Increased Lysine Content Is the Main Characteristic of the Soluble Form of the Polyamide Cyanophycin Synthesized by Recombinant Escherichia coli

Cyanophycin, a polyamide of cyanobacterial or noncyanobacterial origin consisting of aspartate, arginine, and lysine, was synthesized in different recombinant strains of Escherichia coli expressing cphA from Synechocystis sp. strain PCC 6308 or PCC 6803, Anabaena sp. strain PCC 7120, or Acinetobacter calcoaceticus ADP1. The molar aspartate/arginine/lysine ratio of the water-soluble form isolated from a recombinant strain expressing CphA₆₃₀₈ was 1:0.5:0.5, with a lysine content higher than any ever described before. The water-insoluble form consisted instead of mainly aspartate and arginine residues and had a lower proportion of lysine, amounting to a maximum of only 5 mol%. It could be confirmed that the synthesis of soluble cyanobacterial granule polypeptide (CGP) is independent of the origin of cphA. Soluble CGP isolated from all recombinant strains contained a least 17 mol% lysine. The total CGP portion of cell dry matter synthesized by CphA₆₃₀₈ from recombinant E. coli was about 30% (wt/wt), including 23% (wt/wt) soluble CGP, by using terrific broth complex medium for cultivation at 30°C for 72 h. Enhanced production of soluble CGP instead of its insoluble form is interesting for further application and makes recombinant E. coli more attractive as a suitable source for the production of polyaspartic acid or dipeptides. In addition, a new low-cost, time-saving, effective, and common isolation procedure for mainly soluble CGP, suitable for large-scale application, was established in this study.     

Cyanophycin granule polypeptide protease in a unicellular cyanobacterium

An assay was developed to measure the proteolysis of cyanophycin granule polypeptide in crude extracts of a unicellular cyanobacterium. The substrate was radioactively labeled cyanophycin granule polypeptide formed by an unicellular cyanobacterium grown in the presence of chloramphenicol. Substrate polypeptide displayed identical chemical properties with polypeptide isolated from non-chloramphenicol-treated cells. Solubilization of radioactivity as arginine indicated hydrolysis of polypeptide. Radioactively labeled aspartate and arginine from hydrolyzed polypeptide was related to nmol amino acid using a combination of paper chromatography, liquid scintillation analysis, and ninhydrin quantitation. Protease activity was found in extracts of nitrogen-limited cells harvested 16–24 h after a nitrogen source was added back. Optimal pH for protease activity was 8.0 and optimum temperature was 35°C. Protease activity in crude extracts followed Michaelis-Menten kinetics with a V _max of 92 nmol arginine per 15 min/mg protein and a K _m of 2.1×10³ nmol arginine. Protease activity was inhibited by arginine and by high concentrations of aspartate.     

Guanidination of horse methemoglobin.

Reaction of horse methemoglobin with O-methylisourea at pH 10.2 results in 95% conversion of lysine residues to homoarginine. Analysis of the chymotryptic peptides showed that no single ?-amino group was unreactive. Guanidination decreases the dependence of the sedimentation coefficient on hydrogen ion concentration in the range of pH 8 to 11 and did not affect the dependence on protein concentration at pH 7. These results support the conclusion that the lysine side chains involved in subunit contacts have sufficient freedom to accommodate the small changes in bulk and geometry associated with guanidination.     

高赖氨酸蛋白基因在大肠埃希菌中的表达

从四棱豆中克隆高赖氨酸蛋白基因wblys,通过PCR扩增wblys片段,转入原核表达载体pGEX-4T-1,构建pGEX-4T-1/wblys大肠埃希菌工程菌,表达重组蛋白,IPTG诱导后,发现细菌全蛋白在44 ku(含GST标签)处多出1条明显条带。HPLC检测赖氨酸含量。诱导后菌体总赖氨酸含量比正常菌体提高15.84 mg/g。在大肠埃希菌中高效表达植物源高赖氨酸蛋白基因,为该基因在益生菌中表达提供研究工作基础。     

Cyanophycin granule polypeptide in a facultatively heterotrophic cyanobacterium

Cyanophycin granule polypeptide (CGP) formation and degradation was studied in a facultatively heterotrophic, nitrogen-fixing cyanobacterium. CGP was synthesized primarily under photoautotrophic growth conditions in nitrogen-fixing cells. Addition of a utilizable carbon source stopped CGP synthesis, the content of which declined in cultures as the cells grew heterotrophically in light or dark. A high-performance liquid chromatographic method was developed to determine CGP in cyanobacterial extracts and permitted analyses of CGP and simultaneous examination of the protein profile of the cells. The data indicated that, once formed in the photoautotrophs, CGP was not degraded, even by cells in which extensive protein breakdown was occurring.     

