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Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production
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Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production Wen-Chi Tseng a,∗ , Tsuei-Yun Fang b,c , Kai-Chun Chang b , Chorng-Liang Pan b,c a
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10617, Taiwan Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan c Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan b
a r t i c l e
i n f o
Article history: Received 26 September 2012 Received in revised form 17 January 2013 Accepted 20 February 2013 Available online xxx Keywords: Cyanophycin Lactic acid bacteria Recombinant DNA Biopolymer Culture condition
a b s t r a c t Cyanophycin is a natural source of polypetide consisting of aspartic acid as a backbone and arginine as its side chain. After the removal of arginine, the remaining poly-aspartate can be served in numerous industrial and biomedical applications. The synthesis of cyanophycin is catalyzed by cyanophycin synthetase. In this study, we used lactic acid bacteria to produce cyanophycin by nisin-controlled gene expression system (NICE). The cyanophycin synthetase gene cphA of Synechocystis sp. strain PCC6803 was cloned to the vector pNZ8149 followed by transformation into Lactococcus lactis subsp. cremoris NZ3900. The effects of nisin concentrations and the amounts of supplemented aspartic acid and arginine were examined for the production of cyanophycin. Alterations of the terminus of cphA gene were also conducted in an attempt to increase the yield of cyanophycin. An optimal cyanophycin production was noted under a culture condition of log phase induced at 250 ng/mL nisin in M17L medium supplemented with 20 mM arginine and 10 mM aspartic acid. An insertion of glycine residue at the C terminus of cyanophycin synthetase resulted in a yield of 20% of dry cell weight, a 10-fold increase when compared with the wild type. The results showed that recombinant lactic acid bacteria, a GRAS system, could provide an alternative approach of producing cyanophycin suitable for agricultural and biomedical applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Cyanophycin exits as non-membrane granules mostly inside cyanobacteria [1]. The structure of cyanophycin is a linear peptide chain of aspartic acid of which -carboxylic group links to arginine [1,2]. Cyanophycin functions as an intracellular energy reservoir within cyanobacteria under nutrient limitations [3–5]. The synthesis of cyanophycin can be directed by cyanophycin synthetase without the participation of ribosomes [6–8]. The gene coding cyanophycin synthetase, cphA, was identified in 44 prokaryotes by genetic analysis over the genomic sequences of 570 strains [9]. Among them, different cphA genes have been cloned from Anabaena variabilis, Synechoncystis sp., and Acinetobacter baylyi, and expressed in various organisms, such as Escherichia coli [7,10–12], Saccharomyces cerevisiae [13], and Pichia pastoris [14], Corynebacterium glutamicum, Ralstonia, Eutropha, Pseudomonas putida [15], and plants [16–18].
∗ Corresponding author at: Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Road, Taipei 106, Taiwan. Tel.: +886 2 2730 1078; fax: +886 2 2462 2586. E-mail address: [email protected] (W.-C. Tseng).
Cyanophycin has soluble and insoluble forms based on its aqueous solubility. The cyanophycin from cyanobacteria is insoluble in the aqueous condition of physiological pH 7, and is composed of equal moles of aspartic acid and arginine [19,20]. Some recombinant strains can produce at the same time both insoluble and soluble forms of cyanophycin [13,21], the latter of which is soluble in aqueous solution. The recombinant cyanophycin generally contains lysine incorporated into the side chain to substitute arginine [22,23]. Several applications of cyanophycin have been proposed [24]. One is to cleave off the arginine side chains. The remaining polyaspartic backbone resembles poly-aspartate that could be prepared with organic synthesis and has been widely applied in industrial, biomedical, and agricultural areas [25]. Cyanophycin can also be treated by cyanophycinase to obtain peptides which could be further employed as nutritional additives [26]. Because of the low yield of cyanobacteria in nature, recombinant strains will become the major sources of cyanophycin. In order to meet the criteria for biomedical and agricultural applications, the gram positive hosts are favored without the concern of residual endotoxin after purification. In this study, we used lactic acid bacteria [27] as the host which protein expression is regulated under nisin, a Generally Recognized As Safe (GRAS) system, for the
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cyanophycin production. The effects of nisin concentrations and the amounts of supplemented aspartic acid and arginine were examined. Alterations of the terminus of cphA gene were also conducted in an attempt to increase the yield. 2. Materials and methods 2.1. Materials Bacto yeast extract, tryptone, and agar were from BectonDickinson (Franklin Lakes, NJ). Lactococcus lactis subsp. cremoris NZ3900 and pNZ8149 plasmid were obtained from Mobitec (Göettingen, Germany). PfuTurbo DNA polymerase was purchased from Invitrogen (Carlsbad, CA). Restriction enzymes were supplied by Promega (Madison, WI). Phenylisothiocyanate (PITC) and amino acid standards were from Pierce (Rockford, IL). Other chemicals were purchased from Sigma–Aldrich (St. Louis, MO) and were used as received. Water was deionized by a Milli-Q water purification system (Bedford, MA). 