Heterologous Expression of Cyanophycin Synthetase and

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Notes Heterologous Expression of Cyanophycin Synthetase and Cyanophycin Synthesis in the Industrial Relevant Bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida Elsayed Aboulmagd, Ingo Voss, Fred B. Oppermann-Sanio, and Alexander Steinbu1 chel* Institut fu¨r Mikrobiologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstrasse 3, D-48149 Mu¨nster, Germany Received April 19, 2001 Revised Manuscript Received June 29, 2001

Introduction Cyanophycin is a nonribosomally synthesized protein-like polymer, which consists of equimolar amounts of arginine and aspartic acid arranged as a polyaspartate backbone, with arginine moieties linked to the β-carboxyl group of each aspartate by its R-amino group.1 In nature, cyanophycin occurs exclusively in cyanobacteria.2-5 Polymerization of this polyamide is catalyzed by cyanophycin synthetase (encoded by cphA).6 Cyanophycin synthetase had been purified from Anabaena Variabilis ATCC294136 and from the thermophilic Synechococcus sp. strain MA197 and consisted of identical subunits of 100-130 kDa. cphA was sequenced from A. Variabilis ATCC29413, Anabaena sp. strain PCC7120, Synechocystis sp. strain PCC6803, Synechocystis sp. strain PCC6308, and Synechococcus elongatus.6,8-10 In contrast to the authentic cyanophycin isolated from cyanobacteria, which consists of polymer strands with an apparent molecular mass range of 25-100 kDa,1,11 both the in vitro synthesized cyanophycin and the polymer from recombinant Escherichia coli cells harboring cphA exhibited a relatively close molecular mass range of 25-30 kDa.6,8-10 Cyanophycin can be chemically converted to a derivative with reduced arginine content,12 which can be applied in various technical processes as a biodegradable substitute for polyacrylate.13 Because of the low cell yield, the low content of cyanophycin, the long cultivation period, and the highly sophisticated fermentation processing,14 cyanobacteria are unsuitable sources for cyanophycin with respect to cost effectiveness. Cyanophycin synthesis had been established in recombinant E. coli strains harboring cphA from different sources6,8,9 resulting in a maximum content of 26% (w/w) of cell dry mass after growth on rich complex but costly media.9 Unfortunately, attempts to produce cyanophycin during growth on mineral media led to a drastically reduced ability to produce cyanophycin (data not shown). One explanation for this may be the draining of the intracellular * To whom correspondence may be addressed. Tel.: +49 (251) 8339821. Fax.: +49 (251) 8338388. E-mail: [email protected].

pool of amino acids, which is caused by the biosynthesis of cyanophycin and which may lead to a derangement of the host anabolism. Corynebacterium glutamicum is widely used for production of amino acids such as glutamic acid and lysine.15 Due to the relatively high intracellular concentrations of glutamic acid (approximately 200 mM),16,17 recombinant strains of C. glutamicum expressing cphA may be able to open up the potential biosynthetic bottleneck in the route from glutamate to arginine. Similar favorable characteristics are assigned to Pseudomonas putida and Ralstonia eutropha, which have been employed for the industrial scale production of single cell protein18 and polyhydroxyalkanoates (Biopol).19-21 Under reduced aeration, cells of R. eutropha excrete metabolites of the citrate cycle (i.e., 2 oxoglutarate, succinate, fumarate, and malate) into the medium, indicating a surplus of precursors for the biosynthesis of arginine as well as aspartic acid.22 In this paper we describe (i) the transformation of C. glutamicum, R. eutropha, and P. putida with cphA from Synechocystis sp. strain PCC6308 and (ii) the heterologous expression of cphA in the corresponding recombinant strains in order to investigate these bacteria for biotechnological production of cyanophycin. Materials and Methods Bacterial Strains, Plasmids, and Culture Conditions. The bacterial strains and plasmids used in this study are listed in Table 1. E. coli was grown at 37 °C in Luria Bertani (LB) or M9 mineral medium29 containing 1% (v/v) glycerol instead of glucose. C. glutamicum was usually grown at 37 °C in Standard I (StI) nutrient broth (MERCK, Darmstadt, Germany). The medium and conditions for growth, harvest, and storage of electroporation-competent cells of C. glutamicum were exactly as described by van der Rest.30 For cyanophycin production in C. glutamicum, the MCGC medium31 was used as modified by Delaunay et al.32 R. eutropha was cultivated in mineral medium as described by Schlegel et al..33 P. putida was grown at 30 °C in either LB or mineral salts medium supplemented with 2% (w/v) sodium gluconate.33 Antibiotics were added to the media prior to inoculation by the respective strain or plasmid carrying derivative (concentration in µg/mL): ampicillin (75, E. coli), nalidixic acid (30, C. glutamicum), kanamycin (50, E. coli, P. putida, and C. glutamicum; 300, R. eutropha), and streptomycin (500, R. eutropha HF39). Purification and Determination of Cyanophycin. The isolation of cyanophycin from recombinant cells was done according to the procedure of Simon.11 The amino acid constituents of cyanophycin were determined by highperformance liquid chromatography (HPLC) as described before.9

