. variabilis, Synechocystis sp. strainPCC6803, and Synechocystis sp. strain PCC6308 have beencloned and expressed in Escherichia coli (1, 22, 39). Previously,cphA genes have been described for six different strains ofcyanobacteria. These strains are members of species of thegenera Anabaena, Synechococcus, Synechocystis, and Cyano-thece (1, 3, 22, 39; GenBank accession no. AAF97933). Aminoacid sequence analysis revealed high levels of similarity to theATP-dependent carboxylate amine/thiol ligases belonging tothe superfamily of substrate ligases involved in biosynthesis ofbacterial peptidoglycan (murein ligases) and to the capB trans-lation product responsible for poly-�-D-glutamate synthetasein Bacillus anthracis (7, 18, 36). The enzyme cyanophycin syn-thetase contains two ATP and magnesium binding sites andfour other conserved regions (1, 3).

cphB, which encodes the intracellular cyanophycinase, wascloned from Synechocystis sp. strain PCC6803 and expressed inE. coli (23). This serine type of endopeptidase was capable ofhydrolyzing cyanophycin in vitro into arginyl aspartic acid

* Corresponding author. Mailing address: Institut fur Mikrobiologieder Westfalischen Wilhelms-Universitat, Corrensstrasse 3, D-48149Munster, Germany. Phone: 49 251 8339821. Fax: 49 251 8338388.E-mail: steinbu@uni-muenster.de.

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dipeptides (23). cphB and cphA are in the same cluster and areoriented colinearly but do not seem to be cotranscribed (39).

In this paper we describe cloning of cphA and cphB from thethermophilic organism Synechococcus sp. strain MA19. Bio-chemical characterization of the thermostable cyanophycinsynthetase provided information which could be useful for invitro synthesis of cyanophycin on a technical scale.

MATERIALS AND METHODS

Strains, plasmids, and culture conditions. An axenic culture of Synechococcussp. strain MA19 (20), which was also used in a previous study (8), was a gift fromthe culture collection of the Molecular Bioenergetics Laboratory of the NationalInstitute of Bioscience and Human Technology (Tsukuba, Ibaraki, Japan). It wasgrown photoautotrophically at 50°C in liquid BG11 medium (24) with irradiation(100 microeinsteins s�1 m�2) and shaking (100 rpm). Growth of the cyanobac-terium was started with a short chromatic adaptation period (10 h of irradiationat 50 microeinsteins s�1 m�2). The E. coli strains were grown at 37°C in Luria-Bertani medium (26) with shaking (150 rpm). For cultivation of plasmid-carryingE. coli, 75 �g of ampicillin per ml was added. The bacterial strains, plasmids, andDNA fragments used in this study are listed in Table 1. Fresh competent cells ofE. coli were prepared by using the CaCl2 standard method (26).

Isolation of cyanophycin. Cyanophycin was isolated from recombinant cells ofE. coli harboring the cphA gene of Synechococcus sp. MA19 by taking advantageof its solubility properties and using the method described by Simon (31), withtwo modifications (0.4% [vol/vol] Triton X-100 was added to the extractionbuffer, and the sonicated cells were treated with one cycle of heating and cooling[heating at 95°C for 5 min followed by cooling at 0°C for 5 min]). The purity ofthe cyanophycin isolated and its amino acid composition were analyzed bysodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and by re-versed-phase high-performance liquid chromatography (RP18 column; Tech-sphere ODS-2; Kontron Instruments GmbH, Neufahrn, Germany) after precol-umn derivatization with phthaldialdehyde reagent (Merck, Hohenbrunn,Germany) as described recently (8).

Electrophoretic methods. For SDS-polyacrylamide gel electrophoresis, 11.5%(wt/vol) polyacrylamide gels were prepared as described by Laemmli (13). Mo-lecular weight marker proteins were purchased from Bio-Rad. Proteins andcyanophycin were stained with Serva Blue R. For protein determinations we usedthe method of Bradford (6). For analysis of nucleic acids, electrophoresis in 0.8and 1.1% (wt/vol) agarose gels was performed by standard methods (26).

DNA manipulation and construction of a partial genomic library. Totalgenomic DNA of Synechococcus sp. strain MA19 was extracted by a modifiedmethod described recently (9). Protein impurities were removed by using a largeamount of proteinase K (1 mg ml�1). Plasmid DNA was isolated from E. coli bythe alkaline extraction method (4). Purified DNA was digested with restrictionenzymes according to the instructions of the manufacturer (Gibco, BRL). South-ern hybridization was carried out by standard methods. A 1.87-kbp DNA frag-ment harboring part of cphA from Nostoc ellipsosporum NE1 was labeled byusing a DIG-High Prime kit (Boehringer, Mannheim, Germany). HindIII DNArestriction fragments that were 5 to 7 kbp long were cut from a 0.8% (wt/vol)

agarose gel and purified by using a NucleoTrap kit (Machery and Nagel, Duren,Germany). About 400 ng of the fragments was ligated to 400 ng ofHindIII-digested pBluescript SK(�) DNA with T4 DNA ligase at 14°C over-night. The ligation products were transferred into E. coli XL1 Blue by usingstandard methods (26).

