Cyanophycin: A Nitrogen-Rich Reserve Polymer

Cyanophycin is a nitrogen/carbon reserve polymer present in most cyanobacteria as well as in a few heterotrophic bacteria. It is a non-ribosomally synthesized polyamide consisting of aspartate and arginine (multi-l-arginyl-poly-l-aspartic acid). The following chapter provides an overview of the characteristics and occurrence of cyanophycin in cyanobacteria. Information about the enzymes involved in cyanophycin metabolism and the regulation of cyanophycin accumulation is also summarized. Herein, we focus on the main regulator, the PII signal transduction protein and its regulation of arginine biosynthesis. Since cyanophycin could be used in various medical or industrial applications, it is of high biotechnological interest. In the last few years, many studies were published aiming at the large-scale production of cyanophycin in different heterotrophic bacteria, yeasts and plants. Recently, a cyanobacterial production strain has been reported, which shows the highest so ever reported cyanophycin yield. The potential and possibilities of biotechnological cyanophycin production will be reviewed in this chapter

Regulation of the carbon/nitrogen storage polymer cyanophycin by the signal transduction protein PII in Synechocystis sp. PCC 6803

Cyanobacteria are one of the deepest branching bacterial phyla on earth. Today, cyanobacteria occupy almost all illuminated habitats, where they utilize various nitrogen sources. Nitrogen assimilation strictly depends on carbon and nitrogen availability and requires a fine-tuned regulatory network involving the PII signal transduction protein. In the present study I focused on the regulation of the carbon/nitrogen storage polymer cyanophycin by the PII signaling protein. Cyanophycin is a non-ribosomal synthesized polyamide consisting of arginine and aspartate. Cyanophycin accumulation depends on the arginine availability. PII controls the rate limiting step of arginine biosynthesis by regulating the key enzyme N-acetylglutamate kinase (NAGK). A PII variant with a single point mutation (I86N) was previously identified as a NAGK super activator in vitro. By introducing PII(I86N) in Synechocystis sp. PCC 6803, we created a strain which strongly overproduces cyanophycin up to 57 % of the cell dry mass. Since cyanophycin is a bio-polymer with high industrial interest, we performed several process optimization studies. During these studies, we observed that Synechocystis sp. PCC 6803 harboring the PII (I86N) variant showed impaired ammonium utilization. By analyzing this behavior, we could clarify that PII regulates ammonium uptake by interacting the with Amt1 ammonium permease. We could further demonstrate that PII mediates the light and ammonium dependent inhibition of nitrate uptake by interacting with the NrtC and NrtD subunits of the nitrate/nitrite transporter NrtABCD. During this study, we could also identify the UrtE subunit of the ABC-type urea transporter UrtABCDE as novel PII target. The interaction of PII with the UrtE subunit regulates the urea uptake in cyanobacteria. The occurrence of cyanophycin in cyanobacteria was known for more than 100 years; however, the biological function remained largely uninvestigated. During localization studies in Synechocystis sp. PCC 6803, we could show that the cyanophycin synthesizing enzyme CphA resides in an active and inactive state. When CphA was inactive, it localized diffusely in the cytoplasm. When cyanophycin synthesis was triggered, CphA first aggregated into foci and was later localized on the surface of the cyanophycin granules. During degradation, CphA dissociates from the granule surface. Under standard laboratory conditions, the ability to synthesize cyanophycin did not confer a fitness advantage, however with a fluctuating and limiting nitrogen supplementation in combination with day/night cycles, the accumulation of the polymer provides a clear fitness advantage. Furthermore cyanophycin acts as a temporary nitrogen storage which allows nitrogen assimilation during the night. The accumulated cyanophycin can be subsequently used as an internal nitrogen source during the day.In der Domäne der Bakterien bilden die Cyanobakterien eine der ältesten Abteilungen und sind heute in nahezu allen lichtzuganglichen Habitaten angesiedelt. Cyanobakterien können eine Vielzahl organischer und anorganischer Stickstoffquellen verstoffwechseln, wobei die Stickstoffassimilation über die intrazellulare Kohlenstoff- und Stickstoffbalance reguliert wird. Das PII Signaltransduktions-Proteins erfüllt hierbei eine zentrale Rolle. Die vorliegende Arbeit befasst sich mit der Regulation des Kohlenstoff-/Stickstoff- Speicherpolymers Cyanophycin durch das PII Protein. Cyanophycin ist ein nicht-ribosomal synthetisiertes Polyamid bestehend aus Arginin und Asparaginsäure. Die Akkumulation von Cyanophycin ist von der Argininverfügbarkeit abhängig. Das PII Protein reguliert die Argininbiosynthese durch die Interaktion mit der N-Acetylglutamat Kinase (NAGK), dem Schlüssel-Enzym der Argininbiosynthese. Vorangegangene Studien zeigten, dass eine Punktmutation (I86N) in PII die aktivierende Wirkung auf NAGK in vitro deutlich verstärkt. Folglich konnten wir durch das Einbringen dieser PII (I86N) Variante in Synechocystis sp. PCC 6803 einen Stamm kreieren, der eine erhebliche Überproduktion von Arginin und ein Cyanophycinanteil von bis zu 57 % zur Zelltrockenmasse aufwies. Da Cyanophycin von industriellem Interesse ist, wurden verschiedene Prozessoptimierungsstudien angefertigt. Im Zuge dieser Studien stellte sich heraus, dass die PII (I86N) Variante die Ammoniumverwertung von Synechocystis sp. PCC 6803 negativ beeinflusst. Bei näheren Untersuchungen zeigte sich, dass PII die Ammoniumaufnahme durch die Interaktion mit der Amt1 Ammoniumpermease reguliert. Wir konnten ebenfalls demonstrieren, dass PII die licht- und ammoniumabhängige Nitrataufnahme-Inhibition durch die Interaktion mit der NrtC und NrtD Untereinheit des Nitrit/Nitrat Transporter NrtABCD vermittelt. Darüber hinaus konnte nachgewiesen werden, dass PII mit der UrtE Untereinheit des ABC-Typ Urea Transporter UrtABCDE interagiert und hierdurch die Ureaaufnahme reguliert. Das Auftreten von Cyanophycin in Cyanobakterien wurde bereits vor mehr als 100 Jahren beschrieben, jedoch blieb bis heute dessen biologische Funktion ungeklärt. Lokalisationsstudien in Synechocystis sp. PCC 6803, bei denen das Cyanophycin synthetisierende Enzym CphA mit eGFP fusioniert wurde, ergaben, dass CphA eine aktive und inaktive Form aufweist. In seiner inaktiven Form ist CphA diffus im Zytoplasma verteilt. Wird die Cyanophycinakkumulation induziert, aggregiert CphA zunächst in Focis und ist später an der Oberflache der Cyanophycingranula lokalisiert. Während dem Abbau dissoziiert CphA von der Granulaoberfläche und geht wieder in die inaktive Form über. Überraschenderweise hatten Cyanophycin freien Zellen unter Standard-Laborbedingungen einen Wachstumsvorteil gegenüber dem Wild-Typ. Um die Situation eines natürlichen Habitats zu imitieren, kultivierten wir beide Stamme bei schwankender Stickstoffversorgung zusammen mit Tag/Nacht-Zyklen. Unter diesen Bedingungen hatten die Cyanophycin akkumulierenden Zellen einen klaren Vorteil. Darüber hinaus konnten wir vorweisen, dass Cyanophycin als temporarer Stickstoffspeicher dient, der die Stickstoffassimilation in der Nacht ermöglicht. In der Nacht akkumuliertes Cyanophycin kann während des Tages als Stickstoffquelle genutzt werden