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Notes Table 1. Bacterial Strains and Plasmids Used in This Study strains or plasmids Strains Corynebacterium glutamicum DSM 20300 ()ATCC 13032) Ralstonia eutropha HF39 H16-PHB-4 (DSM 541)

Pseudomonas putida KT2440 GPp104 Escherichia coli S17-1 TOP10 Plasmids pSK::cphA pEK0 pEK0::cphA pBBR1-MCS2 pBBR1::cphA a

relevant characteristics

ref or source

wild type, Nxr DSMZa Sm resistant mutant of wild type R. eutropha H16 (DSM 428) mutant of wild type R. eutropha H16 (DSM 428) defective in the synthesis of poly(hydroxyalkanoic acid) (PHA)

23 18

wild type mutant of KT2440 defective in the synthesis of PHA

24 25

recA proA thi-1, harbors the pRP4 tra genes in the chromosome recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 DlacU169 (f80 lacZDM15)

26 Stratagene (San Diego, CA)

pBluescript SK- harboring a 3.3-kbp cphA containing PCR product from Synechocystis sp. strain PCC6308 DNA E. coli /Corynebacterium shuttle vector, Kmr pEK0 harboring cphA from Synechocystis sp. strain PCC6308 Kmr, broad host range vector, lacPOZ′ pBBR1MCS-2 harboring cphA from Synechocystis sp. strain PCC6308

9 27 this study 28 this study

Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. Nx, nalidixic acid; Sm, streptomycin; Km, kanamycin.

Cyanophycin Synthetase Assay. After entering the stationary growth phase, cells were harvested by centrifugation (15 min, 3500g, 4 °C), washed once with 50 mM Tris HCl buffer (pH 8.2), and resuspended with 2 mL of buffer per gram of fresh cell mass. Cells of R. eutropha and E. coli were disintegrated by sonication for 1 min/mL of cell suspension by using a Sonoplus GM200 sonifier (Bandelin electronic, Berlin, Germany), whereas P. putida cells were disintegrated by a 3-fold passage through a French press cell at 96 MPa. Disruption of C. glutamicum cells was achieved by a 10-fold passage through a French press cell at 96 MPa and subsequent sonication for 2 min/mL of suspension. The supernatant of a high-speed centrifugation of the broken cells (1 h, 100000g, 4 °C) was desalted on NAP5 columns (Pharmacia Biotech, Freiburg, Germany) and served as soluble cell fraction. Cyanophycin synthetase activity was measured in the soluble cell fraction by the radiometric procedure, which was described recently.34 Protein concentration was determined by the procedure of Bradford.35 Electrophoresis. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed in 11.5% (w/v) gels as described by Laemmli.36 Staining of proteins and cyanophycin was done with Serva Blue R. Isolation and Manipulation of DNA. Plasmids from E. coli were isolated by the boiling method.37 Plasmids from C. glutamicum were isolated by the alkaline lysis procedure38 with prior incubation of the cells in lysozyme containing buffer at 37 °C for 1 h.16 DNA was digested with various restriction endonucleases under the conditions described by Sambrook et al.29 or by the manufacturer. Other DNAmanipulating enzymes were used as described by the manufacturers. Transfer of Plasmids. Transformation of C. glutamicum was achieved by electroporation after following the optimized transformation protocol of van der Rest et al.30 A complex

