Biology subjects

Forchhammer, K.

Publications and source records attributed to Forchhammer, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Conversion of CO2 into valuable products: Engineering the PirC-PGAM switch in cyanobacteria to direct carbon flux into desired products

BackgroundIn response to rising CO2 emissions driving global warming, there is an urgent need for a transition toward a sustainable bioeconomy. Photo-biotechnological processes based on oxygenic photosynthesis hold high potential for achieving CO2 neutrality and in this regard, cyanobacteria have emerged as promising biocatalysts. Rational metabolic engineering of cyanobacteria depends on a thorough understanding of native regulatory mechanisms governing primary metabolism, which can limit metabolic flux through specific pathways and, consequently, the formation of target products. Recent insights have identified a key regulatory node at the 2,3-bisphosphogylcerate-independent phosphoglycerate mutase (PGAM) reaction, where the metabolic flux from newly fixed carbon is redirected from the Calvin-Benson-Bassham (CBB) cycle towards lower glycolysis. This metabolic valve is controlled by the small inhibitor protein PirC, whose binding to PGAM is determined by the central signal transduction protein PII. ResultsIn this study, we exploit the PirC-PGAM interaction as a novel target for regulatory metabolic engineering in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Chassis strains with engineered control of PGAM, defined as PGAM-ON or PGAM-OFF states, were generated using two complementary approaches: tuning pgam gene expression and modulating PirC abundance to regulate PGAM activity. The effectiveness of this regulatory engineering strategy was demonstrated by redirecting carbon flux toward two representative, naturally occurring products: sucrose, produced via gluconeogenesis fueled by the Calvin-Benson-Bassham (CBB) cycle, and succinate, an intermediate of the tricarboxylic acid (TCA) cycle. Narrowing the PGAM valve resulted in a threefold increase in sucrose accumulation. In contrast, opening the PGAM valve by relieving PGAM inhibition through pirC deletion or separate pgam overexpression resulted in up to an 18-fold increase in succinate excretion. Furthermore, similar genetic configurations were applied to enhance production of a heterologous compound, isoprene, derived from pyruvate. ConclusionsThis study establishes the PGAM valve as a tunable control point for the rational re-direction of carbon flux in Synechocystis and highlights small regulatory proteins as powerful targets for metabolic engineering. Together, these findings provide proof of concept for an advanced level of molecular engineering in cyanobacteria and to fully harness their biocatalytic potential in future photosynthesis-driven biotechnological applications.

bioengineering↗

bioRxiv2025

The ABCs of Cyanobacterial Glycogen: In Vitro Modelling of Glycogen Synthesis and Functional Divergence of Glycogen Synthases in Synechocystis sp. PCC 6803

Glycogen is the principal carbon reserve in Synechocystis sp. PCC 6803. We reconstituted its biosynthetic pathway in vitro--GlgC (Glucose-1-phosphate adenylyltransferase), two glycogen synthase isoenzymes (GlgA1, GlgA2) and the branching enzyme GlgB--to define how supply, polymerisation and branching set flux and product structure. GlgA2 shows higher specific activity and cooperates with GlgB-generated branched primers, whereas GlgA1 has higher substrate affinity and responds more to primer concentration. Product profiling links mechanism to architecture: GlgA1 produces more-branched glycogen, while GlgA2 yields longer, less-branched polymers, with GlgB biasing utilisation towards GlgA2. The complementary behaviours of GlgA1 and GlgA2 provide capacity for rapid accumulation versus steady-state maintenance and offer dynamic metabolic levers to tune glycogen content and architecture in cyanobacteria.

biochemistry↗

bioRxiv2025

Cultivation in a Natural Microbial Community Enhances the Industrial Performance of a Genetically Engineered Cyanobacterium for Bioplastic Production

Large-scale production of polyhydroxybutyrate (PHB), a biodegradable bioplastic, using genetically engineered cyanobacteria offers a sustainable alternative to petrochemical-derived plastics. However, monoculture-based phototrophic systems face major limitations, such poor resilience in large-scale reactors, hindering industrial upscaling. To address these challenges, we established a hybrid photosynthetic microbiome by replacing the native cyanobacterium of a natural microbial consortium with a genetically engineered Synechocystis strain optimized for PHB production. This new community retained the ecological structure and stability of the original microbiome while gaining synthetic production capacity. Compared to the axenic strain, the hybrid system exhibited enhanced robustness under abiotic stress, including light and temperature fluctuations, and improved tolerance to operational instability. These features made it suitable for upscaling and application in non-sterile environments. The hybrid microbiome sustained PHB production in scaled photobioreactors, reaching up to 32% PHB per cell dry weight (CDW) equal to [~]230 mg L-1 under fully photoautotrophic conditions. Production was also achieved under dark conditions with acetate supplementation, highlighting the systems metabolic flexibility. This work demonstrates the successful integration of an engineered phototroph into a stable native microbiome, positioning hybrid communities as powerful platform for industrial biotechnology.