2.2. Restriction-free cloning of the gene coding for cyanophycin synthetase The cphA gene coding for cyanophycin synthetase was cloned into the pNZ8149 vector from a previously constructed plasmid pET-21b-cphA [21] by a restriction-free cloning method using two stages of polymerase chain reaction (PCR) [28]. Primers were designed based on the sequence of pNZ8149 vector and the slr2002 sequence of Synechocystis sp. PCC 6803 which can be accessed from the GenBank by accession number NC 000911 REGION: 1448060.1450681. The primer sequences are as follows: forward primer: 5 -AAA TTA TAA GGA GGC ACT CAC CAT GAA AAT TCT TAA AAC CCT TAC (45 mer); reverse primer: 5 -TAG AAC TAG TGG TAC CGC ATG CCT GCA GTT AAC CAA TGG GTT TAC G-3 (46 mer). The following PCRs were performed by using a TGradient PCR system (Biometra, Göettingen, Germany). The first stage PCR was carried out in 25 L of reaction mixture containing plasmid pET-21b-cphA, forward and reverse primers, dNTP, PfuTurbo DNA polymerase, and PfuTurbo DNA polymerase buffer, and was performed according to the following conditions in sequence: 95 ◦ C for 5 min, 30 cycles of amplification consisting of 1 min at 95 ◦ C, 1 min at 55 ◦ C, and 5 min at 68 ◦ C, and a final extension at 68 ◦ C for 10 min. Then, the second stage PCR was carried out in another 25 L of reaction mixture containing the same components as in the first stage PCR except that the template was replaced by pNZ8149 vector and the primers were replaced by adding the purified PCR products from the first stage (containing the cphA gene), and was performed according to the following conditions in sequence: 95 ◦ C for 5 min, 35 cycles of amplification consisting of 1 min at 95 ◦ C and 15 min at 68 ◦ C, and a final extension at 68 ◦ C for 10 min. The mixture after two PCR stages was Dpn I digested to cleave the pNZ8149 vector, and then was used to transform into competent L. lactis subsp. cremoris NZ3900 (denoted as L. lactis). The resulting recombinant vector was designated as pNZ8149-cphA. The sequence of the entire gene coding for cyanophycin synthetase was confirmed by DNA sequencing which was carried out by Mission Biotech Corp (Taipei, Taiwan). 2.3. Site-directed mutagenesis The cphA gene in pNZ8149-cphA vector was mutated by polymerase chain reaction according to a megaprimed and ligase-free PCR-based site-directed mutagenesis method [29]. The designed mutations were included in the mutagenic primers which have 42–57 bases individually. One universal primer in the forward direction was used together with each mutagenic primer in the reverse direction to produce the mutation-containing
megaprimers. The sequence of the universal primer is 5 -A AAT TAT AAG GAG GCA CTC ACC ATG AAA ATT CTT AAA ACC CTT AC3 , and the sequences of the mutagenic primers were as follows: D1, 5 -GT GGT ACC GCA TGC CTG CAG TTA AAT GGG TTT ACG GGC TTT AAT TAA G-3 , D2: 5 -GT GGT ACC GCA TGC CTG CAG TTA GGG TTT ACG GGC TTT AAT TAA G-3 , D3: 5 -GT GGT ACC GCA TGC CTG CAG TTA TTT ACG GGC TTT AAT TAA G-3 , I1: 5 -GT GGT ACC GCA TGC CTG CAG TTA TCC ACC AAT GGG TTT ACG GGC TTT AAT TAA G-3 , I2: 5 -GT GGT ACC GCA TGC CTG CAG TTA TCC TCC ACC AAT GGG TTT ACG GGC TTT AAT TAA G-3 . The presences of the desired mutations were confirmed by DNA sequencing which was carried out by Mission Biotech Corp (Taipei, Taiwan).
2.4. Production of cyanophycin by recombinant L. lactis For cyanophycin production, the transformed L. lactis was cultured primarily in M17L medium [30] which consists of tryptone 5 g/L, soya peptone 5 g/L, bacteriological peptone 5 g/L, yeast extract 2.5 g/L, ascorbic acid 0.5 g/L, sodium -glycerophosphate 19 g/L, lactose 5 g/L, and 1 mM magnesium sulfate. One single colony from a newly transformed culture plate was inoculated into 10 mL of M17L medium at 30 ◦ C for 8 h, and then 2 mL of the broth was introduced into 48 mL M17L medium at 30 ◦ C for 12 h. After 20 mL of the above broth was inoculated into 480 mL M17L medium, nisin was added to induce the expression of cyanophycin synthetase when OD600 reached around 0.3–0.4. The cells were harvested by centrifugation and stored at −20 ◦ C before further processing. 2.5. Cyanophycin purification The purification of cyanophycin was performed by acid hydrolysis and extraction as previously described [12,13]. Briefly, cell pellets was resuspended at a concentration of 0.3 g wet cell weight/mL in 0.1 N HCl. The suspension was under stirring for 12 h at 40 ◦ C. After centrifugation at 12,500 × g for 30 min, the supernatant was collected, and the pellets were resuspended in 0.1 N HCl and under constant stirring for 6 h at 40 ◦ C. After centrifugation at 12,500 × g for 30 min, the above two portions of supernatants were pooled together and titrated to pH 7 with 2 N NaOH. After centrifugation at 12,500 × g for 30 min, the pellets were insoluble cyanophycin and the supernatant contained soluble cyanophycin. The pellets were washed with 70% ethanol, redissolved in 0.1 N HCl, and then neutralized with 2 N NaOH. The precipitates were isolated by centrifugation followed by drying in vacuum. The soluble cyanophycin was precipitated by an addition of 2-fold volume ethanol. After centrifugation at 12,500 × g for 30 min, soluble cyanophycin were washed with 70% ethanol, redissolved in water, and then precipitated by 2-fold volume of ethanol followed by drying in vacuum.
2.6. Amino acid analysis The amino acid compositions of purified cyanophycin were analyzed by a method as previously described [31]. A weight of 2.5 mg cyanophycin was dissolved in 1 mL of 6 N HCl containing 0.5% phenol. The mixture was hydrolyzed at 108 ◦ C in a dry bath for 24 h under vacuum. An aliquot of 5 L hydrolyzed sample was dried in vacuum and then redissolved in 20 L of solution containing ethanol, triethylamine, and deionized water at a ratio of 2:1:2 followed by drying in vacuum. The dried sample was derivatized with PITC in a mixture of ethanol, triethylamine, deionized water, and PITC at a ratio of 7:1:1:1 for 20 min followed by drying in vacuum. The derivatized mixture was analyzed by a Hitachi HPLC L-2100 system equipped a C-18 column and a UV/VIS detector at 254 nm.
