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Identification of the Anabaena sp. Strain PCC7120 Cyanophycin Synthetase as Suitable Enzyme for Production of Cyanophycin in Gram-Negative Bacteria Like Pseudomonas putida and Ralstonia eutropha Ingo Voss,†,‡ Simone Cardoso Diniz,†,‡ Elsayed Aboulmagd,§ and Alexander Steinbu¨chel*,‡ Institut fu¨r Molekulare Mikrobiologie und Biotechnologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstraβe 3, D-48149 Mu¨nster, Germany, and Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Alexandria University, Alexandria, Egypt Received March 8, 2004; Revised Manuscript Received April 28, 2004
The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp. strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E. coli (6.7 nmol arginine per min and mg protein). CphA1 and cphA genes of Synechocystis sp. strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha. Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P. putida strains and 7.3, 12.6, or 14.1% of CDM for R. eutropha). Furthermore, PHA-negative mutants of P. putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R. eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P. putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R. eutropha strains). Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin. PHA-negative mutants of P. putida and R. eutropha expressing cphA1 of Anabaena sp. strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin. Introduction Cyanophycin [multi-L-arginyl-poly(L-aspartic acid)] is a protein-like cytoplasmic inclusion, which is common to cyanobacteria and was discovered more than 100 years ago.1 This polymer contains aspartic acid and arginine at an about equimolar ratio and is arranged as a polyaspartic acid backbone with arginine moieties linked to the β-carboxyl group of each aspartate by its R-amino group.2 Cyanophycin serves as a temporary nitrogen reserve and usually accumulates during the transition from the exponential to the stationary growth phase.3 At neutral pH and physiological ionic strength, cyanophycin is insoluble. Cyanophycin is of biotechnological interest because a derivative with reduced arginine content can be obtained by partial chemical hydrolysis,4 which can be applied as a biodegradable substitute for polyacrylate in various technical processes.5 Intracellular synthesis and degradation of cyanophycin are catalyzed by cyanophycin synthetase, which is encoded by cphA,6 and cyanophycinase, which is encoded by cphB,7 respectively. The polymerization reaction yields polydisperse * To whom correspondence should be addressed. Phone: +49-2518339821. Fax: +49-251-8338388. E-mail:
[email protected]. † These authors have equally contributed to this work. ‡ Westfa ¨ lische Wilhelms-Universita¨t Mu¨nster. § Alexandria University.
cyanophycin with molecular weights ranging from 25 000 to 100 000 Da.8 The activity of cyanophycin synthetase depends on the presence of L-aspartic acid, L-arginine, ATP, Mg2+, K+, a sulfhydryl compound, and cyanophycin as a primer.6,8-10 In vitro synthesis of cyanophycin was also achieved by using chemically synthesized (β-Asp-Arg) or polyaspartic acid as primer instead of cyanophycin.11,12 Cyanophycin synthetase has been purified from Anabaena Variabilis,6 Synechocystis sp. strain PCC6308,12 and the thermophilic Synechococcus sp. strain MA19.13 CphA was cloned from A. Variabilis,6 Synechocystis sp. strains PCC6803,6,14 and PCC6308,9 Synechococcus sp. strain MA1915 and Synechococcus elongatus.11 More recently, cphA homologous genes were found in strains of Acinetobacter sp., Bordetella sp., Clostridium botulinum, Desulfitobacterium hafniense, Nitrosomonas europea, and others.16,17 Heterologous expression of Acinetobacter sp. cphA in Escherichia coli resulted in cyanophycin accumulation up to 7.5% (w/w) and a specific CphA activity of 1.2 U/mg protein, indicating that CphA of Acinetobacter was functionally active.16 In addition, expression of cphA from D. hafniense in E. coli resulted in the formation of cyanophycin.17 In the genomic sequence of Anabaena sp. PCC7120, two cph clusters, each consisting of a cyanophycinase gene (cphB) and a cyanophycin synthetase gene (cphA), were identified previously.18 However, it was shown
10.1021/bm049861g CCC: $27.50 © 2004 American Chemical Society Published on Web 06/16/2004
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Production of Cyanophycin Table 1. Bacterial Strains and Plasmids Used in This Study strains or plasmids
Anabaena sp. Bacillus megaterium Ralstonia eutropha H16 H16-PHB-4 Pseudomonas putida KT2440 GPp104 Escherichia coli S17-1 TOP10
pSK::cphA7120
pSK::cphAMA19
pBBR1MCS-2 pBBR1MCS-2::cphA6803
pBBR1MCS-2::cphAMA19
pBBR1MCS-2::cphA7120
pWH1520 pWH1520::cphA6308
relevant characteristics
reference or sourcea
Strains wild type mutant of B. megaterium DSM319 defective in Ca2+ -dependent metalloprotease
PCC7120, Rippka et al. 197922 Wittchen and Meinhardt 199538
wild type mutant of H16 defective in synthesis of PHA
DSM 428 Schlegel et al. 197039, DSM 541
wild type mutant of KT2440 defective in synthesis of PHA
DSM 291 Huisman et al. 199140
recA proA thi-1, harbors the RP4 tra genes in the chromosome recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 DlacU169 (f80 lacZDM15)
Simon et al. 198341
Plasmids 3.3-kbp PCR product from Anabaena sp. strain PCC7120 DNA harboring cphA in pBluescript SK- collinear to lacPO 2.98-kbp PCR product from Synechococcus sp. strain MA19 DNA harboring cphA in pBluescript SK- collinear to lacPO Kmr, broad host range vector, lacPOZ’ pBBR1MCS-2 harboring cphA from Synechocystis sp. strain PCC6803 collinear to lacPOZ’ pBBR1MCS-2 harboring cphA from Synechococcus sp. strain MA19 collinear to lacPOZ’ pBBR1MCS-2 harboring cphA from Anabaena sp. strain PCC7120 collinear to lacPOZ’ E. coli/B. megaterium shuttle vector 2.7 kbp SpeI-SphI PCR product from Synechocystis sp. strain PCC6308 DNA harboring cphA collinear to xylA promoter
Stratagene (San Diego, CA) this study
Hai et al. 200215
Kovach et al. 199529 this study
this study
this study
Rygus and Hillen 199142 this study
a DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; PCC, Pasteur Culture Collection of Cyanobacteria, Paris, France. Km, kanamycin; cphAx, source of cyanophycin synthetase gene.
