Production Of Cyanophycin Research Articles

Cyanophycin production in turbidostat cultivation of cyanobacteria under phosphorus limitation on synthetic urine media

The cyanobacterium Synechocystis sp. PCC6803 is capable of using ammonium as nitrogen source which is the main nitrogen compound present in source-separated urine. Under phosphorus limiting conditions, Synechocystis has the ability to accumulate nitrogen in form of cyanophycin, an interesting polymer for the industry of bioplastics with a wide range of potential applications in biotechnology and agroindustry. We studied the accumulation of cyanophycin in Synechocystis cultivated in nutrient supplemented synthetic urine using ammonium as nitrogen source with different concentrations of phosphorus in batch and turbidostat cultivation. A decrease in biomass yield on light was observed in the turbidostat cultivation under strong phosphorus limitation, accompanied by overproduction of carotenoids and accumulation of cyanophycin. Synechocystis accumulated increasing amounts of cyanophycin reaching 10% of dry weight in form of cyanophycin in batch mode cultivation, and 7% in turbidostat cultivation. Higher accumulation of cyanophycin in both, batch and turbidostat cultivation, occurred at the lowest phosphate availability, biomass productivity decreased under strong phosphorus limitation. Mild phosphorus limitation allowed both cyanophycin and biomass production in turbidostat cultivation, and gave the higher titers in batch cultivation and the highest productivity in turbidostat cultivation. Strong phosphorus limitation decreased productivity of both biomass and cyanophycin as well as the nitrogen content in the biomass.

Soluble and stable cyanophycin synthetase expression enhances heterologous cyanophycin production in Escherichia coli

Microbial production of biopolymers represents a promising, sustainable alternative to current approaches for plastic production. Cyanophycin synthetase 1 (CphA1) produces cyanophycin - an attractive biopolymer consisting of a poly-L-aspartic acid backbone decorated with L-arginine side groups. In this work, a series of CphA1 enzymes from different bacteria that were each previously identified to express as soluble and stable enzymes in Escherichia coli were screened for heterologous cyanophycin production in engineered E. coli. In each case, expression of stable CphA1 enzymes resulted in greater insoluble cyanophycin production, and it was found that CphA1 from Tatumella morbirosei (TmCphA1) was especially productive. Under these conditions, TmCphA1 was capable of supporting up to ∼2-fold greater yields of insoluble cyanophycin than any other tested CphA1 enzymes, including 10.8-times more than CphA1 from Synechocystis sp. PCC6308. Finally, using a bench-scale bioreactor, cyanophycin production by TmCphA1-expressing E. coli reached up to 1.9 g per liter of culture by 48 h.

Fixing N2 into cyanophycin: continuous cultivation of Nostoc sp. PCC 7120

Two diazotrophic cyanobacteria (Anabaena cylindrica PCC 7122 and Nostoc sp. PCC 7120) were cultivated to produce cyanophycin, a nitrogen reserve compound, under nitrogen fixing conditions. In preliminary continuous experiments, Nostoc sp. was shown to be more efficient, accumulating a higher amount of cyanophycin and showing a greater capability to fix atmospheric nitrogen in the biomass (67 mgN d−1 of fixed nitrogen per liter of culture). The operating conditions were then optimized to maximize the cyanophycin productivity: the effect of incident light intensity, residence time and nitrogen availability were investigated. Nitrogen availability and/or pH played a major role with respect to biomass production, whereas phosphorus limitation was the main variable to maximize cyanophycin accumulation. In this way, it was possible to achieve a stable and continuous production of cyanophycin (CGP) under diazotrophic conditions, obtaining a maximum cyanophycin productivity of 15 mgCGP L−1 d−1.Key points• Diazotrophic cyanobacteria produce stable amount of cyanophycin in continuous PBR.• Nostoc sp. proved to be more efficient in producing cyanophycin than Anabaena sp.• P deprivation is the major variable to increase cyanophycin productivity in continuous.

Using the design of dynamic experiments to optimize photosynthetic cyanophycin production by Synechocystis sp.