Please cite this article in press as: W.-C. Tseng, et al., Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production, Biochem. Eng. J. (2013), http://dx.doi.org/10.1016/j.bej.2013.02.009
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2.7. SDS polyacrylamide gel electrophoresis A fixed amount of L. lactis broth of 1.0 (OD600 × volume) was centrifuged at 12,500 × g for 5 min followed by an addition of 25 L lysis solution containing 30 mg/mL lysozyme in water. The mixture was incubated in water bath at 30 ◦ C for 10 min, and then was mixed with an equal volume of the loading buffer. The mixture was further heated in boiling water bath for 10 min, and 20 L of the boiled mixture was applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as previously described [32].
3. Results and discussion 3.1. Cyanophycin production under nisin induction Both nisin concentrations and induction timing were examined for cyanophycin production. Various concentrations of nisin were added to induce the production of cyanophycin at an early stage of log phase at OD600 around 0.3–0.4, about an hour after inoculation. The cells were then harvested at the peak of cell density 7 h after inoculation and subjected to the analysis of SDS–PAGE. The expression of cyanophycin synthetase could be clearly identified at the position corresponding to 95 kDa after nisin induction. The levels of cyanophycin increased as the nisin concentration increased from 0 to 50 ng/mL (Fig. 1A). When the concentration was higher than 250 ng/mL, the levels of cyanophycin became hardly distinguishable from each other on the gel, suggesting that the concentration 250 ng/mL could provide a suitable condition for both cell growth and cyanophycin production. The culture was then induced at a final concentration of 250 ng/mL nisin for different induction time periods which lasted for 3, 5, and 7 h, respectively. After cell harvest, both the soluble and insoluble forms of cyanophycin were obtained after purification. The short period of 3 h induction resulted in a high specific yield of 93 mg/g DCW whereas the longer periods of 5 and 7 h induction had lower specific yields of 67 and 63 mg/g DCW, respectively (Fig. 1B). On the other hand, the period of 5 h induction generated a high volumetric productivity of around 81 mg/L (Fig. 1C). Both a longer period of induction for 7 h and a shorter period of induction for 3 h resulted in an approximate level of productivity around 58–67 mg/L (Fig. 1C). The period of 5 h induction was employed for further studies due to the higher volumetric yield. Most of the purified cyanophycin appeared to the insoluble form irrespective of the induction durations. The soluble form only occupied a small portion, less than 13% of the total cyanophycin (Fig. 1B). Some previous studies have reported different proportions of soluble and insoluble cyanophycin by recombinant strains. For E. coli harboring pET-19b with cphA from Desulfitobacterium hafniense [11], the cyanophycin existed mostly in the soluble form. As for E. coli harboring pET-21b with cphA from Synechocystis sp. strain PCC 6803 [21], the cyanophycin appeared to be a mixture of soluble form and insoluble form at a ratio of 2. But for E. coli harboring pPICHOLI-3 with cphA from Synechocystis sp. strain PCC 6308, almost no soluble form of cyanophycin could be found in the product [14]. Some previous studies pointed out that the nitrogen sources of medium and culture conditions could alter the ratio of soluble form to insoluble form [21,33]. In this study, the same cphA gene from Synechocystis sp. strain PCC 6803 was employed, but a minimal amount of soluble form was observed, suggesting that the host might also affect the proportions of soluble and insoluble cyanophycin presumably due to different microbial physiology. A previous report has indicated that the existence of soluble form was related to the employed hosts in spite of the expression of the same cyanophycin synthetase [14].
Fig. 1. Effect of nisin concentrations and induction periods of duration on the cyanophycin synthesis. Panel A shows the cyanophycin production of recombinant L. lactis induced in the presence of various nisin concentrations. Lane M represents molecular weight markers, lane 1 represents the cell lysate before induction, and lanes 2–9 represent the cell extracts under induction at 0, 10, 50, 100, 250, 500, 750, and 1000 ng/mL of nisin, respectively. Different induction periods of 3, 5, and 7 h were further examined for the production of cyanophycin when recombinant L. lactis was induced at 250 ng/mL nisin. Panels B and C show the volumetric yield and specific yield of soluble (white), insoluble (gray) and total cyanophycin (black), respectively (mean ± S.D.; n = three independent experiments).
3.2. SDS–PAGE analysis and amino acid compositions of cyanophycin Unlike the cyanophycin in nature which molecular weight ranges from 20 to 100 kDa, the recombinant cyanophycin has a less dispersed distribution between 15 and 45 kDa depending on the hosts and protein expression systems [23]. The molecular weight distributions of the soluble and insoluble forms were reported to be different. The former generally lies in the range of 14–25 kDa lower than the latter which is in the range of 20–32 kDa [21]. However, the SDS–PAGE analysis indicated that the soluble form had an approximate molecular weight to that of the insoluble form in the range of 22–32 kDa in this study (Fig. 2), suggesting that molecular weight distributions might not be an essential factor to affect the solubility of cyanophycin. Different microbial metabolism of the precursors for the synthesis of cyanophycin might be also related to the modulation of amino acid contents in cyanophycin. In this study, the amino acid
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Fig. 2. Analysis of SDS–PAGE and amino acid compositions of soluble and insoluble forms of cyanophycin. Left panel shows the SDS–PAGE analysis of the soluble form (lane 1) and insoluble form (lane 2) of the purified cyanophycin, and molecular weight standards (lane M). Upper right panel and lower right panel show the corresponding amino acid compositions of soluble form and insoluble form of the purified cyanophycin analyzed by HPLC, respectively.