that cyanophycin synthetase (CphA1) of the first cluster contributed more to synthesis and accumulation of cyanophycin in Anabaena sp. PCC7120 than the second cyanophycin synthetase (CphA2). For technical production of cyanophycin, heterologous expression of cphA was also demonstrated in recombinant strains of E. coli,19 Ralstonia eutropha, Corynebacterium glutamicum, and Pseudomonas putida.20 In addition, the previously used method for isolation of cyanophycin8 was modified, and a fast acid extraction method was applied for downstream processing at large scale.19 In this study, we report on the cloning and heterologous expression of Anabaena sp. strain PCC7120 cphA1 in E. coli and on the biochemical characterization of the enzyme. Furthermore, this and the cyanobacterial cphA genes from Synechocystis sp. PCC6803 and Synechococcus strain MA19 were heterologously expressed in the industrially relevant bacteria Bacillus megaterium, P. putida, and R. eutropha. The recombinant strains and cyanophycin synthetases were compared in detail concerning cyanophycin production rate, composition of the accumulated polymer, and specific
enzyme activity. Consequently, this study provides a good prerequisite for metabolic engineering of the strains regarding formation of substrates of cyanophycin synthetase and optimization of cphA expression level using more specific promoters. Furthermore, possibilities to establish a fermentation process at technical scale employing low cost mineral media were demonstrated. Experimental Section Bacterial Strains and Culture Conditions. The strains used in this study are listed in Table 1. Anabaena sp. strain PCC7120 was cultivated at 30 °C in Allen and Arnon medium21 or in BG-11 medium.22 E. coli was grown at 37 °C in Luria Bertani (LB) medium.23 Strains of P. putida, R. eutropha, and B. megaterium were grown either in complex media like LB or Nutrient Broth (NB) medium (Difco) or in mineral salts medium (MSM) according to Schlegel et al.24 at a temperature of 30 °C. Glucose, fructose, or sodium gluconate were used as carbon sources at concentrations of 1 to 2% (w/v), and aspartic acid and arginine were added as
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supplements at concentrations of 0.2% (w/v). For selection of plasmid carrying strains, antibiotics were added to the media at the following concentrations (µg/mL): ampicillin (75, E. coli), kanamycin (50, E. coli and P. putida; 300, R. eutropha), and tetracycline (12.5, B. megaterium). Preparation of Cell Extracts. After entering the stationary growth phase, cells were harvested by centrifugation (10 min, 2,800 x g, 4 °C), washed once with 50 mM Tris-HCl buffer (pH 8.2), and resuspended with 2 mL buffer per g fresh cell mass. Cells of Anabaena sp., E. coli, R. eutropha, and P. putida were disintegrated by a 3-fold passage through a French press cell at 96 MPa. Cells of B. megaterium instead were disintegrated by a 10-fold passage through a French Press cell after preincubation with lysozyme (2 mg/mL). The supernatants of high-speed centrifugations of the broken cells (1 h, 100 000 × g, 4 °C) were desalted on NAP5 columns (Pharmacia Biotech, Freiburg, Germany) and served as soluble cell fractions. DNA Isolation and Manipulation. Total genomic DNA of Anabaena sp. strain PCC7120 was isolated according to a procedure previously described.25 Plasmid DNA was isolated from E. coli, P. putida, R. eutropha, and B. megaterium by the alkaline extraction procedure.26 DNA was digested with various restriction endonucleases under the conditions described by Sambrook et al.23 or by the manufacturer. Other DNA-manipulating enzymes were used as described by the manufacturers. DNA fragments were isolated from agarose gels by using the NucleoTrap kit (Machery and Nagel, Du¨ren, Germany). For transformation, competent cells of E. coli were prepared employing the calcium chloride procedure.23 Transfer of plasmids into R. eutropha and P. putida was performed by conjugation using E. coli S17-1 as donor strain or by electroporation, respectively. Protoplasts of B. megaterium were transformed using a PEG-mediated procedure as described by Vorobjeva et al.27 and Meinhardt et al.28 Following transformation, regeneration was carried out at 30 °C on LB or NB agar plates containing tetracycline or kanamycin. Cloning of cphA. The cyanophycin synthetase gene of Anabaena sp. strain PCC7120 (cphA1) was amplified by PCR using total genomic DNA of this strain as template and the following oligonucleotides as primers: Primer P1 (5′ATGGGGCATCACATGATTGCTGGCG-3′), which was complementary to the central region of Anabaena sp. strain PCC7120 cphB1 (available in CyanoBase: www.kazusa.or.jp/ cyano/anabaena/), and primer P2 (5′-GGAGATGGGAATCACCACATCTCTAC-3′), which was complementary to a DNA region downstream of the 3′-region of cphA1 (in CyanoBase). Vent DNA polymerase (New England Biolabs, Schwalbach/Taunus, Germany) was used according to the instructions of manufacturer. The PCR products were isolated from agarose gels, ligated into EcoRV-digested pBluescript SK- (Stratagene Cloning Systems, San Diego, CA) and transferred into E. coli. For heterologous expression of cphA1 from Anabaena sp. PCC7120 in P. putida and R. eutropha, the cphA1 gene was subcloned from recombinant pBluescript SK- in the broad host range vector pBBR1MCS-229 and subsequently transferred into the recipients.
