A cyanophycin synthetase from <Emphasis Type="Italic">Thermosynechococcus elongatus</Emphasis> BP-1 catalyzes primer-independent cyanophycin synthesis

Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires l-aspartic acid (Asp), l-arginine (Arg), ATP, Mg²⁺, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg²⁺ or Mn²⁺ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400 ± 9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50°C. In addition, we examined cyanophycin synthesis at 30°C, 40°C, 50°C, and 60°C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.     

Identification of the Anabaena sp. strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha

The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp. strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E. coli (6.7 nmol arginine per min and mg protein). CphA1 and cphA genes of Synechocystis sp. strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha. Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P. putida strains and 7.3, 12.6, or 14.1% of CDM for R. eutropha). Furthermore, PHA-negative mutants of P. putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R. eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P. putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R. eutropha strains). Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin. PHA-negative mutants of P. putida and R. eutropha expressing cphA1 of Anabaena sp. strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin.     

Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production

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.     

Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16

Two different recombinant plasmids both containing the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 but differing concerning the resistance marker gene were tested for their suitability to produce high amounts of cyanophycin in recombinant strains of Ralstonia eutropha. Various cultivation experiments at the 30-L scale revealed very low cyanophycin contents of the cells ranging from 4.6% to 6.2% (w/w) of cellular dry weight (CDW) only, most probably because most cells had lost the corresponding plasmid during cultivation. To establish a cost effective and high efficient system for production of cyanophycin at larger scales using recombinant strains of R. eutropha, we applied two strategies: First, we integrated cphA into the dispensable chromosomal l-lactate dehydrogenase gene (ldh) of R. eutropha. Depending on the cultivation conditions used, relatively low cyanophycin contents between 2.2% and 7.7% (w/w) of CDW were reproducibly detected, which might be due to weak expression or low gene dosage in the single cphA copy strain of R. eutropha. In a second strategy we constructed a KDPG-aldolase gene (eda)-dependent addiction system, which combined features of a multi-copy plasmid with stabilized expression of cphA. Flasks experiments revealed that the cells accumulated extraordinarily high amounts of cyanophycin between 26.9% and 40.0% (w/w) of CDW even under cultivation conditions lacking cyanophycin precursor substrates or plasmid stabilizing antibiotics. Cyanophycin contents of up to 40.0% (w/w) of CDW were also obtained at a 30-L scale or a 500-L pilot-plant scale under such non-selective conditions. This demonstrates impressively that the stabilizing effect of the constructed eda-dependent addiction system can be used for production of enhanced amounts of cyanophycin at a larger scale in recombinant strains of R. eutropha.     

Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp. strain MA19 and in vitro synthesis of cyanophycin and related polyamides.

The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase (cphA) and cyanophycinase (cphB), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50 degrees C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, L-arginine and L-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, alpha-arginyl aspartic acid dipeptide, and poly-alpha,beta-DL-aspartic acid as primers and also incorporated beta-hydroxyaspartic acid instead of L-aspartic acid or L-canavanine instead of L-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.     

Features of the biotechnologically relevant polyamide family “cyanophycins” and their biosynthesis in prokaryotes and eukaryotes

Cyanophycin, inclusions in cyanobacteria discovered by the Italian scientist Borzi in 1887, were characterized as a polyamide consisting of aspartic acid and arginine. Its synthesis in cyanobacteria was analyzed regarding growth conditions, responsible gene product, requirements, polymer structure and properties. Heterologous expression of diverse cyanophycin synthetases (CphA) in Escherichia coli enabled further enzyme characterization. Cyanophycin is a polyamide with variable composition and physiochemical properties dependent on host and cultivation conditions in contrast to the extracellular polyamides poly-γ-glutamic acid and poly-ε-l-lysine. Furthermore, recombinant prokaryotes and transgenic eukaryotes, including plants expressing different cphA genes, were characterized as suitable for production of insoluble cyanophycin regarding higher yields and modified composition for other requirements and applications. In addition, cyanophycin was characterized as a source for the synthesis of polyaspartic acid or N-containing bulk chemicals and dipeptides upon chemical treatment or degradation by cyanophycinases, respectively. Moreover, water-soluble cyanophycin derivatives with altered amino acid composition were isolated from transgenic plants, yeasts and recombinant bacteria. Thereby, the range of dipeptides could be extended by biological processes and by chemical modification, thus increasing the range of applications for cyanophycin and its dipeptides, including agriculture, food supplementations, medical and cosmetic purposes, synthesis of the polyacrylate substitute poly(aspartic acid) and other applications.     

Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308

A 3878-bp genomic region from the cyanobacterium Synechocystis sp. strain PCC6308, amplified by inverse PCR, harbored the structural genes cphA (2625 bp) and cphB (819 bp) encoding cyanophycin synthetase and cyanophycinase, respectively. Both primary structures exhibited a high degree of similarity to the corresponding translational products from other cyanobacteria. Five regions were localized in the cyanophycin synthetase consensus sequence by their resemblance to conserved sites of ATP-dependent carboxylate-amine/thiol ligases and three substrate ligases. The functionality of cphA was proven by heterologous expression of active enzyme and synthesis of cyanophycin in Escherichia coli, which led to a maximum cyanophycin content of 26.6% (w/w) of cell dry mass. Furthermore, a modified radiometric enzyme assay for a more reliable and feasible measurement of cyanophycin synthetase activity was developed and applied to reveal the substrate specificity of the enzyme.     

Investigations on the solubility behavior of cyanophycin. Solubility of cyanophycin in solutions of simple inorganic salts

On the basis of a previous report on the occurrence of water-soluble cyanophycin (CGP, cyanophycin granule polypeptide) in a recombinant strain of Escherichia coli expressing the cyanophycin synthetase (CphA) of Desulfitobacterium hafniense published by others, the conditions of its production were investigated in this study. Although the incubation temperature, aeration level, and NaCl concentration during cultivation had effects on the in vivo production of water-soluble CGP, it could be isolated as a major variant irrespective of the cultivation conditions. The occurrence of the soluble variant was also not dependent on the E. coli host or on the origin of cphA. Furthermore, it was shown that water-insoluble CGP can be in vitro solubilized to extents of up to about 80% (w/w) in solutions of different inorganic salts such as LiCl, NaCl, KCl, RbCl, KBr, MgCl(2), or CaCl(2). Evidence was obtained that the salt ions bind tightly to CGP. If the ions were not removed from the salt solution by dialysis or dilution, the CGP remained stable in solution. This method to solubilize water-insoluble CGP could also be applied to high concentrations of the polymer. CGP that remained insoluble after the first treatment could only marginally be solubilized in following treatments. The polydisperse CGP molecules were solubilized to the same extent over the whole molecular weight range with no preference of a particular molecular weight.     

Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense

Some bacterial genomes were found to contain genes encoding putative proteins with considerable sequence homology to cyanophycin synthetase CphA of cyanobacteria. Such a gene from the Gram-positive, spore-forming anaerobe Desulfitobacterium hafniense was cloned. Expression in Escherichia coli resulted in the formation of a polydispers copolymer of aspartic acid and arginine, with a minor amount of lysine, of about 30 kDa molecular mass. In contrast to cyanophycin, this polymer was water-soluble. The structure of the polymer formed by the synthetase from Desulfitobacterium hafniense was studied by enzymatic degradation with the cyanophycin-specific hydrolase cyanophycinase, and by chemical and mass-spectroscopic analyses. Despite of the differences in solubility, indicating that both polymers cannot be completely identical, the chemical structure was found to be very similar to that of cyanophycin. The results suggest that the use of cyanophycin-like polymers as a nitrogen-rich reserve material is not restricted to cyanobacteria, and that such polymers may not necessarily be stored in granules.     