compositions of insoluble and soluble forms contained low lysine percentages of 1.1% and 10.2%, respectively (Fig. 2). E. coli harboring the same cphA gene produced cyanophycin that contained 8.7% and 25.1% of lysine in the insoluble and soluble forms, respectively. Other reports have showed that a low percentage of 10% lysine could be incorporated into the insoluble cyanophycin [13]. When P. putida was used as a host, citrulline could be incorporated into the side chain as well as lysine [33]. Manipulations on the metabolic pathways confirmed that the metabolism of amino acids affected the amino acid compositions of recombinant cyanophycin [23]. 3.3. Genetic manipulation A previous study reported that alterations on the terminus of cphA gene could affect the productivity of cyanophycin [14]. Five different mutations on the cphA gene were prepared: the cphA genes with three different deletions including one (D1), two (D2), and three (D3) codons removed, respectively, and the cphA genes with two different additions including one (A1) and two (A2) glycine codons added. Three of those mutations, D1, D2, and A2, showed a specific yield approximate to that of wild type (Fig. 3). The mutation of deleting three codons (D3) decreased the specific yield from 67 to 56 mg/g DCW whereas the mutation of adding one glycine codon increased the specific yield from 67 to 113 mg/g DCW (Fig. 3). Most of the cyanophycin still existed in the insoluble form, and the amounts of soluble form were hardly affected by the genetic alterations of deleting codons from or adding codons onto the cphA gene. The crystal structure of cyanophycin synthetase has not been revealed. However, the structure might putatively include two active domains [7,9,34]. By homology analysis, one is located close to the N-terminus, and might participate in the ATP/ADP binding in a way similar to the ATP dependent carboxylate amine/thiol ligase [9]. The other domain close to the C-terminus contains four conserved regions similar to a peptide ligase superfamily, such as murein ligase [9]. A 2.5-fold increase in the productivity has been demonstrated in recombinant S. cerevisiae carrying the cphA gene
from Synechocystis sp. Strain PC6308 by way of deleting one codon plus a point mutation of C595S [14]. In this study, only the addition of one glycine codon resulted in an increase in the specific yield, and the deletions of genes failed to produce positive effects. The reasons for the differences still remained unclear because different sources of cphA genes and different hosts were employed. Different yields of cyanophycin were noted in some commonly employed expression systems, such as E. coli, S. cerevisiae, and P. pastoris. The specific yields were 260, 250, and 345 mg/g DCW for E. coli harboring cphA gene from Synechocytis sp. PCC6308 [10], Synechocytis sp. MA19 [35], and Nostoc ellipsosporum [36], respectively. The same cphA gene from Synechocytis sp. PCC6308 resulted in 69 and 233 mg/g DCW when being expressed in S. cerevisiae [13]
Fig. 3. Effect of genetic manipulation on cphA gene. Recombinant L. lactis harboring either a mutated cphA gene or a wild-type gene (wt) was employed for cyanophycin production. The mutated cphA gene included the deletions of one (D1), two (D2), or three (D3) codons from the terminal sequence, or the additions of one (A1) or two (A2) glycine codons onto the terminal sequence. The specific yields of soluble, insoluble and total cyanophycin are labeled with white, gray, and black, respectively (mean ± S.D.; n = three independent experiments).
Please cite this article in press as: W.-C. Tseng, et al., Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production, Biochem. Eng. J. (2013), http://dx.doi.org/10.1016/j.bej.2013.02.009
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Fig. 4. Effect of additional amino acids on cyanophycin yield. Recombinant L. lactis harboring the wild-type cphA gene was grown in M17L medium (white) and M17L medium containing additional 10 mM aspartic acid plus 10 (light gray) or 20 mM (gray) arginine. Recombinant L. lactis harboring the mutated cphA gene with an extra glycine codon was grown in M17L medium containing additional 10 mM aspartic acid plus 10 (dark gray) or 20 mM (black) (mean ± S.D.; n = three independent experiments).
and P. pastoris [14], respectively. Supplementing amino acids to the medium was carried out in an attempt to examine the productivity and proportions of soluble and insoluble cyanophycin. A low concentration of 10 mM aspartic acid plus 10 mM arginine were sufficient to enhance the yields from 26 to 67 mg/g DCW and to 113 mg/g DCW for the wild type and the A1 mutant, respectively (Fig. 4). When the additional concentration of arginine was doubled to 20 mM, the yield could increase to 132 and 204 mg/g DCW for the wild type and the A1 mutant, respectively. The supplementation of amino acids significantly improved the yield of insoluble form but not the soluble form (Fig. 4). Thus, the ratio of soluble form to insoluble form changed from 0.11 to 0.05 for the wild type grown in the presence of an additional 10 mM arginine. A previous report showed that additional arginine could alter the ratio of insoluble and soluble forms [21]. The similar effect was also observed in this study in spite of different microbial physiology between the hosts. 4. Conclusions In this study, a GRAS system using recombinant lactic acid bacteria was established for the production of cyanophycin suitable for biomedical and agricultural applications. A most favorable induction condition was found to grow the cells in the presence of 250 ng/mL nisin for 5 h after the cell density reached 0.3–0.4. Cyanophycin purified from the harvested cells contained mostly the insoluble form with a low content of lysine. Genetic manipulations on the cphA gene could enhance the yield. The addition of one glycine codon onto the terminus of cphA gene was able to enhance the yield to 204 mg/g DCW, a 10-fold increase as compared with the wild type when the mutant was cultured in the presence of M17L medium supplemented 10 mM aspartic acid and 20 mM arginine. Acknowledgement This research was supported by grant NSC-100-2221-E-011012-MY3 from the National Science Council at Taiwan. References [1] R.D. Simon, P. Weathers, Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in Cyanobacteria, Biochim. Biophys. Acta 420 (1976) 165–176.
