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Applied and Environmental Microbiology, December 2006, p. 7652-7660, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01132-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Engineered Cyanophycin Synthetase (CphA) from Nostoc ellipsosporum Confers Enhanced CphA Activity and Cyanophycin Accumulation to Escherichia coli

Tran Hai, Kay M. Frey, and Alexander Steinbüchel^*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany

Received 16 May 2006/ Accepted 25 September 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cyanophycin (CGP) synthetase gene (cphA_NE1) of the transposon-induced argL mutant NE1 of the cyanobacterium Nostoc ellipsosporum, which exhibits a CGP-leaky phenotype during diazotrophical growth, was cloned and expressed in Escherichia coli strain TOP10. Its amino acid sequence exhibited high similarities to CphAs of other cyanobacteria. Recombinant cells of E. coli, which harbored a fragment comprising the complete cphA_NE1 gene plus 400 bp of its downstream region in colinear orientation to the lacZ promoter, accumulated CGP up to 17 and 8.5% (wt/wt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-ß-D-thiogalactopyranoside, respectively. Two truncated CphAs, lacking 31 (CphA_NE1del96) or 59 (CphA_NE1del180) amino acids of the C-terminal region, were derived from cphA_NE1 by deleting 96 or 180 bp from its 3' region through the introduction of stop codons. In comparison to the wild-type gene, cphA_NE1del96 conferred about 2.1- to 2.2-fold-higher enzyme activity (up to 5.75 U/mg protein) on E. coli. Furthermore, these cells accumulated about twofold more CGP (up to 34.5% [wt/wt] of CDM) than cells expressing the wild-type gene. An engineered CphA possessing significantly enhanced activity and conferring the highest CGP content on E. coli is demonstrated. In contrast, CphA_NE1del180 was inactive and did not confer CGP accumulation on E. coli. Interestingly, a short conserved stretch of 4 to 5 hydrophobic amino acids is located in the protein region present in CphA_NE1del96 but absent in CphA_NE1del180. In addition, CphA_NE1 and CphA_NE1del96 are, besides CphA from Acinetobacter baylyi, the only CphAs exhibiting rigid substrate specificities that do not enable the incorporation of lysine instead of arginine into CGP.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cyanophycin as isolated from cyanophycin-accumulating bacteria consists of L-aspartic acid and L-arginine at an equal molar ratio and is referred to as multi-L-arginine-poly-L-aspartic acid, or cyanophycin grana protein (CGP). CGP is a storage compound for energy, nitrogen, and carbon and is deposited in the cytoplasm as insoluble inclusions. The copolymerization of the two amino acids to the polymer is catalyzed via a nonribosomal pathway by cyanophycin synthetases (CphAs). The structural gene (cphA) of this key enzyme of CGP biosynthesis was identified first in the genome of the cyanobacterium Synechocystis sp. strain PCC6803 (22, 51). In that organism, cphA is located downstream of a cyanophycinase gene (cphB) encoding an endocyanophycinase (CphB) that initiates mobilization of this storage compound. Both genes occur frequently in the genomes of cyanobacteria and also in some heterotrophic bacteria and are often clustered (26, 37, 52). Functional active CphAs are composed of two identical subunits, thus representing homodimeric enzyme proteins (51). The presence of two loops involved in ATP binding, which are referred to as the B loop and the J loop, accelerate the subsequent phosphorylation of the - and ß-carboxylic acid groups of the aspartate residues located in carboxy-terminal regions of the growing CGP polymer molecule, according to a recently proposed mechanism (8). Both active sites of the enzyme were localized in the amino-terminal region of CphA; furthermore, five additional conserved stretches with unknown functions were identified in the carboxy-terminal regions of all cyanobacterial CphA proteins (18). The first four conserved regions also exhibited high similarities to those of noncyanobacterial cyanophycin synthetases and to members of the Mur ligase superfamily, including folyl--glutamate ligase (34).