medium consisting of 5 g/L trypton (Difco), 5 g/L NaCl, 2.5 g/L yeast extract (Difco), and 18.5 g/L Lab-Lemco (Oxoid) was used instead of LBHIS. Plasmids were transferred into E. coli by employing the CaCl2 method,29 whereas the transfer of plasmids into R. eutropha was performed by conjugation.39 using E. coli S17-1 as donor strain. The transfer of the plasmids into P. putida was performed by electroporation.40 Results and Discussion Construction of Plasmids and Transfer into C. glutamicum, R. eutropha, and P. putida Strains. The 3.3-kbp EcoRI insert from pSK::cphA harboring the cyanophycin synthetase gene from Synechocystis sp. strain PCC63089 was ligated to EcoRI-digested E. coli/C. glutamicum shuttle vector pEK0 DNA resulting in pEK0::cphA (Figure 1). Transformands of E. coli TOP10 carrying this vector were clearly recognized on LB medium due to the whitish color of the colonies, indicating cyanophycin production. Restriction analysis revealed a collinear orientation of cphA with respect to the lac promotor of the vector. The hybrid plasmid was further transferred to the final host by electroporation, resulting in transformands of C. glutamicum harboring pEK0::cphA. The 3.3-kbp fragment mentioned above was in addition ligated to EcoRI linearized broad host range vector pBBR1MCS2. The resulting pBBR1::cphA was transferred to P. putida by electroporation and to E. coli S17-1 by transformation. Transformands of the latter were used as donor strain for the conjugative transfer of the plasmid into the strains HF39 and H16-PHB-4 of R. eutropha. Heterologous Expression of cphA and Synthesis of Cyanophycin. To verify whether Synechocystis sp. strain PCC6308 cphA is functionally active in the recombinant strains of C. glutamicum, R. eutropha strains, and P. putida, its expression was examined. HPLC analysis of the polymeric material, which was isolated from pEK0::cphA and pBBR1::

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Notes

Figure 1. Construction of plasmids pBBR1::cphA and pEK0::cphA.

cphA harboring cells, revealed in all cases equimolar amounts of aspartic acid and arginine and identified the polymer as cyanophycin (data not shown). Significant expression of cphA in C. glutamicum (pEK0:: cphA) occurred during growth in complex medium resulting in a cyanophycin content of 2.0% (w/w) of cell dry matter. After growth in MCGC medium under conditions supporting glutamic acid production,31 the cyanophycin content raised 1.8-fold (Table 2). Despite the high intracellular glutamic acid concentration, cphA expression in C. glutamicum did not result in cyanophycin with detectable amounts of glutamic acid. Approximately 1.9- and 2.4-fold higher contents of cyanophycin were obtained with recombinant cells of R. eutropha HF39 and R. eutropha H16-PHB-4 (Table 2). The highest production of cyanophycin (11% (w/w) of cell dry mass)) was obtained with recombinant cells of P. putida KT2440 grown in mineral salts medium supplemented with

aspartic acid and arginine (0.2% (w/v) each) whereas P. putida GPp104 produced only 7% (w/w) (Table 2). Heterologous expression of cphA and production of cyanophycin in the recombinant cells clearly demonstrated the functionality of the cphA translational product in the three bacteria. In contrast to E. coli, the five recombinant strains of C. glutamicum, R. eutropha, and P. putida produced significant amounts of cyanophycin during cultivation on mineral salts media. This is a big step forward toward biotechnological production of cyanophycin employing bacteria which are already very well established in the chemical industry for the production of other bulk chemicals. Although C. glutamicum expressed cphA to specific activities corresponding to 67% of the values obtained from recombinant E. coli after growth on LB, only 14% of the E. coli cyanophycin content was obtained. In comparison to C. glutamicum, lower expression levels of cphA in P. putida and R. eutropha resulted in even higher polymer contents,

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Notes

Table 2. Accumulation of Cyanophycin and Expression of Synechocystis sp. Strain PCC6308 CphA in Recombinant C. Glutamicum, R. Eutropha, P. Putida, and E. Coli organsima

medium

plasmid

cyanophycin content (% of dry cell mass)

specific activity (nmol of arginine incorporated min-1 (mg of protein)-1)

C. glutamicum C. glutamicum R. eutropha HF39 R. eutropha H16-PHB-4 P. putida KT2440 P. putida GPp104 E. coli TOP10

mineral salts complex (StI) mineral salts mineral salts mineral salts mineral salts complex (LB)

pEK0::cphA pEK0::cphA pBBR1::cphA pBBR1::cphA pBBR1::cphA pBBR1::cphA pSK::cphA

3.6 2.0 7.0 8.7 11.0 7.0 26.0

2.0 n.d. 1.1 1.54 1.8 1.0 2.97

a The control organisms harboring the corresponding vector with out cphA exhibited cyanophycin content and specific activity less than 0.1% (w/w) of cell dry mass and 0.05 nmol of arginine‚min-1‚(mg of protein)-1, respectively.

