Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC.

BACKGROUND: PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon-neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW). RESULTS: We have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic. CONCLUSION: The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

Interaction of the Nitrogen Regulatory Protein GlnB (PII) with Biotin Carboxyl Carrier Protein (BCCP) Controls Acetyl-CoA Levels in the Cyanobacterium Synechocystis sp. PCC 6803.

The family of PII signal transduction proteins (members GlnB, GlnK, NifI) plays key roles in various cellular processes related to nitrogen metabolism at different functional levels. Recent studies implied that PII proteins may also be involved in the regulation of fatty acid metabolism, since GlnB proteins from Proteobacteria and from Arabidopsis thaliana were shown to interact with biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase (ACC). In case of Escherichia coli ACCase, this interaction reduces the kcat of acetyl-CoA carboxylation, which should have a marked impact on the acetyl-CoA metabolism. In this study we show that the PII protein of a unicellular cyanobacterium inhibits the biosynthetic activity of E. coli ACC and also interacts with cyanobacterial BCCP in an ATP and 2-oxoglutarate dependent manner. In a PII mutant strain of Synechocystis strain PCC 6803, the lacking control leads to reduced acetyl-CoA levels, slightly increased levels of fatty acids and formation of lipid bodies as well as an altered fatty acid composition.

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, ß-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.

Metabolic pathway engineering using the central signal processor PII.

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 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). 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 cyanophycin. This study furthermore demonstrates the feasibility of metabolic pathway engineering using the PII signaling protein, which occurs in numerous bacterial species.

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 Changes in Synechocystis PCC6803 upon Nitrogen-Starvation: Excess NADPH Sustains Polyhydroxybutyrate Accumulation.

Polyhydroxybutyrate (PHB) is a common carbon storage polymer among heterotrophic bacteria. It is also accumulated in some photoautotrophic cyanobacteria; however, the knowledge of how PHB accumulation is regulated in this group is limited. PHB synthesis in Synechocystis sp. PCC 6803 is initiated once macronutrients like phosphorus or nitrogen are limiting. We have previously reported a mutation in the gene sll0783 that impairs PHB accumulation in this cyanobacterium upon nitrogen starvation. In this study we present data which explain the observed phenotype. We investigated differences in intracellular localization of PHB synthase, metabolism, and the NADPH pool between wild type and mutant. Localization of PHB synthase was not impaired in the sll0783 mutant; however, metabolome analysis revealed a difference in sorbitol levels, indicating a more oxidizing intracellular environment than in the wild type. We confirmed this by directly measuring the NADPH/NADP ratio and by altering the intracellular redox state of wild type and sll0783 mutant. We were able to physiologically complement the mutant phenotype of diminished PHB synthase activity by making the intracellular environment more reducing. Our data illustrate that the NADPH pool is an important factor for regulation of PHB biosynthesis and metabolism, which is also of interest for potential biotechnological applications.

Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichia coli as revealed by LILBID mass spectrometry.

Energy-coupled transporters in the outer membrane of Escherichia coli and other Gram-negative bacteria allow the entry of scarce substrates, toxic proteins, and bacterial viruses (phages) into the cells. The required energy is derived from the proton-motive force of the cytoplasmic membrane, which is coupled to the outer membrane via the ExbB-ExbD-TonB protein complex. Knowledge of the structure of this complex is required to elucidate the mechanisms of energy harvesting in the cytoplasmic membrane and energy transfer to the outer membrane transporters. Here we solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the detergent undecyl maltoside. Using laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), we determined at moderate desorption laser energies the oligomeric structure of ExbB to be mainly hexameric (ExbB(6)), with minor amounts of trimeric (ExbB(3)), dimeric (ExbB(2)), and monomeric (ExbB(1)) oligomers. Under the same conditions ExbB-ExbD formed a subcomplex consisting of ExbB(6)ExbD(1), with a minor amount of ExbB(5)ExbD(1). At higher desorption laser intensities, ExbB(1) and ExbD(1) and traces of ExbB(3)ExbD(1), ExbB(2)ExbD(1), ExbB(1)ExbD(1), ExbB(3), and ExbB(2) were observed. Since the ExbB(6) complex and the ExbB(6)ExbD(1) complex remained stable during solubilization and subsequent chromatographic purification on nickel-nitrilotriacetate agarose, Strep-Tactin, and Superdex 200, and during native blue gel electrophoresis, we concluded that ExbB(6) and ExbB(6)ExbD(1) are subcomplexes on which the final complex including TonB is assembled.