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Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production Wen-Chi Tseng a,∗ , Tsuei-Yun Fang b,c , Kai-Chun Chang b , Chorng-Liang Pan b,c a
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10617, Taiwan Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan c Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan b
a r t i c l e
i n f o
Article history: Received 26 September 2012 Received in revised form 17 January 2013 Accepted 20 February 2013 Available online xxx Keywords: Cyanophycin Lactic acid bacteria Recombinant DNA Biopolymer Culture condition
a b s t r a c t Cyanophycin is a natural source of polypetide consisting of aspartic acid as a backbone and arginine as its side chain. After the removal of arginine, the remaining poly-aspartate can be served in numerous industrial and biomedical applications. The synthesis of cyanophycin is catalyzed by cyanophycin synthetase. In this study, we used lactic acid bacteria to produce cyanophycin by nisin-controlled gene expression system (NICE). The cyanophycin synthetase gene cphA of Synechocystis sp. strain PCC6803 was cloned to the vector pNZ8149 followed by transformation into Lactococcus lactis subsp. cremoris NZ3900. The effects of nisin concentrations and the amounts of supplemented aspartic acid and arginine were examined for the production of cyanophycin. Alterations of the terminus of cphA gene were also conducted in an attempt to increase the yield of cyanophycin. An optimal cyanophycin production was noted under a culture condition of log phase induced at 250 ng/mL nisin in M17L medium supplemented with 20 mM arginine and 10 mM aspartic acid. An insertion of glycine residue at the C terminus of cyanophycin synthetase resulted in a yield of 20% of dry cell weight, a 10-fold increase when compared with the wild type. The results showed that recombinant lactic acid bacteria, a GRAS system, could provide an alternative approach of producing cyanophycin suitable for agricultural and biomedical applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Cyanophycin exits as non-membrane granules mostly inside cyanobacteria [1]. The structure of cyanophycin is a linear peptide chain of aspartic acid of which -carboxylic group links to arginine [1,2]. Cyanophycin functions as an intracellular energy reservoir within cyanobacteria under nutrient limitations [3–5]. The synthesis of cyanophycin can be directed by cyanophycin synthetase without the participation of ribosomes [6–8]. The gene coding cyanophycin synthetase, cphA, was identified in 44 prokaryotes by genetic analysis over the genomic sequences of 570 strains [9]. Among them, different cphA genes have been cloned from Anabaena variabilis, Synechoncystis sp., and Acinetobacter baylyi, and expressed in various organisms, such as Escherichia coli [7,10–12], Saccharomyces cerevisiae [13], and Pichia pastoris [14], Corynebacterium glutamicum, Ralstonia, Eutropha, Pseudomonas putida [15], and plants [16–18].
∗ Corresponding author at: Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Road, Taipei 106, Taiwan. Tel.: +886 2 2730 1078; fax: +886 2 2462 2586. E-mail address: [email protected] (W.-C. Tseng).
Cyanophycin has soluble and insoluble forms based on its aqueous solubility. The cyanophycin from cyanobacteria is insoluble in the aqueous condition of physiological pH 7, and is composed of equal moles of aspartic acid and arginine [19,20]. Some recombinant strains can produce at the same time both insoluble and soluble forms of cyanophycin [13,21], the latter of which is soluble in aqueous solution. The recombinant cyanophycin generally contains lysine incorporated into the side chain to substitute arginine [22,23]. Several applications of cyanophycin have been proposed [24]. One is to cleave off the arginine side chains. The remaining polyaspartic backbone resembles poly-aspartate that could be prepared with organic synthesis and has been widely applied in industrial, biomedical, and agricultural areas [25]. Cyanophycin can also be treated by cyanophycinase to obtain peptides which could be further employed as nutritional additives [26]. Because of the low yield of cyanobacteria in nature, recombinant strains will become the major sources of cyanophycin. In order to meet the criteria for biomedical and agricultural applications, the gram positive hosts are favored without the concern of residual endotoxin after purification. In this study, we used lactic acid bacteria [27] as the host which protein expression is regulated under nisin, a Generally Recognized As Safe (GRAS) system, for the
1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.02.009
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cyanophycin production. The effects of nisin concentrations and the amounts of supplemented aspartic acid and arginine were examined. Alterations of the terminus of cphA gene were also conducted in an attempt to increase the yield. 2. Materials and methods 2.1. Materials Bacto yeast extract, tryptone, and agar were from BectonDickinson (Franklin Lakes, NJ). Lactococcus lactis subsp. cremoris NZ3900 and pNZ8149 plasmid were obtained from Mobitec (Göettingen, Germany). PfuTurbo DNA polymerase was purchased from Invitrogen (Carlsbad, CA). Restriction enzymes were supplied by Promega (Madison, WI). Phenylisothiocyanate (PITC) and amino acid standards were from Pierce (Rockford, IL). Other chemicals were purchased from Sigma–Aldrich (St. Louis, MO) and were used as received. Water was deionized by a Milli-Q water purification system (Bedford, MA). 