microbiology↗

bioRxiv2025

Native metabolomics identifies pteridines as CutA ligands and modulators of copper binding

CutA, a conserved protein across all domains of life, has long been linked to copper tolerance in Escherichia coli, though recent studies question this association. To clarify its function, we studied cutA knockout mutants from two phylogenetically distant species, E. coli and Synechococcus elongatus PCC 7942, using phenotyping combined with targeted and untargeted metabolomics. Native metabolomics of cell extracts revealed the lumazine dehydroxyxanthopterin B2, a previously uncharacterized pteridine, to bind CutA in both species. Based on these results, we identified other pteridines, including the essential cofactor tetrahydrobiopterin, as ligands of CutA proteins. In the presence of pterins, we observed higher affinity of CutA to copper ions. These findings, alongside the known role of pteridines as redox shuttles, suggest a previously unrecognized role for CutA in coordinating copper homeostasis and redox balance via pteridine metabolism. SignificanceWe identified the molecular class of pteridines as natural ligands of CutA, including the so far unknown lumazine dehydroxyxanthopterin B2. Pteridines are known redox shuttles involved in various cellular processes such as cofactors for redox enzymes. Our data showed increased copper binding to CutA in the presence of pteridines. Together, these results suggest that pteridines are physiological ligands of CutA that may modulate copper binding and redox homeostasis.

microbiology↗

bioRxiv2025

Fructose-1,6-bisphosphatase (FBPase) fine-tunes heterotrophic growth in cyanobacteria

Cyanobacteria switch their carbon metabolism between photoautotrophy and heterotrophy during diurnal cycles. In cyanobacteria, the classical glycolytic control point is characterized by two catabolic phosphofructokinases (PFKs) and a bifunctional anabolic fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (F/SBPase; slr2094) catalyzing two key reactions in the Calvin-Benson-Bassham (CBB) cycle. In addition, Synechocystis possesses a fructose-1,6-bisphosphatase (FBPase; slr0952) with yet unknown physiological function and biochemical properties. Hence, our aim was to investigate the FBPase and the interplay of the four enzymes in photoautotrophic and heterotrophic carbon metabolism. We discovered that FBPase is specific for FBP, showing no SBPase activity, and unlike F/SBPase does not exhibit any biochemical regulatory properties. Growth studies with deletion mutants revealed that FBPase and PFKs play a major role under heterotrophic conditions. In contrast to F/SBPase, FBPase is not involved in the CBB cycle, but instead fine-tunes heterotrophic growth. Transaldolase cannot replace the function of SBPase in the CBB cycle. In conclusion, the classical Embden-Meyerhoff-Parnass pathway control point, which is known to be mediated by the antagonistic enzyme pair PFK and FBPase in heterotrophic bacteria and eukaryotes, is also present in Synechocystis. We found redox-insensitive FBPases from plant chloroplasts to be closely related to Synechocystis FBPase, indicating that they might serve a similar function. HighlightSynechocystis fructose-1,6-bisphosphatase (slr0952) is unlike fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (slr2094) monofunctional, not redox-regulated, and supports heterotrophy in darkness, despite catalyzing an anabolic reaction. Thus, presumably Slr2094 alone drives two Calvin-Benson-Bassham cycle key reactions.

plant biology↗

bioRxiv2024

Structural elements of cyanobacterial co-factor-independent phosphoglycerate mutase that mediate regulation by PirC