CGP is of biotechnological interest because the purified polymer can be chemically converted into polymers with reduced arginine contents (21), which might be used, like polyaspartic acid, as biodegradable substitutes for synthetic polyacrylate. For polyaspartic acid, various technical processes, such as use in water treatment and paper processing or as a dispergent in paints (3, 42), as well as biomedical applications (31, 50), have been described. Since CGP may serve as a source of polyaspartic acid, industry is interested in the production of CGP at low cost. In most studies with cyanobacteria, the accumulation of CGP was increased by imposing special cultivation conditions, like the addition of L-arginine or chloramphenicol to the medium as a translation inhibitor (43). Depending on the strain and cultivation conditions, the CGP contents of the cells varied only between 2 and 18% (4, 29, 32, 43). Economically feasible production of CGP on a large scale has been impaired by slow and sparse light-dependent growth of cyanobacteria, low cellular CGP contents (17), or high costs of media if recombinant bacteria, such as Escherichia coli expressing a cyanobacterial cphA gene, are used (2). Thus, CGP production at low cost will become feasible only if cheap complex media, such as protamylasse (14), are used for fermentation or if a more active CphA, which yields higher CGP contents of cells or reduces the production time, is used. Furthermore, downstream processes for the isolation of CGP must be further optimized. Production of CGP was optimized in our pilot plant employing recombinant strains of E. coli, Ralstonia eutropha, or Pseudomonas putida (16, 46, 47), and also Acinetobacter baylyi strain ADP1. Maximum CGP yields of about 24 or 46% (wt/wt) were obtained by using P. putida strain GPp104 harboring pBBR1MCS2::cphA1₇₁₂₀ or A. baylyi strain ADP1, respectively, during growth in mineral salts medium with L-arginine feeding (13, 46). Other strategies for low-cost production of CGP rely on metabolically engineered bacteria and, in particular, on hosts with engineered arginine metabolism to improve the provision of arginine to CphA without requiring arginine in the medium as a supplement (15) or on hosts using an addiction system to prevent loss of a cphA-bearing plasmid (47).

The engineering of CphAs is another approach to improve CGP biosynthesis. However, so far, any changes in cphA genes, such as point mutations (for example, K261G and K497A in cphA of Anabaena variabilis) have caused a complete loss of enzyme activity (7). Alignments of enzymes of the CphA superfamily revealed a stretch of about 30 amino acids in the C-terminal regions, which is strain specific, exhibiting only low or no similarities to the homologues. The enzymes from Synechocystis sp. strain PCC6803 (CphA_syn6803) and Cyanothece sp. strain ATCC 51142 are the smallest cyanobacterial CphAs (comprising only 873 and 872 amino acids, respectively) lacking this region. Thus, this stretch of about 30 amino acids in the C-terminal region may not be essential (18). In this study, we cloned and subsequently truncated the carboxy-terminal region of CphA from a mutant of the heterocystous filamentous cyanobacterium Nostoc ellipsosporum strain NE1 and investigated the truncated CphAs with regard to in vitro and in vivo enzyme activities.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, oligonucleotides, and cultivation conditions.
All bacterial strains and plasmids used in this study are listed in Table 1. Nostoc ellipsosporum strain NE1 is a conditional arginine auxotroph with a Tn5-1058-inactivated argL gene, which prevents nitrogen fixation, germination, and impaired cyanophycin formation in heterocysts and akinets without the addition of L-arginine. N. ellipsosporum NE1 was grown in BG11 medium (39) at 28°C with shaking (100 rpm) and irradiation (150 microeinsteins m^–2 s^–1). Strains of E. coli were grown at 37°C in Luria-Bertani (LB) or Terrific Broth (TB) medium (40) with shaking (150 rpm). For cultivation of recombinant E. coli strains, 100 µg/ml ampicillin was added. Isopropyl-ß-D-thiogalactopyranoside (IPTG) (0.2 mM) was added to the cells after 4 h of growth.

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TABLE 1. Bacterial strains and plasmids used in this study

The oligonucleotides used in this study (Table 2) were synthesized by MWG Biotech (Ebersberg, Germany).

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TABLE 2. Oligonucleotides used in this study as primers for PCRs

DNA isolation and manipulation.
Total DNA from N. ellipsosporum NE1 was prepared according to the method described previously (20). Plasmid isolation and DNA purification were done by standard methods (40) and by applying commercial kits (QIAGEN, Hilden, Germany). Restrictions and ligations of DNA molecules were performed using restriction endonucleases and T4 DNA ligase, respectively, according to the manufacturers' instructions.