2.2. Restriction-free cloning of the gene coding for cyanophycin synthetase The cphA gene coding for cyanophycin synthetase was cloned into the pNZ8149 vector from a previously constructed plasmid pET-21b-cphA [21] by a restriction-free cloning method using two stages of polymerase chain reaction (PCR) [28]. Primers were designed based on the sequence of pNZ8149 vector and the slr2002 sequence of Synechocystis sp. PCC 6803 which can be accessed from the GenBank by accession number NC 000911 REGION: 1448060.1450681. The primer sequences are as follows: forward primer: 5 -AAA TTA TAA GGA GGC ACT CAC CAT GAA AAT TCT TAA AAC CCT TAC (45 mer); reverse primer: 5 -TAG AAC TAG TGG TAC CGC ATG CCT GCA GTT AAC CAA TGG GTT TAC G-3 (46 mer). The following PCRs were performed by using a TGradient PCR system (Biometra, Göettingen, Germany). The first stage PCR was carried out in 25 L of reaction mixture containing plasmid pET-21b-cphA, forward and reverse primers, dNTP, PfuTurbo DNA polymerase, and PfuTurbo DNA polymerase buffer, and was performed according to the following conditions in sequence: 95 ◦ C for 5 min, 30 cycles of amplification consisting of 1 min at 95 ◦ C, 1 min at 55 ◦ C, and 5 min at 68 ◦ C, and a final extension at 68 ◦ C for 10 min. Then, the second stage PCR was carried out in another 25 L of reaction mixture containing the same components as in the first stage PCR except that the template was replaced by pNZ8149 vector and the primers were replaced by adding the purified PCR products from the first stage (containing the cphA gene), and was performed according to the following conditions in sequence: 95 ◦ C for 5 min, 35 cycles of amplification consisting of 1 min at 95 ◦ C and 15 min at 68 ◦ C, and a final extension at 68 ◦ C for 10 min. The mixture after two PCR stages was Dpn I digested to cleave the pNZ8149 vector, and then was used to transform into competent L. lactis subsp. cremoris NZ3900 (denoted as L. lactis). The resulting recombinant vector was designated as pNZ8149-cphA. The sequence of the entire gene coding for cyanophycin synthetase was confirmed by DNA sequencing which was carried out by Mission Biotech Corp (Taipei, Taiwan). 2.3. Site-directed mutagenesis The cphA gene in pNZ8149-cphA vector was mutated by polymerase chain reaction according to a megaprimed and ligase-free PCR-based site-directed mutagenesis method [29]. The designed mutations were included in the mutagenic primers which have 42–57 bases individually. One universal primer in the forward direction was used together with each mutagenic primer in the reverse direction to produce the mutation-containing
megaprimers. The sequence of the universal primer is 5 -A AAT TAT AAG GAG GCA CTC ACC ATG AAA ATT CTT AAA ACC CTT AC3 , and the sequences of the mutagenic primers were as follows: D1, 5 -GT GGT ACC GCA TGC CTG CAG TTA AAT GGG TTT ACG GGC TTT AAT TAA G-3 , D2: 5 -GT GGT ACC GCA TGC CTG CAG TTA GGG TTT ACG GGC TTT AAT TAA G-3 , D3: 5 -GT GGT ACC GCA TGC CTG CAG TTA TTT ACG GGC TTT AAT TAA G-3 , I1: 5 -GT GGT ACC GCA TGC CTG CAG TTA TCC ACC AAT GGG TTT ACG GGC TTT AAT TAA G-3 , I2: 5 -GT GGT ACC GCA TGC CTG CAG TTA TCC TCC ACC AAT GGG TTT ACG GGC TTT AAT TAA G-3 . The presences of the desired mutations were confirmed by DNA sequencing which was carried out by Mission Biotech Corp (Taipei, Taiwan).
2.4. Production of cyanophycin by recombinant L. lactis For cyanophycin production, the transformed L. lactis was cultured primarily in M17L medium [30] which consists of tryptone 5 g/L, soya peptone 5 g/L, bacteriological peptone 5 g/L, yeast extract 2.5 g/L, ascorbic acid 0.5 g/L, sodium -glycerophosphate 19 g/L, lactose 5 g/L, and 1 mM magnesium sulfate. One single colony from a newly transformed culture plate was inoculated into 10 mL of M17L medium at 30 ◦ C for 8 h, and then 2 mL of the broth was introduced into 48 mL M17L medium at 30 ◦ C for 12 h. After 20 mL of the above broth was inoculated into 480 mL M17L medium, nisin was added to induce the expression of cyanophycin synthetase when OD600 reached around 0.3–0.4. The cells were harvested by centrifugation and stored at −20 ◦ C before further processing. 2.5. Cyanophycin purification The purification of cyanophycin was performed by acid hydrolysis and extraction as previously described [12,13]. Briefly, cell pellets was resuspended at a concentration of 0.3 g wet cell weight/mL in 0.1 N HCl. The suspension was under stirring for 12 h at 40 ◦ C. After centrifugation at 12,500 × g for 30 min, the supernatant was collected, and the pellets were resuspended in 0.1 N HCl and under constant stirring for 6 h at 40 ◦ C. After centrifugation at 12,500 × g for 30 min, the above two portions of supernatants were pooled together and titrated to pH 7 with 2 N NaOH. After centrifugation at 12,500 × g for 30 min, the pellets were insoluble cyanophycin and the supernatant contained soluble cyanophycin. The pellets were washed with 70% ethanol, redissolved in 0.1 N HCl, and then neutralized with 2 N NaOH. The precipitates were isolated by centrifugation followed by drying in vacuum. The soluble cyanophycin was precipitated by an addition of 2-fold volume ethanol. After centrifugation at 12,500 × g for 30 min, soluble cyanophycin were washed with 70% ethanol, redissolved in water, and then precipitated by 2-fold volume of ethanol followed by drying in vacuum.
2.6. Amino acid analysis The amino acid compositions of purified cyanophycin were analyzed by a method as previously described [31]. A weight of 2.5 mg cyanophycin was dissolved in 1 mL of 6 N HCl containing 0.5% phenol. The mixture was hydrolyzed at 108 ◦ C in a dry bath for 24 h under vacuum. An aliquot of 5 L hydrolyzed sample was dried in vacuum and then redissolved in 20 L of solution containing ethanol, triethylamine, and deionized water at a ratio of 2:1:2 followed by drying in vacuum. The dried sample was derivatized with PITC in a mixture of ethanol, triethylamine, deionized water, and PITC at a ratio of 7:1:1:1 for 20 min followed by drying in vacuum. The derivatized mixture was analyzed by a Hitachi HPLC L-2100 system equipped a C-18 column and a UV/VIS detector at 254 nm.