Production of cyanophycin in <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> through the expression of a cyanophycin synthetase encoding gene

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid.     

The role of the C-terminal region of cyanophycin synthetase from Nostoc ellipsosporum NE1 in its enzymatic activity and thermostability: a key function of Glu(856)

The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc ellipsosporum NE1, CphA(NE1), was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphA(NE1) in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphA(NE1) as a vector that contained the ochre codon TAA. CphA(NE1DeltaC45), which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu(856), resulted in complete inactivation of CphA(NE1DeltaC46). Replacement of Glu(856) by valine or alanine confirmed the importance of this residue for the activity of CphA(NE1), as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphA(NE1) after removal of the region from Leu(867) to Leu(870). The gel filtration analysis showed that CphA(NE1Delta46C) still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu(856) is critical for CphA(NE1) catalytic activity and that the predicted alpha-helical region that ranges from Val(858) to Leu(870) is important for the thermostability of the enzyme.     

Cyanophycin production from feather hydrolysate using biotechnological methods

Abstract

Cyanophycin is a bacterial storage polymer for carbon, nitrogen and energy with emerging industrial applications. As efficient cyanophycin production is enhanced by peptone, but commercial peptones are very expensive, thereby increasing the overall production cost, an enzymatically produced feather hydrolysate (FH) is assessed as a cheap replacement of peptone to lower the costs and make cyanophycin production more economically feasible. Keratinase production using feather as the sole carbon/nitrogen source by S.pactum 40530 at 30-L fermentation scale was achieved within 93?h with degradation rate of 96.5%. A concentration of 60?g/L of FH, generated by keratinolytic activity (8?×?10³?U?g^?1L^?1d^?1) within 24?h, was used as the main carbon/peptone source to produce cyanophycin. The growth performances of E. coli DapE/L using FH was compared to that of casamino acids (CA) and up to 7.1?±?0.4 and 5.3?±?0.3?g/L of cell mass were obtained after 72?h from FH and CA, respectively. Cyanophycin production yielded 1.4?±?0.1g/L for FH with average molecular mass of 28.8 and 1.4?±?0.2 for CA with average molecular mass of 35.3, after 60?h. For the first time, FH generated by biotechnological methods from environmentally problematic, abundant and renewable feather bioresource was successfully used for cyanophycin biopolymer production.     

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.     

Purification, Characterization of GH11 Endo-β-1,4-xylanase from Thermotolerant Streptomyces sp. SWU10 and Overexpression in Pichia pastoris KM71H

We have previously described two forms of an endo-β-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1–11), more than 80 % of initial activity remained at pH 2–11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.     

Heterologous expression and biochemical characterization of acetyl xylan esterase from Coprinopsis cinerea

Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l^?1. Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc₂Hex_9–16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a K_m of 4.3 mM and a V_max of 2.15 U mg^?1 for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a K_m of 0.11 mM and V_max of 0.78 U mg^?1. The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.     

Molecular cloning and expression of a novel laccase showing thermo- and acid-stability from the endophytic fungus Phomopsis liquidambari and its potential for growth promotion of plants

A novel laccase (LACB3) from the endophytic fungus, Phomopsis liquidambari, was cloned and its potential to promote peanut growth was evaluated. The full-length cDNA is 1,731 bp, encoding a mature protein of 556 amino acids with a molecular mass of 60.1 kDa. Using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), LACB3 exhibited a K _m and k _cat of 85 μM and 92.7 s^?1, respectively. The enzyme had maximal activity at pH 2.5 and 50 °C and retained 50 % of its activity after 20 h at 50 °C. When LACB3 was applied to soil, the peanut biomass was increased by 12 %, and the content of vanillic acid, coumaric acid and 4-hydroxybenzoic acid in soil were decreased by 21, 27 and 40 %, respectively. These results suggest substantial potential for the use of P. liquidambari or its laccase in agriculture.     

Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.