Inverse PCR to identify cphA from N. ellipsosporum strain NE1.
PCR employing primers P-int sense and P-int antisense, which were derived from the consensus amino acid sequences MGHIVEHV and PFMIANA, respectively, of the cyanophycin synthetases from A. variabilis (51) and Synechocystis sp. strain PCC6803 (22) plus total DNA from N. ellipsosporum strain NE1, which was partially digested with PstI, yielded a 1.87-kbp DNA fragment comprising cphA_NE1 (18). This fragment was ligated to pBluescript SK^–, and the resulting hybrid plasmid was used to transform E. coli TOP10 using the CaCl₂ standard method (40) and sequenced. The entire cphA_NE1 sequence, including the 5' and 3' regions, was obtained by inverse PCR according to the PCR application manual (Boehringer Mannheim, Germany) using primers P1 forward and P2 reverse (Table 2), PstI-digested and religated genomic DNA of N. ellipsosporum strain NE1, Pfx DNA polymerase, and a PCR kit (F. Fermentas, Germany) and yielded a 3.150-kbp fragment (Fig. 1) that was purified with the Nucleotrap^CR kit (Machery & Nagel, Düren, Germany).

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FIG. 1. Cloning, truncation, and characterization of cphA genes.

Generation of truncated N. ellipsosporum strain NE1 cphA genes.
The cphA_NE1 gene was amplified by PCR from genomic DNA by using Pfx polymerase plus the CphANE1 sense primer (Table 2), hybridizing at a distance of 400 bp upstream of the start codon of cphA_NE1 (including a stretch of cphB_NE1 and the intergenic sequence between the stop codon of cphB_NE1 and the start codon of cphA_NE1), and the CphANE1 antisense primer (Table 2), hybridizing at the stop codon, by following the procedure described above. Primers NE1del96 and NE1del180 (Table 2) were deduced from the 3' region of cphA_NE1 and yielded in PCR, together with the CphANE1 sense primer (Table 2), truncated cphA genes lacking 96 (cphA_NE1del96) or 180 (cphA_NE1del180) bp of the C-terminal region of cphA_NE1, respectively. Three PCR products, cphA_NE1 (3.1 kbp), cphA_NE1del96 (3.0 kbp), and cphA_NE1del180 (2.95 kbp), were isolated according to standard protocols, and about 400 ng of the PstI-digested PCR products was ligated with 400 ng of PstI- and EcoRV-digested pBluescript SK^– DNA and used to transform E. coli TOP10. Hybrid plasmids harboring the various cphA genes with coorientation to lacPO were selected (Fig. 1). In addition, PCR products of cphA_NE1 and cphA_NE1del96 were also cloned into EcoRV-digested pBR322 (Table 1).

DNA sequencing, sequence analysis, and alignments.
DNA sequences were determined by employing 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, Little Chalfont, Buckinghamshire, United Kingdom) according to the instructions of the respective manufacturers. The sequences obtained were analyzed with the software Genamics Expression (http://genamics.com/expression/index.htm). Sequence comparisons and alignments were performed using the network service programs BLAST (NCBI) and/or ClustalX (EMBL-EBI [http://www.ebi.ac.uk/]). Alignments were done with the program Genedoc (http://www.psc.edu/biomed/genedoc).

Cyanophycin synthetase assay.
The CphA assay followed the method of Simon (44) with slight modification. A reaction mixture (total volume, 100 µl) containing 50 mM Tris (pH 8.2), 20 mM MgCl₂, 20 mM KCl, 10 mM dithiothreitol, 4 mM ATP, 5 mM L-aspartic acid, 495 µM L-arginine, 5 µM of L-[¹⁴C]arginine (Amersham Pharmacia Biotech, Freiburg, Germany), and cell extract with CphA (100 to 200 µg protein) was used. After 15 min of incubation at 30°C, the enzyme reaction was stopped by dilution with 10 volumes of ice water, and the labeled products were collected by centrifugation (14,000 x g; 20 min; room temperature). The final steps were done according to the method described previously (1). Scintillation counting was carried out in a model LS 6500 scintillation counter (Beckman Instruments GmbH, Munich, Germany).

Cultivation of E. coli at the 30-liter scale.
A Biostat DL30 stainless steel reactor with a total volume of 42 liters (28-cm inner diameter and 71-cm height) and a ratio of the stirrer diameter to the vessel diameter of 0.375 and equipped with three stirrers, each containing six paddles and a Funda-Foam mechanical foam destroyer (B. Braun Biotech, Melsungen, Germany), was used for cultivation at the 30-liter scale. Sensors were used to measure the dissolved O₂ (model 25; Mettler Toledo, Steinbach, Switzerland), pH (model Pa/25; Mettler-Toledo), foam level (model L300/Rd.28; B. Braun Biotech), temperature (pt100 electrode; M. K. Juchheim, Fulda, Germany), and optical density at 850 nm (model CT6; Sentex/Monitek Technology, Hayward, CA). Operations were controlled and recorded by a digital control unit in combination with MFCS/win software (B. Braun Biotech). The CO₂ and O₂ concentrations in the spent gas of the reactor were measured by a URAS 10P NDIR spectrometer or a Magnos 6G O₂ analyzer (Mannesmann, Hartmann & Braun, Frankfurt, Germany), respectively. Cultivations were done at 37°C and within a dissolved O₂ range of 0 to 100% saturation in the medium, employing agitation rates between 100 and 800 rpm and aeration rates between 0.4 and 1.0 volume per volume per minute (vvm). The pH of the medium was kept between 7.0 and 7.2 by adding 4 N HCl or NaOH. The antifoam agent Silikon Antischaum Emulsion SLE (Wacker, Germany) was added only if the mechanical foam destroyer was not sufficient. Samples were withdrawn from the culture for analyses and separated into a cell pellet and a cell-free supernatant by 15 min of centrifugation at 3,500 x g.