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp Strain PCC 6803

Cyanobacteria are photoautotrophic microorganisms which fix atmospheric carbon dioxide via the Calvin-Benson cycle to produce carbon backbones for primary metabolism. Fixed carbon can also be stored as intracellular glycogen, and in some cyanobacterial species like Synechocystis sp. strain PCC 6803, polyhydroxybutyrate (PHB) accumulates when major nutrients like phosphorus or nitrogen are absent. So far only three enzymes which participate in PHB metabolism have been identified in this organism, namely, PhaA, PhaB, and the heterodimeric PHB synthase PhaEC. In this work, we describe the cyanobacterial PHA surface-coating protein (phasin), which we term PhaP, encoded by ssl2501. Translational fusion of Ssl2501 with enhanced green fluorescent protein (eGFP) showed a clear colocalization to PHB granules. A deletion of ssl2501 reduced the number of PHB granules per cell, whereas the mean PHB granule size increased as expected for a typical phasin. Although deletion of ssl2501 had almost no effect on the amount of PHB, the biosynthetic activity of PHB synthase was negatively affected. Secondary-structure prediction and circular dichroism (CD) spectroscopy of PhaP revealed that the protein consists of two α-helices, both of them associating with PHB granules. Purified PhaP forms oligomeric structures in solution, and both α-helices of PhaP contribute to oligomerization. Together, these results support the idea that Ssl2501 encodes a cyanobacterial phasin, PhaP, which regulates the surface-to-volume ratio of PHB granules

Metabolic pathway engineering using the central signal processor P-II

Background: PII 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 polyas-partic 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). Results: In 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. Conclusions: The 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 cyano-phycin. This study furthermore demonstrates the feasibility of metabolic pathway engineering using the PII signaling protein, which occurs in numerous bacterial species