Guanidination of a peptide side chain amino group on a solid support

A novel guanidination method of converting a peptide side chain amino group to a guanidino group on a solid support is described. Four guanidinating reagents were evaluated using a model tetrapeptide attached to a polystyrene resin. Experimental data indicate that the two nitroguanidinating reagents, but not the two tosylguanidinating reagents, can be used effectively in solid phase peptide synthesis.     

Guanidination and nitroguanidination of staphylococcal enterotoxin B

Guanidination of the free amino groups of staphylococcal enterotoxin B with 3,5-dimethyl-1-guanylpyrazole converted 31-32 of 33 epsilon-amino groups and 30% of the N-terminal residue. This product, although markedly reduced in solubility, suffered no gross change in conformation and retained full biological activity. A derivative prepared by reaction with O-methylisourea with only one lysyl residue unaltered lost most of its emetic activity. Nitroguanidination with 3,5-dimethyl-1-nitroguanylpyrazole converted up to 28 of the epsilon-amino groups and essentially all of the N-terminus. This material was greatly reduced in ability to produce emesis and like the O-methylisourea prepared guanidinated enterotoxin, gave only a line of partial identity in double diffusion. The loss of activity is attributed to unfolding and it is concluded that the free amino groups of enterotoxin B do not critically participate in either its antigenic determinants or its active center for emesis.     

Cyanophycin production in a phycoerythrin-containing marine synechococcus strain of unusual phylogenetic affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.     

Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases

Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-l-arginyl-poly-l-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA₆₃₀₈) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA₆₃₀₈ and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA₆₃₀₈Δ1, which was truncated by one amino acid at the C terminus; point mutated CphA₆₃₀₈C595S; and the combined double-mutant CphA₆₃₀₈Δ1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA₆₃₀₈ (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA₆₃₀₈Δ2) or three (CphA₆₃₀₈Δ3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA₆₃₀₈. In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA₆₃₀₈Δ1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26°C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA₆₃₀₈ and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA₆₃₀₈Δ1.Since the first isolation of a methylotrophic yeast, Kloeckera sp. strain 2201, in 1969 (43), the two methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have become the most popular methylotrophs in industry and academia (9, 23, 24). The main benefits of these organisms for the production of recombinant proteins are their growth to cell densities as high as 130 g cell dry matter per liter (50, 57) and the availability of strong and tightly regulated promoters that result in a high product yield (13). Viral hepatitis B surface antigen, S. cerevisiae mating factor α, and S. cerevisiae invertase are only a few examples of compounds produced by recombinant P. pastoris (reviewed in reference 9).A variety of strains were optimized for the expression of recombinant proteins (9). Protease-deficient strains such as strain KM71(H) were generated to circumvent the proteolytic degradation of recombinant proteins (17). Three different phenotypes exist that differ in the ability to utilize methanol (reviewed in reference 37). (i) Mut⁺ strains grow on methanol as the sole carbon and energy source at the wild-type rate. (ii) Mut^s strains possess a disrupted alcohol oxidase 1 (AOX1) gene and therefore rely on the weaker AOX2 gene, leading to decreased methanol utilization rates in comparison to those exhibited by Mut⁺ strains. (iii) Mut⁻ strains are not able to utilize methanol as a carbon and energy source; consequently, such strains use the compound as an inducer only and are dependent on the concomitant addition of carbon sources that do not repress the AOX1 promoter (30, 31). Depending on the required product, any of these phenotypes can be optimal (37). The AOX1 promoter is totally repressed during growth on, e.g., glycerol, whereas it is strongly expressed after methanol is supplied (11). Therefore, P. pastoris fermentations are divided into two phases. (i) During growth on glycerol, high cell densities are reached; (ii) subsequent growth on methanol leads to induction of heterologous protein synthesis, resulting in a high product yield (14). Besides glycerol, several other carbon sources, such as, e.g., glucose, acetate, ethanol, or sorbitol, were used for the production of foreign proteins (30, 31). Several fermentation strategies that allow optimal cell and product yields have been established (8, 25, 28).Besides the AOX1 promoter, several other suitable promoters are available (10), e.g., the copper-inducible CUP1 promoter from S. cerevisiae (33, 38), the inducible ICL1 promoter from the isocitrate lyase gene (8), or the constitutive GAP promoter from glyceraldehydes-3-phosphate dehydrogenase (56).Synthesis of cyanophycin (multi-l-arginyl-poly-l-aspartic acid [CGP]) was only recently established in the yeast S. cerevisiae. Recombinant strains harboring cphA from Synechocystis sp. strain PCC 6308 but otherwise with a wild-type background accumulated CGP up to 6.9% (wt/wt) (52), whereas recombinant strains with a mutation in arginine metabolism accumulated CGP even up to 15.3% (wt/wt) of the cell dry mass (CDM) (54). All of the strains synthesized the polymer in soluble and insoluble forms, which was also observed in transgenic plants (29, 42); the soluble type of CGP was first observed in Escherichia coli expressing the cphA gene from Desulfitobacterium hafniense (59). Several cyanobacterial and heterotrophic CGP synthetase genes were expressed heterologously in the past (16, 26, 29, 52, 59). To unravel structurally or catalytically relevant residues of the enzyme, a few site-directed mutations were generated in cyanobacterial cphA genes (26, 27, 35, 53). In addition, several variations in the amino acid composition of the polymer were recently obtained; while cyanobacterial CGP or CGP synthesized by specific CphA proteins exhibiting a narrow substrate range contained aspartate and arginine only (18, 51); lysine was observed as a component replacing arginine at up to 18 mol% in recombinant strains of E. coli and S. cerevisiae harboring CphA with a broader substrate range (34, 54). Moreover, citrulline and ornithine were also detected as constituents replacing arginine in mutants of S. cerevisiae expressing CphA from Synechocystis sp. strain PCC 6308 (54). The soluble CGP contained up to 20 mol% citrulline or up to 8 mol% ornithine instead of arginine. The latter enzyme also revealed a wide substrate range in vitro comprising agmatine and canavanine besides arginine, lysine, citrulline, and ornithine (2, 58).A multitude of technical or pharmaceutical applications are known for degradation products of CGP (44, 48, 49). Dipeptides obtained after α cleavage of the polymer by cyanophycinases are employed as high-value pharmaceuticals (45, 46). Through β cleavage of the polymer, polyaspartic acid can be obtained, which serves as a biodegradable alternative to the persistent polyacrylic acid (9). Finally, research on the synthesis of bulk chemicals such as urea or acrylonitrile from CGP has become of special interest (40, 48, 49).In this study, the methylotrophic yeast P. pastoris was, for the first time, employed for synthesis of the polyamide CGP to analyze if this organism provides a perspective for the production of the polymer. For further optimization of polymer yields, mutated CphA proteins were generated by site-directed mutagenesis and characterized and optimal growth parameters were determined in parallel fermentations.     