Isolation and characterization of CGP.
Cells from the 30-liter-scale fermentations were harvested by centrifugation at 4°C in a CEPA type Z41 continuous centrifuge (Carl Padberg Zentrifugenbau GmbH, Lahr, Germany). CGP was isolated on a small scale from recombinant cells by employing the procedure described by Simon and Weathers (45) for Anabaena cylindrica. The amino acid composition of CphA was analyzed by high-performance liquid chromatography (HPLC) of ortho-phthaldialdehyde derivatives of the amino acids, applying a reversed-phase RP18 techsphere ODS-2 column (0.46 cm by 12.5 cm) as described previously (16). Calibration was done with samples from an amino acid reference kit (Kollektion AS-10 from Serva Feinbiochemica, Heidelberg, Germany).

Putative contaminants in isolated CGP samples were quantified as follows. Quantification of carbohydrate was done by following the colorimetric reaction with Anthrone reagent using glucose as a standard (19). Protein and nucleic acid contents were determined spectrophotometrically at 260 and 280 nm in clear solutions of CGP samples in 100 mM HCl, employing the following equations: (i) protein (mg · ml^–1) = 1.55 x A₂₈₀ – 0.76 x A₂₆₀ (48) and (ii) nucleic acids (mg · ml^–1) = 0.05 x A₂₆₀ (40). The protein contents were determined by utilizing the principle of protein-dye binding (9).

Electrophoretic methods.
Proteins were separated by electrophoresis in 11.5% (wt/vol) sodium dodecyl sulfate (SDS)-polyacrylamide gels according to the method of Laemmli (27). Molecular weight standard proteins were purchased from Amersham Biosciences (Little Chalfont, United Kingdom). Proteins and CGP were stained with Serva Blue R. Nucleic acids were analyzed by electrophoresis in 0.8% and 1.1% (wt/vol) agarose gels according to standard methods (40).

Nucleotide sequence accession number.
The nucleotide sequence of cphA_NE1 and part of the adjacent cphB_NE1 was submitted to GenBank under accession no. DQ168807.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of cphA from N. ellipsosporum NE1.
Employing PstI-digested and religated genomic DNA from N. ellipsosporum strain NE1 and primers P1 forward and P2 reverse, which were deduced from the upstream and downstream regions of the 1,870-bp cphA fragment of the bacterium (18), a 3,150-bp DNA fragment was obtained by inverse PCR (Fig. 1). The entire nucleotide sequence of cphA_NE1, and also part of the adjacent cphB_NE1, was subsequently revealed. Based on this information, the primers CphANE1 sense and CphANE1 antisense were designed, and a 3,120-bp fragment comprising the entire cphA_NE1 gene plus 400 bp of its upstream region was obtained by PCR. This PCR product, which comprised the entire cphA_NE1 gene downstream of and colinear to the lacZ promoter-operator region, part of cphB (239 bp), and the cphB-cphA intergenic region (164 bp), was ligated to pBluescript SK^– and pBR322, yielding pSK::cphA_NE1 and pBR322::cphA_NE1 (Table 1), respectively, and was then used to transform E. coli TOP10.