Screening of genomic libraries. Genomic libraries were screened by colonyhybridization at 53°C by using standard procedures (26). To visualize the che-moluminescent substrate chloro-5-substituted adamantyl 1,2-dioxetane phos-phate (CSPD), a kit (Boehringer) was used. Transformants that gave the stron-gest signals were analyzed further. One positive clone, which carried a 6.5-kbpinserted fragment, was chosen for sequencing.

Inverse PCR to obtain the 3� region of cphA. Since cphA of Synechococcus sp.strain MA19 contained a HindIII restriction site in the 3� region, an inverse PCRwas performed as follows. Genomic DNA was digested with KpnI. About 150 ngof the purified KpnI restriction fragments was religated with T4 DNA ligaseincubation for 6 h at 14°C. The circular DNA molecules formed were used for aninverse PCR performed with primer P1 (5�-AGAAAACAGCTCGGCTACACACTTT-3�) and primer P2 (5�-TTTAAGACGAAACACAAGCGATTAA-3�),whose sequences were deduced from the 3�-terminal region of the sequencedregion of cphA from the positive clone with the 6.5-kbp HindIII fragment in-serted. The primers were purchased from MWG-Biotech AG (Ebersberg, Ger-many). The PCR were performed by using Vent DNA polymerase (New EnglandBiolabs, Schwalbach/Taunus, Germany) according to the instructions in the PCRapplications manual (Biochemica 1995; Boehringer), and the 1.7-kbp productwas purified with a Nucleotrap kit by following the instructions of the manufac-turer (Machery and Nagel).

DNA sequencing and sequence analysis. DNA sequences were determined byusing a model 4000L DNA sequencer (LI-COR Inc., Biotechnology Division,Lincoln, Nebr.) and a Thermo Sequenase fluorescence-labeled primer cyclesequencing kit (Amersham Life Science, Buckinghamshire, United Kingdom)according to the instructions of the manufacturers. The nucleic acid sequencesobtained from both strands were analyzed with a computer program from Hei-delberg Unix Sequence Analysis Resources (HUSAR, release 4.0.; based on theWisconsin Genetics Computer Group program package, version Unix-8.1, 1995).Sequences were compared and aligned by using the network service programsBLAST provided by the National Center for Biotechnology Information andClustalW provided by the European Bioinformatics Institute at EMBL. Thealignments used for construction of a phylogenetic tree were obtained by usingthe Genedoc and TreeView service programs (25) (http://www.psc.edu/biomed/genedoc or http://taxonomy.zoology.gla.ac.uk).

Cloning of cphA from Synechococcus sp. strain MA19. The cphA gene wasamplified from the genomic DNA by PCR as described above. The followingprimers were used for this reaction and were deduced from nucleotide sequencesupstream and downstream of cphA (except for the nucleotides in italics, whichwere inserted to generate an NdeI restriction site plus a histidine-encoding tripletand a BamHI restriction site, respectively): primers cphAMAsense (5�-GTGTTTCCCATATGCATCACCATCACCATCACATGACACTGATTTGACCGCTA-3�) and cphAMAanti (5�-AAATTCCAGTGAGGATCCAGTCC-3�). For PCR weused a temperature program consisting of one denaturation cycle (95°C for 2min) and 32 amplification cycles (95°C for 30 s, 56°C for 30 s, and 72°C for 2min). For transformation, about 400 ng of the purified PCR product was ligated

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Genotype and/or phenotype Source

Synechococcus sp. strain MA 19 Thermophilic, optimal growth at 50°C NIBHTa

Escherichia coli strainsTOP10 recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 �lacU169 (�80 lacZ�M15) Invitrogen (San Diego,

Calif.)XL1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F� proAB lac1qZ�M15Tn10(Tetr)] Stratagene

PlasmidspBluescript SK(�) Ampr lac POZ�, T7 and T3 promotor StratagenepSK::H6.5 6.5-kbp HindIII fragment from Synechococcus sp. strain MA19 genomic DNA harboring

complete cphB and 5� part of cphA inserted in pBluescript SK(�)This study

pSK::Kp1.7 1.7-kbp inverse PCR product from Synechococcus sp. strain MA19 genomic DNA con-taining 3� region of cphA inserted in pBluescript SK(�)

This study

pSK::cphAMA19co 2.98-kbp PCR product from Synechococcus sp. strain MA19 genomic DNA carryingcphA inserted in pBluescript SK(�) colinear with respect to lacPO

This study

pSK::cphAMA19anti 2.98-kbp PCR product from Synechococcus sp. strain MA19 genomic DNA carryingcphA inserted in pBluescript SK(�) antilinear with respect to lacPO

This study

a NIBHT, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki, Japan.

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to 400 ng of EcoRV-linearized pBluescript SK(�) DNA. Transformation wasperformed with competent cells of E. coli TOP10 obtained by the CaCl2 method(26). Recombinant plasmids harboring cphA with a colinear orientation(pSK::cphAMA19co) and an antilinear orientation (pSK::cphAMA19anti) with re-spect to lacPO were obtained.