Voss et al.
The Synechocystis sp. PCC6803 cphA gene was amplified from genomic DNA by PCR. The following primers, which were deduced from the nucleotide sequence upstream and downstream of cphA, were used: P3 (5′P-ATGGGGCATCACATGATCGCCGG-3′) and P4 (5′P-TGACTCCAGCAAGGGGGAAGAAAACCT-3′). PCR was performed using Pfx DNA polymerase (MBI Fermentas, Germany), and the resulting 3.2-kbp product was purified from agarose gels and subsequently ligated to SmaI-linearized pBBR1MCS-2. The cphA gene from Synechococcus sp. strain MA19 was subcloned from pSK::cphAMA1915 in pBBR1MCS-2. Primers were purchased from MWG-Biotech AG (Ebersberg, Germany). DNA Sequence Analysis. DNA sequences were determined with a model 4000L DNA sequencer (LI-COR Inc., Biotechnology Division, Lincoln, NE) and a Thermo Sequenase fluorescence-labeled primer cycle sequencing kit (Amersham Life Science, UK) according to the instructions of the manufacturer. Purification and Determination of Cyanophycin. Cyanophycin was isolated according to the method described by Simon8 or by the acid extraction procedure described by Frey et al.19 The amino acid constituents of cyanophycin were determined by HPLC as described before.9 Determination of Carbohydrate, Protein, and Nucleic Acid Impurities in Cyanophycin. Quantification of carbohydrate was done by following the colorimetric reaction of samples with anthrone reagent using glucose as the standard as described by Hanson and Philips.30 Protein and nucleic acid contents were determined spectrophotometrically in clear solutions of cyanophycin preparations in 100 mM HCl by using the following equations: (i) protein (mg mL-1) ) 1.55A280 - 0.76A260,31 (ii) nucleic acids (mg mL-1) ) 0.05A260.23 Polyacrylamide Gel Electrophoresis. SDS-PAGE was performed in 11.5% (w/v) gels as described by Laemmli.32 Staining of proteins and cyanophycin was done with Serva Blue R. Protein concentration was determined by the procedure of Bradford.33 Cyanophycin Synthetase Assay. Cyanophycin synthetase activity was determined in the soluble cell fraction following the radiometric procedure described before,9 which was slightly modified by using 5 mM L-aspartic acid, 0.5 mM (U-14C) L-arginine (Amersham Pharmacia Biotech, specific activity: 10.3 Gbq/mmol) and 3 mM ATP in the reaction mixture. The activity was measured by following the incorporation of (U-14C) L-arginine into insoluble cyanophycin. Cultivation at the 30-L Scale. Cultivations at the 30-L scale were done in a Biostat UD30 stainless steel reactor (B. Braun Biotech International, Melsungen, Germany) which had a total volume of 42 L (28 cm inner diameter and 71 cm height). This bioreactor was equipped with three stirrers each containing six paddles and a Funda-Foam mechanical foam destroyer (B. Braun Biotech International, Melsungen, Germany). In addition, ports for sterilizeable probes to measure the dissolved oxygen concentration (model 25, Mettler Toledo GmbH, Steinbach, Switzerland), pH (model Pa/25, Mettler-Toledo GmbH, Steinbach, Switzerland), foam
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Table 2. Expression of Anabaena sp. Strain PCC7120 CphA1 in E. coli and Accumulation of Cyanophycin strain
cyanophycin content [%, w/w, of CDM]
specific activitya [nmol arginine‚(min‚mgprotein) -1]
Anabaena sp. strain PCC7120 E. coli (pBluescript SK-) E. coli (pSK::cphA17120)
n.d.b