The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been identified as a crucial regulating key point in the carbon storage metabolism of cyanobacteria. Upon nitrogen starvation, the iPGAM is inhibited by the PII-interacting regulator PirC, released from its interaction partner PII due to elevated 2-oxoglutarate levels. In-silico analysis of 338 different iPGAMs revealed a deep-rooted distinctive evolution of iPGAMs in cyanobacteria. Remarkably, cyanobacterial iPGAMs possess a unique loop structure and an extended C-terminus. Our analysis suggests that iPGAM forms a complex with three individual PirC monomers. Complex affinity is affected by the unique loop and the C-terminal structural elements. A C-terminal truncated enzyme showed loss of control by PirC and two-fold increased enzymatic activity compared to the iPGAM-WT. By contrast, deleting the loop structure drastically reduced the activity of this variant. By replacing the WT iPGAM in Synechocystis with different iPGAM variants, in which these structural elements were deleted, it became apparent that deletion of the C-terminal element showed a similar overproduction of polyhydroxybutyrate as deletion of the iPGAM-regulator PirC. However, in contrast to the latter, these strains showed higher over-all biomass accumulation, making them a better chassis for a production strain for PHB or other valuable substances than the PirC-deficient mutant. These findings significantly contribute to our understanding of the metabolic pathways in cyanobacteria and open up new avenues for further research in this field, inspiring future investigations and discoveries. ImportanceThe primordial cyanobacteria were responsible for developing oxygenic photo-synthesis early in evolution. Through endosymbiosis, they further evolved into the chloroplasts found in the plant kingdom. Many metabolic pathways within chloroplasts originated from cyanobacteria. However, differences emerged during their long separate evolution, providing insights into the endosymbiotic process. In the metabolic pathways involving fixed CO2, the co-factor-independent phosphoglycerate mutase (iPGAM) plays a crucial role by directing the first CO2 fixation product, 3-phosphoglycerate, towards critical anabolic path-ways. Our findings reveal a distinct evolution of iPGAM within oxygenic photo-synthetic organisms. We have identified two specific segments in cyanobacterial iPGAMs that tightly control the cellular carbon/nitrogen state through a specific protein interactor (PiC). This understanding of iPGAM has allowed us to engineer cyanobacterial strains with altered carbon fluxes. Since cyanobacteria can directly convert CO2 into valuable products, our results demonstrate a novel approach for developing a chassis for biotechnical use.

microbiology↗

bioRxiv2024

A novel recombinant PHB production platform in filamentous cyanobacteria avoiding nitrogen starvation while preserving cell viability

During the past decades, the importance of developing sustainable, carbon dioxide (CO2)-neutral and biodegradable alternatives to conventional plastic has become evident in the context of global pollution issues. Therefore, heterotrophic bacteria such as Cupriavidus sp. have been intensively explored for the synthesis of the biodegradable polymer polyhydroxybutyrate (PHB). PHB is also naturally produced by a variety of phototrophic cyanobacteria, which only need sunlight and CO2, thereby allowing a CO2 negative, eco-friendly synthesis of this polymer. However, a major drawback of the use of cyanobacteria is the need of a two-stage production process, since relevant amount of PHB synthesis only occurs after transferring the cultures to conditions of nitrogen starvation, which hinders continuous, large-scale production. This study aimed at generating, by means of genetic engineering, a cyanobacterium that continuously produces PHB in large amounts. We choose a genetically amenable filamentous cyanobacterium of the genus Nostoc sp., which is a diazotrophic cyanobacterium, capable of atmospheric nitrogen (N2) fixation but naturally does not produce PHB. We transformed this Nostoc strain with various constructs containing the PHB synthesis operon from Cupriavidus necator H16. In fact, while the transformants initially produced PHB, the PHB-producing strains rapidly lost cell viability. Therefore, we next attempted further optimization of the biosynthetic gene cluster. Finally, we succeeded in stabilized PHB production, whilst simultaneously avoiding decreasing cell viability. In conclusion, the recombinant Nostoc strain constructed in the present work constitutes the first example of a continuous and stable PHB production platform in cyanobacteria, which has been decoupled from nitrogen starvation and, hence, harbours great potential for sustainable, industrial PHB production.

microbiology↗

bioRxiv2024

Structural basis of the allosteric regulation of cyanobacterial glucose-6-phosphate dehydrogenase by the redox sensor OpcA