Purification of cyanophycin synthetase. Cells of E. coli TOP10 harboringpSK�::cphAMA19co were cultivated as described above until the optical density at578 nm of the cell suspension was 1.0 to 1.1. Then, 1.0 mM isopropyl-�-D-thiogalactopyranoside (IPTG) was added to the culture. After the stationaryphase was reached, the cells were spun down (4,000 g, 10 min, 4°C) and werewashed once with extraction buffer containing 50 mM Tris-HCl (pH 8.2), 20 mMKCl, 1 mM MgCl2, 1 mM EDTA, and 5 mM 2-mercaptoethanol. The washedcells (approximately 30 g [wet weight]) were dissolved in 100 ml of the samebuffer and were disintegrated by using a Sonoplus GM200 Sonifier (BandelinElectronic, Berlin, Germany). The cell debris was removed by centrifugation(30,000 g, 4°C, 15 min). The soluble protein fraction was obtained as super-natant after a second centrifugation (100,000 g, 4°C, 1 h).

Enzyme purification was carried out by a three-step procedure, which wasdescribed recently (8). All steps were carried out at 4 to 7°C. A buffer composedof 50 mM Tris-HCl (pH 8.2), 20 mM KCl, 5 mM �-mercaptoethanol, and 1 mMEDTA was used throughout the purification procedure. The soluble proteinfraction was dialyzed against this buffer and was applied to a Q Sepharose Hiloadcolumn (2.6 by 10 cm; bed volume, 53 ml; Amersham Pharmacia Biotech,Freiburg, Germany) equilibrated with the buffer. After the protein was washedwith 100 ml of buffer, it was eluted with a KCl gradient (0 to 600 mM in 750 ml)at a flow rate of 2.5 ml min�1. Fractions containing high enzyme activity werecombined, desalted, and concentrated by ultrafiltration with a YM 30 membrane(Amicon Corp., Lexington, Ky.). The concentrated preparation was applied to atriazine dye Procion Blue HE-RD Sepharose CL-4B column. The combinedfractions exhibiting high activity were concentrated and applied to a Superdex200 HR column (2.6 by 60 cm; bed volume, 330 ml; Amersham PharmaciaBiotech) equilibrated with buffer which contained 150 mM KCl. Proteins wereeluted at a constant flow rate of 1.0 ml min�1. The most active fractions weredesalted and concentrated to a concentration of 0.2 mg of protein ml�1.

Cyanophycin synthetase assay. For the cyanophycin synthetase assay we useda previously described protocol (31) modified as described recently (1). Tosimplify the calculations and minimize the radioactive waste, a reaction mixturecontaining 50 mM Tris (pH 8.2), 20 mM MgCl2, 20 mM KCl, 10 mM dithio-threitol, 3 mM ATP, 5 mM L-aspartic acid, 0.495 mM L-arginine, 5 �M radio-labeled L-[14C]arginine (Amersham Pharmacia Biotech), and enzyme solution (4to 50 �g of protein) was used. The reaction mixture was overlaid with 30 �l ofwax to prevent evaporation. The enzyme reaction was carried out at 30°C for 15min and was stopped by diluting the mixture with 10 volumes of ice-cold water.The labeled products were collected by centrifugation (14,000 g, room tem-perature, 20 min). The final steps were performed by using the method describedpreviously (1). Scintillation counting was carried out with a model LS 6500scintillation counter (Beckman Instruments GmbH, Munich, Germany).

Primers and chemicals used for in vitro synthesis of cyanophycin. The primersand chemicals used for in vitro synthesis of cyanophycin were purchased fromSigma (-arginyl aspartic acid dipeptide, lots 197FO334 and 125HO515); Cal-biochem (�-hydroxyaspartic acid, lot 511235); Acros Organics (N-acetylglu-cosamine, 2-acetamido-2-deoxy-alpha-D-glucopyranose, lot A009910201); Bayer,Leverkusen, Germany (chemosynthetic poly-,�-DL-aspartic acid LPOC618);and Biotop Gesellschaft fur biotechnische Optimierung mbH, Witten, Germany[ectoine, (S)-2-methyl-1,4,5,6-tetrahydroxypyrimidine-4-carboxylic acid (purity,�97%)]. Aspartic acid-rich biopolymer was isolated from capsules of a Klebsiellasp. isolate which was capable of forming slimy colonies on agar mineral medium,by precipitation in 80% (vol/vol) ethanol. Cyanophycin-free cell membranes ofSynechococcus sp. strain PCC7942, which probably contains no cyanophycinsynthetase (39), were obtained by triplicate extraction of a cell debris fractionwith 0.1 N HCl. Since this strain contained no cyanophycin (data not shown), thepreparation obtained was considered a cyanophycin-free cyanobacterial mem-brane particle preparation. Modified cyanophycin was obtained by hydrolyzing10 mg of cyanophycin in 30 mM acetic acid at 105°C for 4 h. In this way argininewas partially removed from the polymer chain (30). Cyanophycin with a reducedarginine content was purified by titration with NaOH and was washed with colddistilled water. The modified cyanophycin consisted of aspartic acid and arginineat a molar ratio of 1:0.6, which was determined by amino acid analysis byhigh-performance liquid chromatography with precolumn derivatization as de-scribed recently (8).