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2.7. SDS polyacrylamide gel electrophoresis A fixed amount of L. lactis broth of 1.0 (OD600 × volume) was centrifuged at 12,500 × g for 5 min followed by an addition of 25 L lysis solution containing 30 mg/mL lysozyme in water. The mixture was incubated in water bath at 30 ◦ C for 10 min, and then was mixed with an equal volume of the loading buffer. The mixture was further heated in boiling water bath for 10 min, and 20 L of the boiled mixture was applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as previously described [32].
3. Results and discussion 3.1. Cyanophycin production under nisin induction Both nisin concentrations and induction timing were examined for cyanophycin production. Various concentrations of nisin were added to induce the production of cyanophycin at an early stage of log phase at OD600 around 0.3–0.4, about an hour after inoculation. The cells were then harvested at the peak of cell density 7 h after inoculation and subjected to the analysis of SDS–PAGE. The expression of cyanophycin synthetase could be clearly identified at the position corresponding to 95 kDa after nisin induction. The levels of cyanophycin increased as the nisin concentration increased from 0 to 50 ng/mL (Fig. 1A). When the concentration was higher than 250 ng/mL, the levels of cyanophycin became hardly distinguishable from each other on the gel, suggesting that the concentration 250 ng/mL could provide a suitable condition for both cell growth and cyanophycin production. The culture was then induced at a final concentration of 250 ng/mL nisin for different induction time periods which lasted for 3, 5, and 7 h, respectively. After cell harvest, both the soluble and insoluble forms of cyanophycin were obtained after purification. The short period of 3 h induction resulted in a high specific yield of 93 mg/g DCW whereas the longer periods of 5 and 7 h induction had lower specific yields of 67 and 63 mg/g DCW, respectively (Fig. 1B). On the other hand, the period of 5 h induction generated a high volumetric productivity of around 81 mg/L (Fig. 1C). Both a longer period of induction for 7 h and a shorter period of induction for 3 h resulted in an approximate level of productivity around 58–67 mg/L (Fig. 1C). The period of 5 h induction was employed for further studies due to the higher volumetric yield. Most of the purified cyanophycin appeared to the insoluble form irrespective of the induction durations. The soluble form only occupied a small portion, less than 13% of the total cyanophycin (Fig. 1B). Some previous studies have reported different proportions of soluble and insoluble cyanophycin by recombinant strains. For E. coli harboring pET-19b with cphA from Desulfitobacterium hafniense [11], the cyanophycin existed mostly in the soluble form. As for E. coli harboring pET-21b with cphA from Synechocystis sp. strain PCC 6803 [21], the cyanophycin appeared to be a mixture of soluble form and insoluble form at a ratio of 2. But for E. coli harboring pPICHOLI-3 with cphA from Synechocystis sp. strain PCC 6308, almost no soluble form of cyanophycin could be found in the product [14]. Some previous studies pointed out that the nitrogen sources of medium and culture conditions could alter the ratio of soluble form to insoluble form [21,33]. In this study, the same cphA gene from Synechocystis sp. strain PCC 6803 was employed, but a minimal amount of soluble form was observed, suggesting that the host might also affect the proportions of soluble and insoluble cyanophycin presumably due to different microbial physiology. A previous report has indicated that the existence of soluble form was related to the employed hosts in spite of the expression of the same cyanophycin synthetase [14].
Fig. 1. Effect of nisin concentrations and induction periods of duration on the cyanophycin synthesis. Panel A shows the cyanophycin production of recombinant L. lactis induced in the presence of various nisin concentrations. Lane M represents molecular weight markers, lane 1 represents the cell lysate before induction, and lanes 2–9 represent the cell extracts under induction at 0, 10, 50, 100, 250, 500, 750, and 1000 ng/mL of nisin, respectively. Different induction periods of 3, 5, and 7 h were further examined for the production of cyanophycin when recombinant L. lactis was induced at 250 ng/mL nisin. Panels B and C show the volumetric yield and specific yield of soluble (white), insoluble (gray) and total cyanophycin (black), respectively (mean ± S.D.; n = three independent experiments).
3.2. SDS–PAGE analysis and amino acid compositions of cyanophycin Unlike the cyanophycin in nature which molecular weight ranges from 20 to 100 kDa, the recombinant cyanophycin has a less dispersed distribution between 15 and 45 kDa depending on the hosts and protein expression systems [23]. The molecular weight distributions of the soluble and insoluble forms were reported to be different. The former generally lies in the range of 14–25 kDa lower than the latter which is in the range of 20–32 kDa [21]. However, the SDS–PAGE analysis indicated that the soluble form had an approximate molecular weight to that of the insoluble form in the range of 22–32 kDa in this study (Fig. 2), suggesting that molecular weight distributions might not be an essential factor to affect the solubility of cyanophycin. Different microbial metabolism of the precursors for the synthesis of cyanophycin might be also related to the modulation of amino acid contents in cyanophycin. In this study, the amino acid
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Fig. 2. Analysis of SDS–PAGE and amino acid compositions of soluble and insoluble forms of cyanophycin. Left panel shows the SDS–PAGE analysis of the soluble form (lane 1) and insoluble form (lane 2) of the purified cyanophycin, and molecular weight standards (lane M). Upper right panel and lower right panel show the corresponding amino acid compositions of soluble form and insoluble form of the purified cyanophycin analyzed by HPLC, respectively.