The oxidative pentose phosphate (OPP) pathway is a fundamental carbon catabolic route for generating reducing power and metabolic intermediates for biosynthetic processes. In addition, its first two reactions form the OPP shunt, which replenishes the Calvin-Bassham cycle under certain conditions. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting reaction of this metabolic route. In photosynthetic organisms, G6PDH is redox-regulated to allow fine-tuning and to prevent futile cycles while carbon is being fixed. In cyanobacteria, regulation of G6PDH requires the redox protein OpcA, but the underlying molecular mechanisms behind this allosteric activation remain elusive. Here, we used enzymatic assays and in vivo interaction analyses to show that OpcA binds G6PDH under different environmental conditions. However, complex formation enhances G6PDH activity when OpcA is oxidized and inhibits it when OpcA is reduced. To understand the molecular basis of this regulation, we used cryogenic electron microscopy to determine the structure of cyanobacterial G6PDH and the G6PDH-OpcA complex. OpcA binds the G6PDH tetramer and induces conformational changes in the active site of G6PDH. The redox sensitivity of OpcA is achieved by intramolecular disulfide bridge formation, which influences the allosteric regulation of G6PDH. In vitro assays reveal that the level of G6PDH activation depends on the number of bound OpcA molecules, which implies that this mechanism allows delicate fine-tuning. Our findings unveil a novel and unique molecular mechanism governing the regulation of the OPP pathway in cyanobacteria.

molecular biology↗

bioRxiv2023

Diurnal rhythm causes metabolic crises in the cyanobacterial mutants of c-di-AMP signalling cascade

In nature, the photoautotrophic lifestyle of cyanobacteria has to cope with the successive diurnal changes in light supply. Light supply throughout the day enables photosynthesis and glycogen biosynthesis, while night phases require the switch to a heterotrophic-like lifestyle relying on glycogen catabolism. We previously highlighted a unique function of the carbon control protein, SbtB, and its effector molecule c-di-AMP, for the nighttime survival of cyanobacteria through the regulation of glycogen anabolism. However, the extent to which c-di-AMP and SbtB impact the cellular metabolism for day-night survivability remained elusive. To gain better understanding of cellular processes regulated by SbtB or c-di-AMP, we compared the metabolomic and proteomic landscapes of {Delta}sbtB and the c-di-AMP-free ({Delta}dacA) mutants of the model strain Synechocystis sp. PCC 6803. While our results indicate that the cellular role of SbtB is restricted to carbon/glycogen metabolism, the diurnal lethality of {Delta}dacA seems to be a sum of dysregulation of multiple metabolic processes. These processes include photosynthesis and redox regulation, which lead to elevated levels of intracellular ROS and glutathione. Further, we show an impact of c-di-AMP on central carbon as well as on nitrogen metabolism. Effects on nitrogen metabolism are linked to reduced levels of the global nitrogen transcription regulator NtcA and highlighted by an imbalance of the glutamine to glutamate ratio as well as reduced metabolite levels of the arginine pathway. We further identified the HCO3- uptake systems, BicA and BCT1 as novel SbtB targets, in agreement with its broader role in regulating carbon homeostasis.

microbiology↗

bioRxiv2023

PII signal transduction superfamily acts as a valve plug to control bicarbonate and ammonia homeostasis among different bacterial phyla

Life on Earth relies on carbon and nitrogen assimilation by RubisCO and GS-GOGAT enzymes, respectively, whose activities depend on a constant supply of inorganic carbon (Ci) and nitrogen (N). Members of the PII signal transduction superfamily are among the most ancient and widespread cell signaling proteins in nature. One of their most highly conserved functions is controlling Ci- and N-transporters, a feature found in different phyla of the Archaea and in both Gram-positive and Gram-negative bacteria. Recently, we identified the PII-like protein SbtB as Ci-sensing module, mainly controlling the HCO3- transporter SbtA in cyanobacteria. Similar to canonical PII proteins, SbtB is able to bind the adenine nucleotides ATP and ADP. Unlike those, it also binds AMP and preferentially binds the second messenger cAMP and c-di-AMP. The functional significance of the binding of different adenyl-nucleotides to SbtB has remained elusive, particularly in the context of the interaction of SbtB with SbtA. By a combination of structural, biochemical and physiological analysis, we revealed that by binding to SbtA, SbtB acts as unidirectional valve, preventing the reverse transport of intracellular enriched bicarbonate. This mechanistic principle holds true for the PII protein from Bacillus acting on the ammonium transporter AmtB, suggesting an evolutionary conserved role for PII superfamily proteins in controlling unidirectional flow of different transporters.

microbiology↗

bioRxiv2022

On the role and regulation of glycogen catabolic isoenzymes in Synechocystis sp. PCC6803