Nucleotide sequence accession number. The nucleotide and amino acid se-quence data determined in this study have been deposited in the GenBankdatabase under accession no. AF329282.

RESULTS

Cloning of cphA and cphB. In order to identify and clone thecyanophycin biosynthesis genes from the thermophilic organ-ism Synechococcus sp. strain MA19, we used a strategy forconstruction of a partial genomic library based on Southernhybridization with HindIII-digested MA19 DNA and a labeledDNA probe harboring cphA of N. ellipsosporum NE1 (seeMaterials and Methods) (unpublished data). Fragments rang-ing from 5 to 7 kbp long from HindIII-digested genomic DNAwere ligated to HindIII-linearized pBluescript SK(�) DNAand transformed into E. coli XL1 Blue. Approximately 1,000transformants were selected and used for colony hybridizationwith the heterologous DNA probe, and 24 of these transfor-mants produced signals which revealed that they harbored a6.5-kbp DNA fragment insertion.

DNA sequencing of the 6.5-kbp HindIII insert revealed a4.193-kbp region harboring two colinear open reading frames,ORF1 and ORF2. ORF1 consisted of 834 bp, and its transla-tion product consisted of 277 amino acids. As the translationproduct exhibited levels of similarity of 86, 69, 63, and 57% tothe cphB translation products of A. variabilis (39), Synechocystissp. strain PCC6308 (1), Synechocystis sp. strain PCC6803 (22,39), and Synechococcus elongatus (3), respectively, ORF1 wasidentified as cphB encoding a cyanophycinase. In ORF2, whichwas located 174 bp downstream from and colinear with cphB,the 3� region was not present on the 6.5-kbp HindIII fragment.In order to clone this missing region, inverse PCR (37) wasemployed. Total genomic DNA was completely digested withKpnI, for which a corresponding restriction site was localizedin the previously sequenced region of ORF2, and ligated toobtain circular DNA molecules, which were then employed astemplates for PCR performed with primers P1 and P2 (seeMaterials and Methods). The 1.7-kbp PCR product includedthe 3� region of ORF2. ORF2 was then completely sequenced.ORF2 consisted of 2.712 kbp, which encoded 903 deducedamino acids. Alignment of the CphA proteins from A. variabilisATCC 29413 (� strain FD), S. elongatus, Synechocystis sp.strain PCC6803, Cyanothece sp. strain ATCC 51142, and Syn-echocystis sp. strain PCC6308 and the ORF2 translation prod-uct revealed high levels of similarity, especially in the amino-terminal regions (Fig. 1). The amino acid sequence deducedfrom cphA (903 amino acids) also exhibited striking levels ofsimilarity in almost complete overlaps with the cyanophycinsynthetases encoded by the cphA genes of A. variabilis ATCC29413 (� strain FD), S. elongatus, Synechocystis sp. strainPCC6803, Cyanothece sp. strain ATCC 51142, and Synechocys-tis sp. strain PCC6308 (85, 67, 67, 68, and 64%, respectively). Inaddition, these sequences exhibited high levels of similaritywith the ATP-grasp fold-containing glutathione synthetase andthe murein and folyl poly-�-D-glutamate ligase superfamilies ofE. coli (7, 36). Region V in the carboxy-terminal amino acidsequences of CphA contained characteristic aspartic acid-richstretches (Fig. 1).

Heterologous expression of cphA and characterization of theenzyme products. In order to determine whether Synechococ-cus sp. strain MA19 cphA is functionally active, expression ofthis gene in E. coli was examined. To do this, the cphA-carryingregion was amplified from genomic DNA by using primerscphAMAsense and cphAMAanti. The 2.98-kbp PCR product, con-

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FIG. 1. Similarities among amino acid sequences deduced from the sequences of six cphA genes. The sequences of A. variabilis ATCC 29173(GenBank accession no. AAF97933) (line i), Synechocystis sp. strain PCC6803 (12) (line ii), Synechocystis sp. strain PCC6308 (1) (line iii),Cyanothece sp. strain ATCC 51142 (GenBank accession no. AAF97933) (line iv), S. elongatus (3) (line v), and Synechococcus sp. strain MA19 (thisstudy) (line vi) were compared. The consensus sequence is shown on line vii. The amino acids are indicated by standard one-letter abbreviations.Amino acid residues which are identical to the residues in the gene product of cphAMA19 are highlighted. The B-loop and J-loop regionscorresponding to the ATP- and Mg2�-binding sites of cphA (3) and five other important regions are indicated by arrows and/or are underlined.