Heterologous expression of the wild-type cphA_NE1 gene in E. coli.
When cells of recombinant strains of E. coli harboring plasmid pSK::cphA_NE1 were analyzed with respect to heterologous expression, high CphA enzyme activities were measured not only in cells cultivated in the presence (2.60 U/mg protein), but also in those cultivated in the absence (0.85 U/mg protein), of IPTG (Table 3). CGP accumulated by IPTG-induced cells of the recombinant strain contributed up to 17% (wt/wt) of the cell dry matter, whereas the CGP content was much lower in noninduced cells. The CGP isolated from either strain consisted of approximately equimolar amounts of aspartate and arginine, as revealed by HPLC analysis; only trace amounts of lysine (<0.3% [wt/wt]) could be detected. SDS-polyacrylamide gel electrophoresis of the isolated CGP revealed an average molecular mass between 28 and 30 kDa (Fig. 2, lanes 1 and 2) and polydispersities similar to those of CGPs isolated from other recombinant E. coli sources (36, 51). Therefore, the cloned cphA gene encoded a functional active CphA. In comparison to E. coli harboring plasmid pSK::cphA_NE1, cells with pBR322::cphA_NE1 contained only 2.5% (wt/wt) CGP and showed a low but significant specific enzyme activity (Table 3). Therefore, the amplified cphA upstream region (400 bp) contained its own cyanobacterial promoter, which is active in E. coli.

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TABLE 3. Expression of N. ellipsosporum strain NE1 CphA and of truncated derivatives and accumulation of CGP in recombinant cells of E. coli strains

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FIG. 2. SDS-polyacrylamide gel electrophoresis of purified CGP and whole-cell extracts of recombinant E. coli. Lanes 1 and 2, CGP isolated from E. coli Top10 pSK::cphANE1 (lane 1) and E. coli pSKcphANE1del96 (lane 2) purified by the method of Simon and Weathers (45); lanes 3 to 5, cell lysates of E. coli Top10 pBluescript SK– (lane 3), E. coli Top10 pSK::cphANE1 (lane 4), and E. coli pSK::cphANE1del96 (lane 5). The sizes of molecular mass standard proteins (lane Std) are provided on the left. CGP and protein bands were visualized by staining them with Serva Blue R.

Generation of truncated cphA_NE1 genes.
The sequence of CphA_NE1 was aligned to those of the other 12 known CphAs, which were available in the database. The lowest similarity was exhibited to the noncyanobacterial cphA of Acinetobacter baylyi strain ADP1, with 41% identity. With cyanobacterial cphA genes, homologies of 56% for Gloebacter violaceus PCC 7421 to 99% sequence identity for Anabaena sp. strain PCC 7120, in comparison to Nostoc ellisposporum NE1, were found. The homology levels of the C-terminal regions, comprising a stretch of about 30 amino acids, of all CphAs were very low (Fig. 3), indicating that this region is rather variable and strain specific. Only a short stretch consisting of four or five hydrophobic amino acids, like leucine, valine, and isoleucine, at about position 855 was conserved in most CphA proteins. No CphA activity was detectable if deletions or point mutations were introduced in the amino-terminal region of CphA from Anabaena variabilis strain ATCC 29413 (7). Deletion of 200 bp of the 3' cphA_MA19 region of the Synechococcus sp. strain MA19 cphA gene led to an inactive enzyme (18). Based on these observations, we generated truncations in the 5' region close to the stop codon of cphA_NE1 to reveal the influence of the C-terminal region on activity and to eventually obtain CphAs with interesting properties. Two truncated cphA_NE1 DNA fragments were obtained by PCR employing genomic DNA isolated from N. ellipsosporum strain NE1 and the primer CphANE1 sense in combination with the deletion primer NE1del96 or NE1del180, lacking 96 or 180 bp of the 3' region, respectively (Table 2 and Fig. 1). These modified cphA genes encoded truncated CphAs lacking 31 or 59 amino acids, comprising regions with no or little homology to known CphA sequences, respectively. In addition to pBluescript SK^–, pBR322 was also used for the cloning of cphA_NE1del96, yielding pSK::cphA_NE1del96, pSK::cphA_NE1del80, and pBR322::cphA_NE1del96, respectively (Table 1).

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FIG. 3. Alignment of the C-terminal regions of 13 CphA proteins. Sequences: 1, Acinetobacter baylyi strain ADP1 (NCBI accession no. YP_045974 [6]); 2, Anabaena variabilis strain ATCC 29413 (accession no. CAA06440 [51]); 3, Anabaena sp. strain PCC 7120 (accession no. P58572 [23]); 4, Crocosphaera watsonii strain WH 8501 (accession no. ZP_00519191 [12]); 5, Cyanothece sp. strain ATCC 51142 (accession no. Q9KGY4); 6, Gloeobacter violaceus strain PCC 7421 (accession no. NP_927272 [35]); 7, Nostoc ellipsosporum strain NE1 (accession no. DQ168807 [this study]); 7a, N. ellipsosporum strain NE1 deleted to amino acid position 870; 7b, N. ellipsosporum strain NE1 deleted to amino acid position 842; 8, Nostoc punctiforme strain PCC 73102 (accession no. ZP_00107371 [NCBI Microbial Genome Annotation Project]); 9, Synechococcus elongates (accession no. CAC07987 [8]); 10, Synechococcus sp. strain MA19 (accession no. AAL56990 [18]); 11, Synechocystis sp. strain PCC 6308 (accession no. P56947 [1]); 12, Synechocystis sp. strain PCC 6803 (accession no. NP_441210 [22]); 13, Trichodesmium erythraeum strain IMS101 (accession no. ZP_00672194 [11]). The first amino acid shown is amino acid no. 820.