The production of cyanophycin by photosynthetic microorganisms, as a high-value bio-based compound, is getting increasing interest. The aim of this work is to maximize the production of this compound by the cyanobacterium Synechocystis sp. in semi-batch cultivation systems, by applying a data-driven modeling approach based on the Design of Dynamic Experiments (DoDE) and Response Surface Model (RSM) methodologies. A first set of experiments, carried out inside an initially defined domain, was used to find a preliminary RSM model describing cyanophycin concentration as a function of incident light intensity profile, temperature, and phosphorus supply profile. The model was then improved, according to an evolutionary optimization approach, by carrying out additional experiments in a modified domain, exploiting information derived by the initial model. The updated model was used to identify the optimal operating conditions resulting in maximum cyanophycin concentration at the end of the batch. The cyanophycin concentration found experimentally (228.2 ± 20.0 mg/L) in these conditions fell within the confidence interval of the model prediction. Remarkably, this experimentally obtained value represents a significant (about 20 %) increase in the cyanophycin production with respect to the highest value found in the experiments before the optimization step (184.3 ± 0.8 mg/L).

A cryptic third active site in cyanophycin synthetase creates primers for polymerization

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.

Modeling the production of cyanophycin in Synechocystis sp. PCC 6803 cultivated in chemostat reactors

Cyanophycin is a molecule produced by cyanobacteria under stressful conditions, and used as a renewable biodegradable feedstock in various industrial applications. A correlation between the amount of cyanophycin in the biomass and the internal cell phosphorus reserve has been demonstrated for Synechocystis sp. PCC 6803. Here, a mathematical model based on Droop kinetics was developed and validated for this species, describing biomass growth, phosphorus uptake and accumulation, and cyanophycin production as a function of operating variables in continuous photobioreactors. The proposed model was able to estimate process performances in a reliable way with respect to experimental data. Therefore, it was used to find the optimal operating conditions that maximize cyanophycin productivity. The results indicated that the highest cyanophycin productivity can be obtained at a residence time of 2.2 days and inlet phosphorus concentration of 1.7 gP m−3. This result was validated experimentally, thus obtaining a productivity of 28.46 ± 3.7 gCP m−3 d−1.

Stabilizing autotrophic cyanophycin production in continuous photobioreactors

Cyanophycin, an intracellular reserve molecule synthetized by cyanobacteria, is considered as a potential biobased raw material for the industrial sector. It is usually produced through the cultivation of photosynthetic microorganisms in batch systems, which are affected by the high variability of cyanophycin accumulation due to varying growth phases, which lower the overall productivity. In this work, Synechocystis sp. PCC 6803 was cultivated in a continuous photobioreactor to increase cyanophycin productivity and to assess the feasibility of large-scale application. A stable production of the compound was obtained at steady-state. The effect of residence time and inlet phosphorus concentration on the biomass and cyanophycin productivity was evaluated, to identify the optimal conditions for its accumulation. A maximum value of cyanophycin productivity of about 30 mg L−1 d−1 was obtained, doubling the one achieved in the batch system. A quantitative correlation between the phosphorus quota and the cyanophycin produced was eventually proposed, highlighting a threshold of 4 mg of phosphorus per g of biomass for cyanophycin accumulation.

Cyanobacterial growth and cyanophycin production with urea and ammonium as nitrogen source

Several technologies have aimed to recover nitrogen directly from urine. Nitrogen recovery in these technologies was limited by the mismatch of the nitrogen-phosphorus molar ratio (N:P) of urine, being 30–46:1, and that of the final product, e.g., 1:1 in struvite and 16–22:1 in microalgae biomass. Additionally, the high nitrogen concentrations found in urine can be inhibitive for growth of microorganisms. Cyanobacteria were expected to overcome phosphorus (P) limitation in urine given their ability to store an N-rich polymer called cyanophycin. In this study, it was found that the model cyanobacterium Synechocystis sp. PCC6803 did not experience significant growth inhibition when cultivated in synthetic medium with concentrations of 0.5 g ammonium-N L−1. In the case of urea, no inhibition was observed when having it as sole nitrogen source, but it resulted in chlorosis of the cultures when the process reached stationary phase. Synechocystis was successfully cultivated in a medium with 0.5 g ammonium-N L−1 and a N:P ratio of 276:1, showing the N:P flexibility of this biomass, reaching biomass N:P ratios up to 92:1. Phosphorus starvation resulted in cyanophycin accumulation up to 4%. Dilution of the culture in fresh medium with the addition of 118 mg N L−1 and 1.5 mg P L−1 (N:P of 174:1) resulted in a rapid and transient cyanophycin accumulation up to 11%, after which cyanophycin levels rapidly decreased to 3%.