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taining cphA plus the intergenic region between cphB andcphA, was ligated into EcoRV-restricted pBluescript SK(�).Recombinant strains of E. coli harboring cphA either in acolinear orientation (pSK::cphAMA19co) or in an antilinear ori-entation (pSK::cphAMA19anti) with respect to lacPO were ana-lyzed for heterologous expression. Strong expression of cphAoccurred in cells harboring pSK::cphAMA19co in the presenceof IPTG. No activity was detected when the gene was tran-scribed from pSK::cphAMA19anti (Table 2). Polymeric materialthat accounted for up to 25% (wt/wt) of the cell dry matter wasisolated from cells harboring pSK::cphAMA19co. The polymericmaterial was composed of aspartic acid and arginine at a molarratio 1:0.98, which is typical for cyanophycin molecules. Theelectrophoretic mobility of this polymer is shown in Fig. 2b(lane 1).

Purification of cyanophycin synthetase. Cyanophycin syn-thetase was enriched 123-fold to electrophoretic homogeneityfrom the soluble protein fraction of E. coli(pSK::cphAMA19co)by anion-exchange chromatography, affinity chromatography,and gel filtration chromatography (Table 3) and accounted for0.8% (wt/wt) of the total protein in the soluble cell fraction.SDS-PAGE of the purified preparation produced one proteinband, which corresponded to a molecular mass of 100 15kDa (Fig. 2a, lane 7). This value was in good agreement withthe calculated molecular mass of the translation product ofcphA (100.6 kDa).

Characterization of the in vitro polyamide synthesis cata-lyzed by cyanophycin synthetase. Purified cyanophycin syn-thetase requires cyanophycin as a primer and is almost com-pletely inactive without this compound (reference 1 andreferences therein). Cyanophycin synthetase purified from re-combinant cells of E. coli harboring pSK::cphAMA19co usedonly cyanophycin, modified cyanophycin, and the -arginyl as-partic acid dipeptide as primers for elongation of the cyano-phycin polyamide chain (Table 4). Impure enzyme also cata-lyzed in vitro cyanophycin synthesis by using noncyanophycinprimers, such as N-acetylglucosamine and chemosyntheticpoly-,�-DL-aspartic acid, as well as cyanophycin-free cellmembrane fragments of Synechococcus sp. strain PCC7942 andaspartic acid-rich exobiopolymer from Klebsiella sp. (Table 4).While 45% activity was expressed when lot 107FO334 -argi-nyl aspartic acid dipeptide was used as the primer, only 2%

activity was expressed with lot 125HO515 dipeptide (Table 4).The reason for the difference is not known. The molecular sizeof the biopolymers obtained when lot 107FO334 dipeptide wasused was similar to the molecular size of cyanophycin isolatedfrom the E. coli recombinant strain harboring cphAMA19co

(Fig. 2a, lane 6, and Fig. 2b, lane 1).The products obtained from the enzyme reactions in which

noncyanophycin substances were used as primers were ana-lyzed by SDS-PAGE (Fig. 2a). When chemosynthetic poly-,�-DL-aspartic acid (Fig. 2a, lane 2), N-acetylglucosamine (Fig. 2a,lane 3), or lot 107FO334 -arginyl aspartic acid dipeptide (Fig.2a, lane 4) was used as the primer, the cyanophycin synthetase-containing soluble cell extracts synthesized polymeric materialthat had a molecular mass of 28 to 30 kDa, which corre-sponded to the molecular mass of cyanophycin purified fromrecombinant E. coli (Fig. 2b, lane 1) and clearly differed fromthe molecular mass of an authentic cyanobacterial sample (Fig.2a, lane 5). A reduced amount of polymer was also detectedafter elongation of -arginyl aspartic acid dipeptide when pu-rified enzyme was used (Fig. 2a, lane 6). In a complete 2-mlreaction mixture containing excess ATP (9 �mol) when cya-nophycin was the primer, the purified cyanophycin synthetase(40 �g ml�1) incorporated approximately 5 �mol of arginineand 5 �mol of aspartic acid into the growing cyanophycin chainduring incubation for 20 h. There was no L-[U-14C]arginine

FIG. 2. SDS-PAGE analysis of the in vitro-synthesized cyanophy-cin-like polymers. (a) Reactions catalyzed by the cyanophycin syn-thetase-containing soluble cell fraction (0.5 mg of protein ml�1) and bythe purified cyanophycin synthetase (0.04 mg of protein ml�1) at 30°Covernight in the presence of different primers at a concentration of 1mg ml�1 as described in Materials and Methods. Lane 1, soluble cellfraction with cyanophycin purified from Synechococcus sp. strainMA19; lane 2, soluble cell fraction with chemosynthetic poly-,�-DL-aspartic acid; lane 3, soluble cell fraction with N-acetylglucosamine;lane 4, soluble cell fraction with -arginyl aspartic acid dipeptide (lot197FO334); lane 5, cyanophycin purified from Synechococcus sp. strainMA19; lane 6, purified cyanophycin synthetase with the -arginyl as-partic acid dipeptide (lot 197FO334); lane 7, purified cyanophycinsynthetase; lane 8, soluble cell fraction. (b) Lane 1, cyanophycin puri-fied from E. coli (cphAMA19co). The sizes of molecular mass standardproteins (lane Std) are indicated on the left.