tis sp. strain PCC6308 cphA and heterologous synthesis of cyanophycin in the recombinant cells clearly proved the functionality of the cphA translational product in these five bacteria. Due to the high cyanophycin productivity of the recombinant strains of C. glutamicum, R. eutropha, and P. putida during cultivation on mineral salts media in comparison with E. coli., these recombinant strains could be a suitable candidates for the biotechnological production of cyanophycin. The lower molecular weight of cyanophycin synthesized in these five recombinant strains as well as in E. coli in comparison to cyanophycin isolated from cyanobacteria may be due to the absence of a certain cyanobacterial factor. Further studies will focus on factors, which restrict the productivity of the cyanophycin synthesis in the recombinant strains and the dispersity and the molecular weight of the product. Figure 2. Synthesis and accumulation of cyanophycin in recombinant C. glutamicum, R. eutropha, P. putida, and E. coli. (A) Samples of purified cyanophycin from the following strains (60 µg each) were separated in an SDS-polyacrylamide gel and stained as described in Materials and Methods: Synechocystis sp. strain PCC6308, lane 1; E. coli (pSK::cphA), lane 2; C. glutamicum (pEK0::cphA), lane 3; R. eutropha HF39 (pBBR1::cphA), lane 4. (B) Total protein of P. putida KT2440 harboring (pBBR1::cphA) (45 µg), lane 5; total protein of P. putida KT2440 (40 µg), lane 6. The sizes of molecular mass standard proteins (Std) are provided on the left and right margin in kDa.

which, however, was still below the values obtained from E. coli. These data showed that also other endogenic or exogenic factors than (i) the expression level of cphA and (ii) the provision with precursors of the biosynthetic pathways to arginine and/or aspartic acid mainly affect the final cyanophycin content. Examination of the molecular weights of the cyanophycin revealed similar values of the polyamides isolated from recombinant C. glutamicum, R. eutropha, and P. putida (Figure 2), indicating that the polymer strands produced by recombinant cphA harboring cells are of more reduced length and polydispersity than the authentic material from cyanobacteria. This phenomenon was also reported for the polymers isolated from cphA expressing cells of E. coli.6,8,9 Conclusions Employing the E. coli/C. glutamicum shuttle vector pEK0 and the broad host range vector pBBR1-MCS2, Synechocystis sp. strain PCC6308 cphA was transferred to C. glutamicum, to two strains of R. eutropha, and to two strains of P. putida, respectively. Heterologous expression of Synechocys-

Acknowledgment. The authors thank Dr. Eikmanns (Universita¨t Ulm, Abtlg. Angewandte Mikrobiologie) for providing plasmid pEK0. This work was supported by a grant provided by the BAYER AG and by a fellowship provided by the government of the Arabic Republic of Egypt to Elsayed Aboulmagd. References and Notes (1) Simon, R. D.; Weathers, P. Biochim. Biophys. Acta 1976, 420, 165176. (2) Lawry, N. H.; Simon, R. D. J. Phycol. 1982, 18, 391-399. (3) Simon, R. D. In The cyanobacteria; Fay, P.; van Baalen, C.; Eds., Elsevier: Amsterdam, New York, 1987; pp 199-225. (4) Allen, M. M. Annu. ReV. Microbiol. 1984, 38, 1-25. (5) Allen, M. M. Methods Enzymol. 1988, 167, 207-213. (6) Ziegler, K.; Diener, A.; Herpin, C.; Richter, R.; Deutzmann, R.; Lockau, W. Eur. J. Biochem. 1998, 254, 154-159. (7) Hai, T.; Oppermann-Sanio, F. B.; Steinbu¨chel, A. FEMS Microbiol. Lett. 1999, 181, 229-236. (8) Oppermann-Sanio, F. B.; Hai, T.; Aboulmagd, E.; Hezayen, F. F.; Jossek, S.; Steinbu¨chel, A. In Biochemical principles and mechanisms of biosynthesis and biodegradation of polymers; Steinbu¨chel A., Ed.; Wiley-VCH: Weinheim, 1999; pp 185-193. (9) Aboulmagd, E.; Oppermann-Sanio, F. B.; Steinbu¨chel, A. Arch. Microbiol. 2000, 174, 297-306. (10) Berg, H.; Ziegler, K.; Piotukh, K.; Baier, K.; Lockau, W.; VolkmerEngert, R. Eur. J. Biochem. 2000, 267, 5561-5570. (11) Simon, R. D. Biochim. Biophys. Acta 1976, 422, 407-418. (12) Joentgen, W.; Groth, T.; Steinbu¨chel, A.; Hai, T.; Oppermann, F. B. International patent application WO 98/39090, 1998. (13) Schwamborn, M. Polym. Degrad. Stabil. 1998, 59, 39-45. (14) Hai, T.; Ahlers, H.; Gorenflo, V.; Steinbu¨chel, A. Appl. Microbiol. Biotechnol. 2000, 53, 383-389. (15) Eggeling. L.; Sahm. H. Appl. Microbiol. Biotechnol. 1999, 52, 146153.

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