Glycogen serves as the main carbon storage polymer in many organisms and is widespread across all domains of life. In cyanobacteria, glycogen degradation is crucial for metabolic transitions during dark phases or during resuscitation from nitrogen starvation. Like many other cyanobacteria, Synechocystis sp. PCC 6803 possesses multiple homologues of glycogen catabolizing enzymes, though their specific roles and regulatory mechanisms are only partially understood. Here we demonstrate through biochemical analysis that the glycogen phosphorylase GlgP1, known to promote high temperature acclimation, is uniquely regulated by a C-terminal redox switch found certain cyanobacteria. This is the first evidence of redox regulation of a prokaryotic glycogen degrading enzyme. Notably, GlgP1 is activated via oxidation by reactive oxygen species and inactivated by reducing agents, with thioredoxin being the most effective inhibitor tested. The physiological implications of this redox regulation are discussed. Additionally, a biochemically characterization of the two glycogen debranching isoenzymes GlgX1 and GlgX2 revealed that only GlgX1 exhibits debranching activity, while GlgX2 does not. Mutant analysis confirmed that GlgX1 plays an essential role in glycogen mobilization, being crucial for resuscitation from chlorosis and survival during extended dark periods. In contrast the physiological function of GlgX2 remains unclear.

microbiology↗

bioRxiv2022

Carbon signaling protein SbtB possesses redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is expressed with the conserved PII-like protein, SbtB, to augment intracellular Ci levels for efficient CO2-fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cAMP, known as carbon-status indicator, and c-di-AMP, involved in global cellular homeostasis. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge. We previously implied a redox-regulatory function of this extension, which we now call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of the hydrolysis reactions from ATP over ADP to AMP in crystal-lographic snapshots and reveal the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin TrxA, suggesting that it is generally linked to cellular metabolism. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch-point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding, but also from the day/night status reported by the C-terminal redox-switch.

molecular biology↗

bioRxiv2021

Glucose-1,6-bisphosphate, a key metabolic regulator, is synthesized by a distinct family of α-D-phosphohexomutases widely distributed in prokaryotes

The reactions of -D-phosphohexomutases (PHM) are ubiquitous, key to primary metabolism and essential for several processes in all domains of life. The functionality of these enzymes relies on an initial auto-phosphorylation step which requires the presence of -D-glucose-1,6-bisphosphate (Glc-1,6-BP). While well investigated in vertebrates, the origin of this activator compound in bacteria is unknown. Here we show that the Slr1334 protein from the unicellular cyanobacterium Synechocysitis sp. PCC 6803 is a Glc-1,6-BP-synthase. Biochemical analysis revealed that Slr1334 efficiently converts fructose-1,6-bisphosphate (Frc-1,6-BP) and -D-glucose-1-phosphate/-D-glucose-6-phosphate into Glc-1,6-BP and also catalyzes the reverse reaction. As inferred from phylogenetic analysis, the slr1334 product belongs to a primordial subfamily of PHMs that is present especially in deeply branching bacteria and also includes human commensals and pathogens. Remarkably, the homologue of Slr1334 in the human gut bacterium Bacteroides salyersiae catalyzes the same reaction, suggesting a conserved and essential role for the members of this PHM subfamily.

microbiology↗

bioRxiv2021

Regulatory phosphorylation site tunes Phosphoglucomutase 1 as a metabolic valve to control mobilization of glycogen stores.

Regulation of glycogen metabolism is of vital importance in organisms of all three kingdoms of life. Although the pathways involved in glycogen synthesis and degradation are well known, many regulatory aspects around the metabolism of this polysaccharide remain undeciphered. Here, we used the unicellular cyanobacterium Synechocystis as a model to investigate how glycogen metabolism is regulated in nitrogen-starved dormant cells, which entirely rely on glycogen catabolism to resume growth upon nitrogen repletion. We identified phosphoglucomutase 1 (PGM1) as a key regulatory point in glycogen metabolism, and post-translational modification as an essential mechanism for controlling its activity. We could show that PGM1 is phosphorylated at a peripheral residue (Ser 47) during nitrogen starvation, which inhibits its activity. Inactivation of PGM1 by phosphorylation at Ser 47 prevents premature degradation of the glycogen stores and appears to be essential for survival of Synechocystis in the dormant state. Remarkably, this regulatory mechanism seems to be evolutionary conserved in PGM1 enzymes, from bacteria to humans. Significance statementIn this study, we identified phosphoglucomutase 1 (PGM1) as a central metabolic valve that regulates the utilization of the glycogen reserves. We showed that post-translational modification of PGM1 via phosphorylation at a peripheral residue is a key, evolutionary-conserved regulatory mechanism that controls PGM1 activity and the mobilization of the glycogen stores.