TABLE 2. Expression of Synechococcus sp. strain MA19 cphA andaccumulation of cyanophycin in recombinant E. colia

Plasmid Orientationb IPTGcCyanophycin content

(% [wt/wt] of celldry matter)

Sp actd

pSK::cphAMA19co Colinear � 25.0 2.0 1.81 0.1� 11.5 2.0 0.66 0.1

pSK::cphAMA19anti Antilinear � 0.5 0.2 0.05 0.1� 0.5 0.1 0.05 0.1

pBluescript SK(�) Not relevant � 0.1 0.1 0.05 0.1

a The experiments and enzyme assays were done in triplicate, and the standarderrors were calculated by using standard statistics (P � 0.05).

b Orientation with respect to lacPO.c Recombinant E. coli TOP10 cells were grown in Luria-Bertani medium in the

presence (�) or in the absence (�) of 1.0 mM IPTG as described in Materialsand Methods.

d Specific activity is expressed as the rate of incorporation of L-arginine innanomoles per minute per milligram of protein.

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incorporation if CTP or GTP was used instead of ATP (datanot shown).

To investigate the role of phosphorylation steps in the en-zyme reaction catalyzed by cyanophycin synthetase, the effectof two acidic phosphatases on incorporation of arginine wasexamined. We found that [14C]arginine incorporation wasstopped rapidly when we added the acidic pyrophosphatase(EC 3.6.1.1) or orthophosphoric phosphohydrolase-phospha-tase (acid optimum) (EC 3.1.3.2) (Boehringer) at a concentra-tion of approximately 5 �g ml�1 to the reaction mixtures.

Substrate specificity. Table 5 shows the activities of theSynechococcus sp. strain MA19 cyanophycin synthetase in re-sponse to various substrates. The specific activities of the cya-nophycin synthetase were almost identical whether L-[14C]argi-nine or L-[14C]aspartic acid was used as the labeled substrate(Table 5, reactions 1 and 13). The ratio of the incorporationrate of L-aspartic acid to the incorporation rate of L-arginine(1:0.95) corresponded to the ratio of the two amino acids inauthentic cyanophycin. Only negligible activities were foundwhen L-aspartic acid or L-arginine was omitted. No activity wasdetected when arginine was replaced by L-glutamic acid,-arginyl aspartic acid dipeptide, citrulline, ornithine, arginineamide, agmatine, or norvaline. In the reaction in which lysinereplaced arginine, negligible enzyme activity was detected (Ta-ble 5, reaction 9). However, when �-hydroxy aspartic acid wasused instead of L-aspartic acid, about 83% enzyme activity wasobtained (Table 5, reaction 6).

Replacement of L-arginine by 0.5 mM L-canavanine, whichwas shown to be an arginine analogue and a competitive in-hibitor of the cyanophycin synthetase from Synechocystis sp.strain PCC6308 (1), resulted in a residual activity of 62%,which corresponded to a rate of incorporation of [3H]canava-nine of 58%. As incorporation of canavanine is competitivewith incorporation of arginine, it can be totally overcome byequal amounts of arginine (Table 5, reaction 12). On the otherhand, the rate of incorporation of L-aspartic acid in a completereaction mixture in the presence of additional canavanine (Ta-ble 5, reaction 14) was 159%, which corresponded to the sumof the activities in reactions 13 and 11a (Table 5).

Cyanophycin synthetase isolated from recombinant E. colihad the parental thermostable character. Maximum activitywas obtained at an assay temperature of 50°C; this was alsoobserved previously for the enzyme isolated from the parentalcyanobacterium Synechococcus sp. strain MA19 (8). The activ-ity decreased at higher assay temperatures. If the enzyme waspreincubated for 30 min at an elevated temperature and thenactivity was assayed at 30°C, 84 and 72% of the activity re-mained after preincubation at 50 and 60°C, respectively. Alevel of enzyme activity of 100% at an incubation temperature

of 30°C corresponded to a specific activity of 136 U mg ofprotein�1 (Fig. 3).

Ectoine, which is a nontoxic amphoterous protector andstabilizer of enzymes and proteins (16), significantly stabilizedthe cyanophycin synthetase. In the presence of 1 M ectoine theenzyme retained approximately 90% of its activity even after120 min of incubation at 50°C. In the absence of ectoine only45% of the enzyme activity was retained. The rate of incorpo-ration of L-[U-14C]arginine at the beginning of incubation rep-resented 100% enzyme activity (128 U mg of protein�1).