The Formation of Spore-Like Akinetes: A Survival Strategy of Filamentous Cyanobacteria

Some cyanobacteria of the order Nostocales can form akinetes, spore-like dormant cells resistant to various unfavorable environmental fluctuations. Akinetes are larger than vegetative cells and contain large quantities of reserve products, mainly glycogen and the nitrogen storage polypeptide polymer cyanophycin. Akinetes are enveloped in a thick protective coat containing a multilayered structure and are able to germinate into new vegetative cells under suitable growth conditions. Here, we summarize the significant morphological and physiological changes that occur during akinete differentiation and germination and present our investigation of the physiological function of the storage polymer cyanophycin in these cellular processes. We show that the cyanophycin production is not required for formation and germination of the akinetes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

Cyanophycin production from feather hydrolysate using biotechnological methods

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.

Mercury Methylation Capacity and Removal of Hg Species from Aqueous Medium by Cyanobacteria

Most technologies used for decontamination presents good results for high concentrations, but limitations for lower ones. The desirable Hg concentration in the water is extremely low because of its toxicity. The aims of this study were to evaluate inorganic mercury (Hg2+) and methylmercury (CH3Hg+) toxicity in Nostoc paludosum, to assess the potential of this cyanobacteria strain to remove these Hg species from aqueous medium and also to investigate Hg methylation by the cyanobacteria. CH3Hg+ determination was performed by gas chromatography-pyrolysis-atomic fluorescence spectrometry in cultures exposed to a concentration of 20 μg L−1 for 30 days. Both Hg species were removed from the supernatant, ranging from 73 to 96% of Hg2+ and from 73 to 95% of CH3Hg+. Ultrastructural Hg2+ effects in the cyanobacteria cells investigated by transmission electron microscopy revealed higher production of glycogen, cyanophycin, and intrathylacoidal spaces than the control group. When Hg was added to the culture in the form of CH3Hg+, a decrease corresponding to approximately 60% of the initial concentration due to Hg volatilization was observed. The production of CH3Hg+ by the cyanobacteria was detected in concentrations near the limit of detection (0.0025%) of the bioaccumulated THg. This is an advantage for biotechnological decontamination applications, as CH3Hg+ is a very toxic specie and can be bioaccumulated and biomagnified. The results demonstrated that cyanobacteria cells are an efficient alternative to retain and/or remove Hg at low concentrations and they constitute a potential tool for a “final cleaning” of contaminated waste water.

Exploring the potential of high-density cultivation of cyanobacteria for the production of cyanophycin

Photoautotrophic cyanobacteria and microalgae offer significant potential for the renewable synthesis of high-value products. As yet, however, the productivity of phototrophic cultures is limited due to the low cell densities that are typically obtained in current pilot scale photobioreactors. Here, we explore the use of ultrahigh-density cultivation of cyanobacteria for the production of cyanophycin, a non-ribosomally synthesized biopolymer of high biotechnological interest. We demonstrate that ultrahigh-density cultivation using a two-tier vessel with membrane-mediated CO2 supply yields a cyanophycin content per cellular dry weight similar to previously reported values, while the volumetric productivity per culture volume is significantly increased. Already after 96 h of cultivation, the engineered production strain BW86 reached up to 1 g cyanophycin per liter culture, approximately a 4-fold increase over the previously reported maximal yield obtained after 12 days of cultivation. Under phosphate-limiting growth conditions, the wild-type strain Synechcocystis sp. PCC 6803 accumulates up to 0.6 g cyanophycin per L culture. Our results demonstrate that ultrahigh-density cultivation is a suitable strategy towards the development of viable phototrophic production processes for cyanophycin and possibly other products of interest.

Comparative statistical component analysis of transgenic, cyanophycin-producing potatoes in greenhouse and field trials.