Solubility Behavior of Cyanophycin Depending on Lysine Content

Study of the synthesis of cyanophycin (CGP) in recombinant organisms focused for a long time mostly on the insoluble form of CGP, due to its easy purification and its putative use as a precursor for biodegradable chemicals. Recently, another form of CGP, which, in contrast to the insoluble form, was soluble at neutral pH, became interesting due to its high lysine content, which was also assumed to be the reason for the solubility of the polymer. In this study, we demonstrate that lysine incorporated into insoluble CGP affected the solubility of the polymer in relation to its lysine content. Insoluble CGP can be separated along a temperature gradient of 90°C to 30°C, where CGP showed an increasing lysine content corresponding to a decreasing temperature needed for solubilization. CGP with less than 3 to 4 mol% lysine did not become soluble even at 90°C, while CGP with 31 mol% lysine was soluble at 30°C. In lysine fractions at higher than 31 mol%, CGP was soluble. The temperature separation will be suitable for improving the downstream processing of CGP synthesized in large-scale fermentations, including faster and more efficient purification of CGP, as well as enrichment and separation of dipeptides and CGP with specific amino acid compositions.     

Cyanophycin Production in a Phycoerythrin-Containing Marine Synechococcus Strain of Unusual Phylogenetic Affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.     

Accumulation of Cyanophycin Granules as a Result of Phosphate Limitation in Agmenellum quadruplicatum

Phosphate-limited growth of the blue-green alga Agmenellum quadruplicatum resulted in the accumulation of cyanophycin granule polypeptide (CGP), which is a 1:1 co-polymer of aspartic acid and arginine. The progressive accumulation of CGP began after depletion of phosphate from the medium. CGP increased in concentration much faster than the increase in cell number. Electron microscopy indicated that both the number of cyanophycin granules per cell section and the diameter of each granule increased as phosphate starvation progressed. A marked decrease in the electron density of the inter-thylakoidal areas took place concurrently with the accumulation of CGP. At the same time a progessive decrease in the pigment concentration of cells and in the rate of nitrate uptake was observed. Thirty-two hours after phosphate depletion from the medium up to 28% of total cellular nitrogen was found in CGP.     