DISCUSSION

By combining the methods of colony hybridization with PCRand inverse PCR, the complete cphBA locus encoding cyano-phycinase and cyanophycin synthetase was identified andcloned. As in other cyanobacteria, cphA was located down-stream of and colinear with cphB in the genome of Synecho-coccus sp. strain MA19. All regions which had been identified

TABLE 3. Purification of cyanophycin synthetase from recombinant E. coli harboring cphA of Synechococcus sp. strain MA19

Purification step Vol (ml) Protein(mg)

Total activity(U)a

Sp act (U mg ofprotein�1)

Purification(fold)

Yield(%)

Soluble protein fraction 90.0 1,703 1,566 1.5 0.9 0.5 1.0 100Q-Sepharose 95.0 164 1,166 2.0 7.1 1.0 7.7 74.5Procion Blue 10.0 11 254 1.5 23.1 0.5 25.1 15.8Superdex S200 10.5 1.8 203 1.0 113.0 1.0 123.0 13.0

a One unit was defined as incorporation of 1 nmol of L-arginine min�1.

TABLE 4. Primer specificity of the purified cyanophycin synthetaseexpressed from E. coli haboring pSK::cphAMA19co

a

Primer added

Relative activity (%)b

Soluble proteinfraction

Purifiedenzyme

Cyanophycin 100.0 0.2 100.0 0.5Modified cyanophycinc 120.0 1.5 140.0 2.5None 4.0 2.0 0.1 0.1Poly-,�-DL-aspartic acidd 8.7 0.5 6.9 1.5Poly--L-aspartic acid 0.8 0.5 NDe

N-Acetylglucosamine 90.0 2.5 1.8 2.0-Arginyl aspartic acid dipeptide

(lot 197FO334)75.0 1.0 45.0 2.5

-Arginyl aspartic acid dipeptide(lot 125HO515)

10.0 1.5 2.0 0.5

Cell membrane fraction fromSynechococcus sp. strain PCC7942

14.0 1.0 ND

Aspartic acid-rich exobiopolymerfrom Klebsiella sp.

80.0 0.5 ND

a The reaction conditions were those described in Materials and Methods,except that the primer was varied. The primers were each added at a finalconcentration of 1 mg ml�1. The experiments were done in triplicate, andmeans standard errors (P � 0.05) were determined.

b The relative activities were calculated with respect to the control (whenpurified cyanophycin from Synechococcus sp. strain MA19 was used as the prim-er); 100% activity for the unpurified enzyme corresponded to a specific activityof 1.1 U mg of protein�1, and 100% activity for the purified enzyme corre-sponded to a specific activity of 128 U mg of protein�1. One unit was defined asincorporation of 1 nmol of arginine per min.

c The modified cyanophycin contained aspartic acid and arginine at a molarratio of approximately 1:0.6.

d The exact structure of polyaspartic acid LPOC618 obtained by thermal con-densation is not known.

e ND, not detectable.

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previously in the CphA consensus sequence as possible impor-tant sites of cyanophycin synthetase (1, 3) were also located inSynechococcus sp. strain MA19 CphA. The multiple alignmentidentified only 71 positions in the Synechococcus sp. strainMA19 cphA translation product that were unique to this se-quence. In the resulting phylogram constructed for six avail-able cyanophycin synthetases, Synechococcus sp. strain MA19grouped with S. elongatus and A. variabilis in a distinct cluster.

Polymeric material that accounted for up to 25% (wt/wt) ofthe cellular dry matter was isolated from cells harboringpSK::cphAMA19co. This polyamide was composed of only as-partic acid and arginine, which is in accordance with the com-position of the authentic cyanobacterial polymer, and it dif-fered from cyanophycin-like polymers isolated from otherrecombinant strains of E. coli, which contained lysine in addi-tion to these two amino acids (39).

The cyanophycin synthetase activities in the recombinantstrains of E. coli depended on the orientation of the gene withrespect to the lacPO region. The CphA enzyme was over-expressed and active when the orientation of the corre-sponding cphA gene was colinear with respect to lacPO. Noactivity was detected when the gene was transcribed frompSK::cphAMA19anti. Hence, the cloned DNA fragment con-tained a functional active cphA gene but no promoter whichcan be recognized in E. coli.

The cyanophycin synthetase was purified to electrophoretichomogeneity from E. coli harboring pSK::cphAMA19co. Thepurified enzyme exhibited more restricted primer selectivitythan the impure enzyme in the crude protein soluble fraction.The purified form accepted only cyanophycin, modified cyano-phycin, and the -arginyl aspartic acid dipeptide as primers forelongation of the cyanophycin polyamide chain. However, the

unpurified enzyme catalyzed in vitro polymerization by usingnoncyanophycin primers such as N-acetylglucosamine andpoly-,�-DL-aspartic acid, as well as cyanophycin-free cellmembrane fragments of Synechococcus sp. strain PCC7942 andaspartic acid-rich exobiopolymer from Klebsiella sp. Perhapsthese macromolecules fulfill a function as nuclear crystals forgrowth of the cyanophycin polyamide chain by acting as bind-ing agents for the cyanophycin oligomers, including dimers and

FIG. 3. Temperature profile (}) and thermostability (F) of thepurified cyanophycin synthetase (40 �g ml�1). To determine the tem-perature profile, the enzyme activity was assayed at 20, 30, 40, 50, 60,70, and 80°C for 15 min. The thermostability of the enzyme wasdetermined by incubating the enzyme solution at different tempera-tures (30, 40, 50, 60, 70, and 80°C) for 30 min and then measuring theactivity at 30°C as described in Materials and Methods.