Potatoes are a promising system for industrial production of the biopolymer cyanophycin as a second compound in addition to starch. To assess the efficiency in the field, we analysed the stability of the system, specifically its sensitivity to environmental factors. Field and greenhouse trials with transgenic potatoes (two independent events) were carried out for three years. The influence of environmental factors was measured and target compounds in the transgenic plants (cyanophycin, amino acids) were analysed for differences to control plants. Furthermore, non-target parameters (starch content, number, weight and size of tubers) were analysed for equivalence with control plants. The huge amount of data received was handled using modern statistical approaches to model the correlation between influencing environmental factors (year of cultivation, nitrogen fertilization, origin of plants, greenhouse or field cultivation) and key components (starch, amino acids, cyanophycin) and agronomic characteristics. General linear models were used for modelling, and standard effect sizes were applied to compare conventional and genetically modified plants. Altogether, the field trials prove that significant cyanophycin production is possible without reduction of starch content. Non-target compound composition seems to be equivalent under varying environmental conditions. Additionally, a quick test to measure cyanophycin content gives similar results compared to the extensive enzymatic test. This work facilitates the commercial cultivation of cyanophycin potatoes.

Material properties of the cyanobacterial reserve polymer multi-l-arginyl-poly-l-aspartate (cyanophycin)

Bio-sourced macromolecules such as cyanophycin are an attractive source for alternative, sustainable plastics. While the chemical structure and biological function of cyanophycin have been previously investigated, its material properties remain largely unexplored. This study investigates the structural, thermal, mechanical, and solution properties of cyanophycin produced from recombinant Escherichia coli. Unplasticized, it has an elastic compression modulus of about 560 MPa and undergoes brittle failure at 78 MPa. Cyanophycin exhibits thermal stability in air up to 200 °C and does not undergo glass transition within its limit of thermal stability. The polypeptide is amorphous and has no long-range ordering in the solid state. In solution, water-soluble cyanophycin is thermoresponsive, exhibiting both upper and lower critical solution temperatures. Because the feasibility of industrial scale cyanophycin production through fermentation has been well studied, an expanded understanding of its materials properties should contribute to the development of new applications for this biopolymer.

Effect of phosphate availability on cyanophycin accumulation in Synechocystis sp. PCC 6803 and the production strain BW86

The cyanobacterial wild-type strain Synechocystis sp. PCC 6803 and its engineered strain BW86 were cultivated under defined conditions in photobioreactors to investigate the effect of phosphate availability on cyanophycin accumulation. Cyanophycin, multi-l-arginyl-poly-l-aspartate, can be deployed as amino acid source or can be chemically converted into polyaspartic acid, a biodegradable polymer. In previous studies it was demonstrated that a single point mutation in the PII signaling protein from the Synechocystis wild type is sufficient to unlock the arginine pathway causing an over accumulation of the biopolymer cyanophycin in BW86. One process strategy to evoke cyanophycin synthesis in Synechocystis is nutrient starvation. Therefore, different phosphate concentrations from 17.5 to 175μM were tested. Progressive phosphate starvation resulted in an increased cyanophycin accumulation. The highest obtained cyanophycin amounts in g cyanophycin per g cell dry mass were 18% and 40% for Synechocystis sp. PCC 6803 wild type and BW86 respectively, demonstrating that phosphate starvation is an effective route for biotechnological cyanophycin production. By evaluating cyanophycin and phosphor quotas per cell dry mass, it was possible to determine the specific required amount of phosphor to accumulate cyanophycin and to initialize stationary growth phase. Phosphor quotas in the range of 4 to 1mg phosphor per g cell dry mass triggered cyanophycin biosynthesis while Synechocystis entered the stationary phase at phosphor quotas of 1mg phosphor per g cell dry mass or less. Additionally, light kinetics was determined. Photon flux densities exceeding 46μmol photons m−2s−1 result in a maximum growth rate of 1.32d−1.

Enhanced cyanophycin production by Escherichia coli overexpressing the heterologous cphA gene from a deep sea metagenomic library

Cyanophycin is a non-ribosomally synthesized polymer and its microbial production has attracted increased attention due to its pharmaceutical and chemical values. For the characterization and production of cyanophycin, the cphA49 gene was cloned and expressed in Escherichia coli. Soluble cyanophycin was isolated from the cultures and characterized. The results showed that it was composed of 50% of aspartic acid, 45% of arginine, and 3.5% of lysine, and exhibited a homogenous molecular mass of 35kDa. To improve soluble cyanophycin production, the induction conditions for cphA49 gene expression were optimized. Meanwhile, the effects of medium content and induction duration on soluble cyanophycin production were also investigated, and the soluble cyanophycin yield reached a maximum at 72h. Finally, to further increase the soluble cyanophycin production, the cultivation was carried out by supplement of arginine, aspartic acid, lysine and glucose into the minimal resource medium after cphA49 gene expression level was improved under optimized conditions, and the maximum concentration of soluble cyanophycin reached 1.72g/L.