Activity and CGP accumulation in recombinant cells expressing truncated CphAs.
Colonies of the recombinant cells of E. coli TOP10 harboring pSK::cphA_NE1del96 on LB agar medium appeared whiter and exhibited a much higher opacity than colonies of cells expressing the wild-type cphA_NE1 gene (pSK::cphA_NE1), indicating a higher CGP content of the cells. The results of the cultivation experiments, summarized in Table 3, demonstrated that truncation of a stretch of 31 amino acids of the CphA_NE1 C-terminal region starting from the amino acid valine at position 871 accelerated biosynthesis and accumulation of CGP in the recombinant cells. In comparison to the wild-type enzyme, the truncated enzyme gave about 2.2- and 2.1-fold-higher CphA activity in the presence (5.75 U/mg protein) or absence (1.80 U/mg protein) of IPTG, respectively (Table 3). Consistent with this, cells expressing the truncated CphA_NE1del96 accumulated about 2.0- and 1.6-fold-higher concentrations, respectively, of CGP in the cells, amounting to 34.5% (wt/wt) more cell dry matter than cells expressing the wild-type enzyme. CGP isolated from cells expressing the truncated CphA was similar to the polymer synthesized by the wild-type enzyme, with an almost equal molar ratio of aspartic acid and arginine, a lack of lysine, and molecular weight and polydispersity like those revealed for the isolated CGP.

In contrast, recombinant cells of E. coli TOP10 harboring pSK::cphA_NE1del180 did not accumulate any detectable CGP. In addition, no CphA activity was detectable in the cells (Table 3). Therefore, truncation of a 59-amino-acid stretch of the carboxy-terminal region of CphA_NE1 from the amino acid aspartic acid at position 843 onward led to a totally inactive enzyme.

Production of CGP in recombinant E. coli employing cphA_NE1del96 at pilot scale.
Due to the rather low CGP contents of cells harboring the pBR322-based constructs (Table 3), only pSK::cphA_NE1 and pSK::cphA_NE1del96 were used for further experiments. To obtain larger amounts of the polymer for further analysis, E. coli Top10, harboring the wild-type cphA gene from N. ellipsosporum strain NE1 (pSK::cphA_NE1) or the truncated cphA gene (pSK::cphA_NE1del96), was cultivated at the 30-liter scale in TB complex medium. The fermentation was performed at 37°C, the airflow was kept low at 0.6 vvm to avoid foam formation, and the stirrer speed was adjusted in order to avoid an insufficient supply of oxygen during the exponential growth phase. The influence of the expression level on CGP production was investigated. Induction of the lac promoter was done by addition of IPTG in the exponential phase after 4 h of cultivation. The time course of the fermentation with truncated cphA and IPTG induction, which gave the highest CGP content and enzyme activity, is shown in Fig. 4. As can be seen from the time course, formation of CGP occurred mainly in the late exponential and early stationary growth phases. After only 18 h of cultivation, CGP contents of the cells as high as 34% (wt/wt) were obtained. Further cultivation led to a slight decrease in the CGP content. Similar to cultivation in flasks, the induced cultures expressed higher specific CphA activities (5.15 U/mg protein) than the noninduced cultures (4.22 U/mg protein) at the end of fermentation.

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FIG. 4. Batch fermentation of E. coli TOP10 harboring truncated cphANE1del96. The cultivation was done in a 30-liter stirred tank reactor containing 26 liters of TB complex medium. The fermentation parameters were as follows: pH 7.0, 37°C, aeration at 0.6 vvm, and agitations ranging from 100 to 550 rpm. For induction of CphA formation, IPTG was added, as indicated by the arrow. The concentrations of O2 and CO2 were determined in the exhaust gas. The experiment was done in triplicate. Standard deviations of the CGP contents were below 3%.