TABLE 5. Substrate specificity of purified cyanophycin synthetase from recombinant E. coli (pSK::cphAMA19co)

Reaction Variation of substrate mixturea Labeled substrate usedb Relative activity (%)c

1 Completec L-[U-14C]arginine 100 2.52 Without L-aspartic acid L-[U-14C]arginine 2.0 1.03 Without L-arginine L-[U-14C]aspartic acid 5.0 0.54 L-Glutamic acid instead of L-aspartic acid L-[U-14C]arginine 3.0 0.55 L-Glutamic instead of L-arginine L-[U-14C]glutamic acid NDd

6 �-Hydroxy-L-aspartic acid instead of L-aspartic acid L-[U-14C]arginine 83.0 2.57 -Arginyl aspartic acid dipeptide lot 107FO334

instead of L-aspartic acidL-[U-14C]arginine 4.0 1.5

8 -Arginyl aspartic acid dipeptide lot 107FO334instead of L-arginine

L-[U-14C]aspartic acid ND

9 L-Lysine instead of L-arginine L-[3H]lysine 2.7 1.010 L-Lysine instead of L-aspartic acid L-[3H]lysine ND11a L-Canavanine instead of L-arginine L-[U-14C]aspartic acid 62.0 3.011b L-Canavanine instead of L-arginine L-[3H]canavanine 58.0 2.012 L-Canavanine added L-[U-14C]arginine 100.6 2.513 Complete L-[U-14C]aspartic acid 105.7 2.014 L-Canavanine added L-[U-14C]aspartic acid 158.6 1.515 L-Citrulline instead of L-arginine L-[U-14C]aspartic acid ND16 L-Ornithine instead of L-arginine L-[U-14C]aspartic acid ND17 L-Arginine amide instead L-arginine L-[U-14C]aspartic acid ND18 L-Agmatine instead of L-arginine L-[U-14C]aspartic acid ND19 L-Norvaline instead of L-arginine L-[U-14C]aspartic acid ND

a The complete reaction mixture is described in Materials and Methods.b The concentrations of the radiolabeled compounds used were 10 �M for L-[U-14C]aspartic acid and 5 �M for the other compounds. The experiments were done

in triplicate, and means standard errors (P � 0.05) were determined.c A relative activity of 100% corresponded to a specific activity of 125.3 U mg of protein�1. One unit was defined as incorporation of 1 nmol of arginine per min.d ND, not detectable.

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trimers, which presumably were present in the soluble proteinfraction of the recombinant cells, thus permitting the elonga-tion reaction. This would be consistent with the reaction mech-anism postulated recently (3). No de novo cyanophycin syn-thesis occurred in the reactions catalyzed by purifiedcyanophycin synthetase in the absence of cyanophycin primersor in the presence of noncyanophycin primers. The use of-arginyl aspartic acid dipeptide, in which both carboxylicgroups of the aspartic acid residue are free, as a primer forpolyamide de novo synthesis produced different results, whichdepended on the batch of dipeptide purchased. Since no de-tailed analysis of the two batches was available from the man-ufacturer, the reason for the difference and the catalytic mech-anism for this primer type remain unknown.

Compared to other cyanophycin synthetases described re-cently (3), the enzyme from a recombinant strain of E. coliharboring cphAMA19co exhibited differences not only in primerselectivity but also in substrate specificity. Only one of theintermediates of the urea cycle and arginine analogues, cana-vanine, served as an alternative substrate for cyanophycin syn-thetase and was incorporated instead of L-arginine. Whetherincorporation of canavanine occurred in one growing polymerchain or occurred independently at separate primer sites (i.e.,whether a copolymer or a blend of homopolymers was formed)remains to be elucidated. The in vivo influence of canavanineon cyanophycin accumulation was demonstrated in a previousstudy (21). After addition of the arginine analogue to themedium, formation of akinets increased fivefold and there wasa concomitant complete loss of cyanophycin granules. The dataobtained by us showed that canavanine affected in vitro cya-nophycin synthesis by competitive incorporation.

The purified cyanophycin synthetase had a temperature pro-file and a thermostability like those of the enzyme preparationwhich was obtained from the parental cyanobacterium, Syn-echococcus sp. strain MA19 (8). The stability could be im-proved by addition of ectoine, thus making the enzyme fromSynechococcus sp. strain MA19 a promising candidate for invitro cyanophycin and polyamide production on a technicalscale.

ACKNOWLEDGMENTS

We thank M. Miyake and Y. Asada (Molecular Laboratory of theNational Institute of Bioscience and Human Technology, Tsukuba,Ibaraki, Japan) for providing Synechococcus sp. strain MA19 and E.Galinski (Institut fur Biochemie, Westfalische Wilhelms-Universitat,Munster, Germany) for providing ectoine. The assistance of D. Rehderwith inverse PCR is gratefully acknowledged.

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