microbiology↗

bioRxiv2020

5-Deoxyadenosine Salvage by Promiscuous Enzyme Activity leads to Bioactive Deoxy-Sugar Synthesis in Synechococcus elongatus

7-Deoxysedoheptulose is an unusual deoxy-sugar, which acts as antimetabolite of the shikimate pathway thereby exhibiting antimicrobial and herbicidal activity. It is produced by the unicellular cyanobacterium Synechococcus elongatus PCC 7942, which has a small, stream-lined genome, assumed to be free from gene clusters for secondary metabolite synthesis. In this study, we identified the pathway for the synthesis of 7-deoxysedoheptulose. It originates from 5-deoxyadenosine, a toxic byproduct of radical S-adenosylmethionine (SAM) enzymes, present in all domains of life. Thereby we identified a novel 5-deoxyadenosine salvage pathway, which first leads to the synthesis and excretion of 5-deoxyribose and subsequently of 7-deoxysedoheptulose. Remarkably, all reaction steps are conducted by promiscuous enzymes. This is a unique example for the synthesis of a bioactive compound without involving a specific gene cluster. This challenges the view on bioactive molecule synthesis by extending the range of possible compounds beyond the options predicted from secondary metabolite gene clusters.

biochemistry↗

bioRxiv2020

The essential role of sodium bioenergetics and ATP homeostasis in the developmental transitions of a cyanobacterium

The ability to resume growth after a dormant period is an important strategy for the survival and spreading of bacterial populations. Energy homeostasis is critical in the transition into and out of a quiescent state. Synechocystis sp. PCC 6803, a non-diazotrophic cyanobacterium, enters metabolic dormancy as a response to nitrogen starvation. We used Synechocystis as a model to investigate the regulation of ATP homeostasis during dormancy and unraveled a critical role for sodium bioenergetics in dormant cells. During nitrogen starvation, cells reduce their ATP levels and engage sodium bioenergetics to maintain the minimum ATP content required for viability. When nitrogen becomes available, energy requirements rise, and cells immediately increase ATP levels employing sodium bioenergetics and glycogen catabolism. These processes allow them to restore the photosynthetic machinery and resume photoautotrophic growth. Our work reveals a precise regulation of the energy metabolism essential for bacterial survival during periods of nutrient deprivation.

microbiology↗

bioRxiv2020

The novel PII-interacting protein PirA regulates flux into the cyanobacterial ornithine-ammonia cycle

Among prokaryotes, cyanobacteria have an exclusive position due to the fact that they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g. they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here we reveal another small, 51 amino acid protein, which is encoded by the ssr0692 gene, to regulate flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls the entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK). We suggest that Ssr0692 competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it PII-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen-control transcription factor NtcA. Consistently, deletion of PirA affects the cell to balance metabolite pools of the OAC in response to ammonium shocks. Moreover, its interaction with PII requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein-interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell. ImportanceCyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g. as major primary producers. Due to their photosynthetic lifestyle cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis in the cyanobacterial model strain Synechocystis sp. PCC 6803. Beside its role as proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. The obtained results will therefore not only enhance our understanding of flux control in these organisms, it will also help to provide a scientific fundament for targeted metabolic engineering and hence the design of photosynthesis-driven biotechnological applications.

microbiology↗

bioRxiv2020

Maximizing PHB content in Synechocystis sp. PCC 6803: development of a new photosynthetic overproduction strain.

PHB (poly-hydroxy-butyrate) represents a promising bioplastic variety with good biodegradation properties. Furthermore, PHB can be produced completely carbon-neutral when synthesized in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This model strain has a long history of various attempts to further boost its low amounts of produced intracellular PHB of ~15 % per cell-dry-weight (CDW). We have created a new strain that lacks the regulatory protein PirC (gene product of sll0944), which causes a rapid conversion of the intracellular glycogen pools to PHB under nutrient limiting conditions. To further improve the intracellular PHB content, two genes from the PHB metabolism, phaA and phaB from the known production strain Cupriavidus necator, were introduced under the regime of the strong promotor PpsbA2. The created strain, termed PPT1 ({Delta}sll0944-REphaAB), produced high amounts of PHB under continuous light as well under day-night rhythm. When grown in nitrogen and phosphor depleted medium, the cells produced up to 63 % / CDW. Upon the addition of acetate, the content was further increased to 81 % / CDW. The produced polymer consists of pure PHB, which is highly isotactic. The achieved amounts were the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

microbiology↗