Isolation and characterization of CGP.
CGP was isolated from lyophilized cells harvested from the broth of the 30-liter-scale fermentation by employing the isolation method of Simon and Weathers (45). The same tendencies in CGP formation as shown for the flask experiments (Table 3) were obtained during upscaling to the 30-liter scale. The highest yield, with CGP at 30% of cell dry mass at the end of fermentation, was found in E. coli harboring pSK::cphA_NE1del96 upon IPTG induction; the lowest yield (11.5% [wt/wt] of cell dry mass) was obtained with E. coli pSK::cphA_NE1 without induction. Analyses of CGP isolated by this procedure revealed only small amounts of DNA, carbohydrates, and protein; all contaminants were in the same low range as in CGP previously obtained from other recombinant E. coli strains (16). The HPLC analysis of amino acid constituents of the isolated CGP revealed only two major amino acids, aspartate and arginine. This was in contrast to CGP produced with other cphA genes in recombinant E. coli, all of which contained in addition significant amounts of lysine (16, 51). The only other known CphA producing lysine-free CGP is the enzyme of A. baylyi strain ADP1 (13, 15).

Top Abstract Introduction Materials and Methods Results Discussion References

 
CphAs are widespread among cyanobacteria (18) and were recently also found in other bacteria (26, 52). All CphA enzymes characterized so far perform the same biochemical reaction: they catalyze the elongation of preexisting CGP primers in the presence of L-aspartate, L-arginine, Mg²⁺ ions, and ATP. Members of this enzyme family are functionally and evolutionarily related and possess a highly conserved primary structure. Sequence analyses revealed particularly high degrees of similarities in two regions of the CphA proteins. The N-terminal to central regions exhibit similarities to carboxylate-amine/thiol ligases, whereas in the central to C-terminal regions, strong similarities to another family of peptide ligases, which includes murein ligases and poly(-glutamate) ligases, were found. Since the argL knockout mutant N. ellipsosporum strain NE1 could grow in BG11 medium supplemented with neomycin and exhibited some differences in CGP accumulation properties (30), CphA of that strain was of interest for further investigations. The cphA_NE1 gene was identified and cloned. Like all other cyanobacterial cphA genes, cphA_NE1 was located downstream of and colinear to a cyanophycinase-encoding cphB_NE1 gene (2, 18, 36, 51).

CphA_NE1 is composed of 901 amino acid residues, and it exhibits high homology to other cyanobacterial CphAs. A multiple alignment of CphA proteins revealed that their sizes differed, ranging from 872 to 903 amino acids. The C-terminal region starting from amino acid 872 is not conserved and seems to be highly variable in different strains. It was recently shown that the removal of 200 bp of the C-terminal region of cphA from Synechoccocus sp. strain MA19, which has a size (903 amino acids) similar to that of CphA from N. ellipsosporum NE1, led to complete inactivation of the enzyme and that the recombinant cells could not accumulate detectable CGP (18). A deletion of 180 bp in cphA_NE1 also yielded a completely inactive enzyme (CphA_NE1del180), whereas a deletion of 96 bp yielded an enzyme (CphA_NE1del96) which retained its activity. The latter had a total length of 870 amino acids, which is in the range of the smallest cyanobacterial CphAs, e.g., that of Synechocystis sp. strain PCC6803. Cultivation experiments in complex medium revealed unexpectedly that the truncated CphA_NEdel96 conferred significantly higher enzyme activities (5.75 U/mg protein) and higher CGP contents (up to 34% [wt/wt]) on E. coli than the wild-type CphA_NE. The maximum CGP yields for recombinant E. coli harboring plasmids containing cphA genes from different cyanobacteria, like A. variabilis, Synechocystis sp. strain PCC 6803, Synechococcus sp. strain MA19, and Synechocystis sp. strain PCC 6308, were in the range of 20% to 26% using LB or TB complex medium (1, 8, 16, 18). The CGP yield in this study with recombinant E. coli harboring pSK::cphA_NE1del96, which was constructed in the same way as cphA genes from Synechococcus sp. strain MA19 or Synechocystis sp. strain PCC 6308, was 34%, which is the highest ever obtained in a recombinant strain of E. coli. Upscaling of the batch cultivation in complex medium was also done successfully in 30-liter bioreactors, as described previously (16), yielding similarly high CGP contents within a cultivation period of only 16 h. With both constructs, the wild type and the truncated CphA, the highest activity was found in IPTG-induced cultures. However, because the pBR322-derived constructs (pBR322::cphA_NE1 and pBR322::cphA_NE1del96) harbored only the cphA gene and 400 bp of the upstream intergenic region without a vector-based promoter, it was demonstrated that the native cyanobacterial promoter is active in E. coli, as proposed for the cphA promoter of Synechocystis sp. strain PCC 6308 (1).