compositions of insoluble and soluble forms contained low lysine percentages of 1.1% and 10.2%, respectively (Fig. 2). E. coli harboring the same cphA gene produced cyanophycin that contained 8.7% and 25.1% of lysine in the insoluble and soluble forms, respectively. Other reports have showed that a low percentage of 10% lysine could be incorporated into the insoluble cyanophycin [13]. When P. putida was used as a host, citrulline could be incorporated into the side chain as well as lysine [33]. Manipulations on the metabolic pathways confirmed that the metabolism of amino acids affected the amino acid compositions of recombinant cyanophycin [23]. 3.3. Genetic manipulation A previous study reported that alterations on the terminus of cphA gene could affect the productivity of cyanophycin [14]. Five different mutations on the cphA gene were prepared: the cphA genes with three different deletions including one (D1), two (D2), and three (D3) codons removed, respectively, and the cphA genes with two different additions including one (A1) and two (A2) glycine codons added. Three of those mutations, D1, D2, and A2, showed a specific yield approximate to that of wild type (Fig. 3). The mutation of deleting three codons (D3) decreased the specific yield from 67 to 56 mg/g DCW whereas the mutation of adding one glycine codon increased the specific yield from 67 to 113 mg/g DCW (Fig. 3). Most of the cyanophycin still existed in the insoluble form, and the amounts of soluble form were hardly affected by the genetic alterations of deleting codons from or adding codons onto the cphA gene. The crystal structure of cyanophycin synthetase has not been revealed. However, the structure might putatively include two active domains [7,9,34]. By homology analysis, one is located close to the N-terminus, and might participate in the ATP/ADP binding in a way similar to the ATP dependent carboxylate amine/thiol ligase [9]. The other domain close to the C-terminus contains four conserved regions similar to a peptide ligase superfamily, such as murein ligase [9]. A 2.5-fold increase in the productivity has been demonstrated in recombinant S. cerevisiae carrying the cphA gene
from Synechocystis sp. Strain PC6308 by way of deleting one codon plus a point mutation of C595S [14]. In this study, only the addition of one glycine codon resulted in an increase in the specific yield, and the deletions of genes failed to produce positive effects. The reasons for the differences still remained unclear because different sources of cphA genes and different hosts were employed. Different yields of cyanophycin were noted in some commonly employed expression systems, such as E. coli, S. cerevisiae, and P. pastoris. The specific yields were 260, 250, and 345 mg/g DCW for E. coli harboring cphA gene from Synechocytis sp. PCC6308 [10], Synechocytis sp. MA19 [35], and Nostoc ellipsosporum [36], respectively. The same cphA gene from Synechocytis sp. PCC6308 resulted in 69 and 233 mg/g DCW when being expressed in S. cerevisiae [13]
Fig. 3. Effect of genetic manipulation on cphA gene. Recombinant L. lactis harboring either a mutated cphA gene or a wild-type gene (wt) was employed for cyanophycin production. The mutated cphA gene included the deletions of one (D1), two (D2), or three (D3) codons from the terminal sequence, or the additions of one (A1) or two (A2) glycine codons onto the terminal sequence. The specific yields of soluble, insoluble and total cyanophycin are labeled with white, gray, and black, respectively (mean ± S.D.; n = three independent experiments).
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Fig. 4. Effect of additional amino acids on cyanophycin yield. Recombinant L. lactis harboring the wild-type cphA gene was grown in M17L medium (white) and M17L medium containing additional 10 mM aspartic acid plus 10 (light gray) or 20 mM (gray) arginine. Recombinant L. lactis harboring the mutated cphA gene with an extra glycine codon was grown in M17L medium containing additional 10 mM aspartic acid plus 10 (dark gray) or 20 mM (black) (mean ± S.D.; n = three independent experiments).
and P. pastoris [14], respectively. Supplementing amino acids to the medium was carried out in an attempt to examine the productivity and proportions of soluble and insoluble cyanophycin. A low concentration of 10 mM aspartic acid plus 10 mM arginine were sufficient to enhance the yields from 26 to 67 mg/g DCW and to 113 mg/g DCW for the wild type and the A1 mutant, respectively (Fig. 4). When the additional concentration of arginine was doubled to 20 mM, the yield could increase to 132 and 204 mg/g DCW for the wild type and the A1 mutant, respectively. The supplementation of amino acids significantly improved the yield of insoluble form but not the soluble form (Fig. 4). Thus, the ratio of soluble form to insoluble form changed from 0.11 to 0.05 for the wild type grown in the presence of an additional 10 mM arginine. A previous report showed that additional arginine could alter the ratio of insoluble and soluble forms [21]. The similar effect was also observed in this study in spite of different microbial physiology between the hosts. 4. Conclusions In this study, a GRAS system using recombinant lactic acid bacteria was established for the production of cyanophycin suitable for biomedical and agricultural applications. A most favorable induction condition was found to grow the cells in the presence of 250 ng/mL nisin for 5 h after the cell density reached 0.3–0.4. Cyanophycin purified from the harvested cells contained mostly the insoluble form with a low content of lysine. Genetic manipulations on the cphA gene could enhance the yield. The addition of one glycine codon onto the terminus of cphA gene was able to enhance the yield to 204 mg/g DCW, a 10-fold increase as compared with the wild type when the mutant was cultured in the presence of M17L medium supplemented 10 mM aspartic acid and 20 mM arginine. Acknowledgement This research was supported by grant NSC-100-2221-E-011012-MY3 from the National Science Council at Taiwan. References [1] R.D. Simon, P. Weathers, Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in Cyanobacteria, Biochim. Biophys. Acta 420 (1976) 165–176.
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Please cite this article in press as: W.-C. Tseng, et al., Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production, Biochem. Eng. J. (2013), http://dx.doi.org/10.1016/j.bej.2013.02.009