Enhanced Yields of Iron-Oxidizing Bacteria by In Situ Electrochemical Reduction of Soluble Iron in the Growth Medium

An electrochemical apparatus for culturing chemolithotrophic bacteria that respire aerobically on ferrous ions is described. Enhanced yields of the bacteria were achieved by the in situ electrochemical reduction of soluble iron in the growth medium. When subjected to a direct current of 30 A for 60 days, a 45-liter culture of Thiobacillus ferrooxidans grew from 6 × 10⁷ to 9.5 × 10⁹ cells per ml. Growth of the bacterium within the electrolytic bioreactor was linear with time. A final cell density corresponding to 4.7 g of wet cell paste per liter was achieved, and a total of 320 g of wet cell paste was harvested from one culture. The apparatus was designed to deliver protons concomitantly with electrons; therefore, the pH of the culture remained stable at 1.6 ± 0.1 for the duration of growth. This laboratory-scale apparatus may be readily adapted to pilot or production scale. It is thus anticipated that abundant numbers of iron-oxidizing bacteria may be obtained for both fundamental and applied studies.     

Cyanophycin and glycogen synthesis in a cyanobacterial Scytonema species in response to salt stress

The response to salinity of a Scytonema species isolated from the central Australian desert was studied. Under nitrogen-fixing conditions the addition of increasing concentrations of salt (NaCl) caused progressive inhibition of growth, with growth ceasing at 150 mM NaCl. This correlated with a progressive loss of nitrogenase activity, a low level of activity being retained at 150 mM NaCl. The inhibition of growth was overcome when KNO₃ (10 mM) was added to the growth medium. In response to the salt stress, cells accumulated the reserve compounds cyanophycin and glycogen. Time course experiments showed that they were steadily synthesized over 48 h, after which the concentrations stabilized. Cyanophycin synthesis was enhanced in salt-stressed cells grown in nitrate. When cells were restored to their normal growth medium the content of these substances decreased towards control levels.     

Guanidination of ovine luteinizing hormone and effects on activity.

The free amino groups in oLH, oLHalpha and oLHbeta were guanidinated by O-methylisourea. The epsilon-NH2 groups of lysine residues reacted bo substitute these positions in the sequence with the more basic homoarginine residue. The alpha-NH2 groups did not react under the conditions used. Guanidinated oLH or the products of guanidinated oLHalpha + native oLHbeta or guanidinated oLHalpha + guanidinated oLHbeta were inactive in two bioassay systems. Native oLHalpha + guanidinated oLHbeta, however, showed potencies of 39% to 55% of that observed with the native subunit recombinant or native oLH. Possible structural implications for hormone-receptor site interactions are discussed.     

Protamylasse, a Residual Compound of Industrial Starch Production, Provides a Suitable Medium for Large-Scale Cyanophycin Production

Protamylasse is a residual compound occurring during the industrial production of starch from potatoes. It contains a variety of nutrients and all necessary minerals and could be used as a carbon, nitrogen, and energy source for the growth of bacteria and also for cyanophycin (CGP) biosynthesis. Media containing protamylasse as the sole compound diluted only in water were therefore examined for their suitability in CGP production. Among various bacterial strains investigated in this study, a recombinant strain of Escherichia coli DH1 harboring plasmid pMa/c5-914::cphA₆₈₀₃, which carries the cyanophycin synthetase structural gene (cphA) from Synechocystis sp. strain PCC6803, was found to be most suitable. Various cultivation conditions for high CGP contents were first optimized in shake flask cultures. The optimized conditions were then successfully applied to 30- and 500-liter fermentation scales in stirred tank reactors. A maximum CGP content of 28% (wt/wt) CGP per cell dry matter was obtained in 6% (vol/vol) protamylasse medium at an initial pH of 7.0 within a cultivation period of only 24 h. The CGP contents obtained with this recombinant strain employing protamylasse medium were higher than those obtained with the same strain cultivated in mineral salts medium or in expensive commercial complex media such as Luria-Bertani or Terrific broth. It was shown that most amino acids present in the protamylasse medium were almost completely utilized by the cells during cultivation. Exceptions were alanine, tryptophan, tyrosine, and most interestingly, arginine. Furthermore, CGP was easily isolated from protamylasse-grown cells by applying the acid extraction method. The CGP exhibited a molecular mass of about 26 to 30 kDa and was composed of 50% (mol/mol) aspartate, 46% (mol/mol) arginine, and 4% (mol/mol) lysine. The use of cheap residual protamylasse could contribute in establishing an economically and also ecologically feasible process for the biotechnological production of CGP.     

Guanidination of notexin alters its membrane-damaging activity in response to sphingomyelin and cholesterol

To elucidate the contribution of phospholipase A₂ (PLA₂) activity of notexin to its ability to perturb membranes, comparative studies on the interaction of notexin and guanidinated notexin (Gu-notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of notexin against EYPC vesicles, but had an insignificant influence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA₂ activity by EDTA led to a reduction in the ability of Gu-notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of notexin and Gu-notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of notexin and Gu-notexin differed in either Ca²⁺-bound or metal-free states. These results indicate that notexin and Gu-notexin could induce membrane permeability without the involvement of PLA₂ activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.