More active CphAs yielding larger amounts of CGP in the cells, as obtained in this study, are of great interest with regard to biotechnological production of CGP. The results of this study indicate that the short stretch of hydrophobic amino acids around amino acid position 855, which is conserved in CphAs, is important for enzyme activity. For many proteins, like polyhydroxyalkanoate (PHA) synthases (PhaC), proteases, permeases, regulator proteins, transporter proteins, and several others, truncation experiments or other modifications of N- or C-terminal domains have been described in the literature in the last several years (2, 5, 10, 24, 25, 28, 33, 38, 41, 49). N- and C-terminal regions are often essential for enzyme assembly and allosteric and/or catalytic properties of an enzyme, and modifications in these regions cause different effects. In vitro evolution of other polymer-synthesizing enzyme systems, such as PhaC, employing error-prone PCR random mutagenesis, produced enhanced polymer accumulation and also changed the monomer composition of the accumulated PHA. For example, due to the exchange of a single amino acid in the N-terminal region of the Aeromonas caviae PhaC (N149S), the PHA content of the cells could be increased 6.5-fold in comparison to the wild type (5, 25). Removal of the first 36 or 100 amino acid residues of the R. eutropha PhaC led to only partially reduced activities of the truncated enzyme proteins (41). Whereas the removal of a stretch of 10 amino acids from the N terminus of the small subunit of ADP-glucose pyrophosphorylase increased its resistance to the allosteric inhibitor P_i and also its sensitivity to heat treatment, the removal of the putative C-terminal allosteric binding region abolished the formation of the enzyme ADP-glucose pyrophosphorylase (28). Deletion of the C-terminal peptide sequence of the Cel5Z endoglucanase significantly increased its specific activity (38). The secA gene encodes an ATPase of a preprotein translocase of E. coli. The SecA homologues show C-terminal sequence variations like those of the cyanobacterial cphA genes. Experiments demonstrated that deletion of up to 70 amino acids revealed no significant influence on the stability or activity of the protein (24). However, more extended deletions prevented dimerization of the SecA protein and caused loss of activity. In contrast to SecA, a recent study by Weber and Jung (49) on the stress protein UspG of E. coli revealed that upon short truncations of about 18 amino acids, further reductions by cellular proteases occurred, inhibiting the formation of dimers and causing significant losses of activity.

An influence of the length of CphA on the accumulation of CGP in the analyzed cyanobacteria has not been described in the literature. Hypothetically, the variation in the C terminus of cphA may be important for enzyme stability in cyanobacteria. However, the truncated CphA_NE1del96 did not exhibit reduced enzyme stability or activity in comparison to the wild-type CphA_NE1 in recombinant E. coli. The increased enzyme activity and higher CGP yields were unexpected and may be important for further optimization of strains suitable for biotechnological production of this interesting polymer.

The apparent molecular masses of CGPs isolated from cyanobacteria range widely between 25 and 125 kDa. The molecular masses and polydispersities of the CGPs isolated from E. coli harboring pSK::cphA_NE1 or pSK::cphA_NE1del96 exhibited no differences and closely resembled the CGP previously isolated from recombinant E. coli (16, 51), as revealed in Fig. 2. One remarkable feature of CphA_NE1 is its substrate specificity. Whereas recombinant strains of E. coli expressing CphAs from any other cyanobacterium, like Anabaena variabilis and Synechocystis sp. strain PCC 6308 or PCC6803 (1, 16, 51), contain up to 10% (wt/wt) lysine, which was not detected in the CGP accumulated by the cyanobacteria itself, only traces of lysine were detectable in the polymer produced by the recombinant E. coli strain expressing CphA_NE1. Therefore, CphA from N. ellipsosporum NE1 seems to have a much higher substrate specificity than other cyanobacterial CphAs. Such stringent substrate specificity was otherwise found only in Acinetobacter sp. strain 587 (26).

 
We thank F. Leganés (Universidad Autonoma de Madrid, Spain) for providing Nostoc ellipsosporum strain NE1.

 
* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Corrensstrasse 3, D-48149 Münster, Germany. Phone: 49 251 8339821. Fax: 49 251 8338388. E-mail: steinbu{at}uni-muenster.de.

Published ahead of print on 29 September 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, December 2006, p. 7652-7660, Vol. 72, No. 12
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