Tobacco as platform for a commercial production of cyanophycin

Cyanophycin (CP) is a proteinogenic polymer that can be substituted for petroleum in the production of plastic compounds and can also serve as a source of valuable dietary supplements. However, because there is no economically feasible system for large-scale industrial production, its application is limited. In order to develop a low-input system, CP-synthesis was established in the two commercial Nicotiana tabacum (N. tabacum) cultivars ‘Badischer Geudertheimer’ (BG) and ‘Virginia Golta’ (VG), by introducing the cyanophycin-synthetase gene from Thermosynecchococcus elongatus BP-1 (CphATe) either via crossbreeding with transgenic N. tabacum cv. Petit Havana SR1 (PH) T2 individual 51-3-2 or by agrobacterium-mediated transformation. Both in F1 hybrids (max. 9.4% CP/DW) and T0 transformants (max. 8.8% CP/DW), a substantial increase in CP content was achieved in leaf tissue, compared to a maximum of 1.7% CP/DW in PH T0 transformants of Hühns et al. (2008). In BG CP, yields were homogenous and there was no substantial difference in the variation of the CP content between primary transformants (T0), clones of T0 individuals, T1 siblings and F1 siblings of hybrids. Therefore, BG meets the requirements for establishing a master seed bank for continuous and reliable CP–production. In addition, it was shown that the polymer is not only stable in planta but also during silage, which simplifies storage of the harvest prior to isolation of CP.

Peculiarities and impacts of expression of bacterial cyanophycin synthetases in plants.

Cyanophycin (CP) can be successfully produced in plants by the ectopic expression of the CphA synthetase from Thermosynechococcus elongatus BP-1 (Berg et al. 2000), yielding up to 6.8 % of dry weight (DW) in tobacco leaf tissue and 7.5 % in potato tubers (Huehns et al. 2008, 2009). Though, high amounts of the polymer lead to phenotypical abnormalities in both crops. The extension of abnormalities and the maximum amount of CP tolerated depend on the compartment that CP production is localized at the tissue/crop in which CP was produced (Huehns et al. 2008, 2009; Neumann et al. 2005). It cannot be ascribed to a depletion of arginine, lysine, or aspartate, the substrates for CP synthesis.

Metabolic pathway engineering using the central signal processor PII.

BackgroundPII signal processor proteins are wide spread in prokaryotes and plants where they control a multitude of anabolic reactions. Efficient overproduction of metabolites requires relaxing the tight cellular control circuits. Here we demonstrate that a single point mutation in the PII signaling protein from the cyanobacterium Synechocystis sp. PCC 6803 is sufficient to unlock the arginine pathway causing over accumulation of the biopolymer cyanophycin (multi-l-arginyl-poly-l-aspartate). This product is of biotechnological interest as a source of amino acids and polyaspartic acid. This work exemplifies a novel approach of pathway engineering by designing custom-tailored PII signaling proteins. Here, the engineered Synechocystis sp. PCC6803 strain with a PII-I86N mutation over-accumulated arginine through constitutive activation of the key enzyme N-acetylglutamate kinase (NAGK).ResultsIn the engineered strain BW86, in vivo NAGK activity was strongly increased and led to a more than tenfold higher arginine content than in the wild-type. As a consequence, strain BW86 accumulated up to 57 % cyanophycin per cell dry mass under the tested conditions, which is the highest yield of cyanophycin reported to date. Strain BW86 produced cyanophycin in a molecular mass range of 25 to >100 kDa; the wild-type produced the polymer in a range of 30 to >100 kDa.ConclusionsThe high yield and high molecular mass of cyanophycin produced by strain BW86 along with the low nutrient requirements of cyanobacteria make it a promising means for the biotechnological production of cyanophycin. This study furthermore demonstrates the feasibility of metabolic pathway engineering using the PII signaling protein, which occurs in numerous bacterial species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0384-4) contains supplementary material, which is available to authorized users.

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 250ng/mL nisin in M17L medium supplemented with 20mM arginine and 10mM 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.