The transcriptional regulator RbcR controls ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes in the cyanobacterium Synechocystis sp. PCC 6803.

Oxygenic photosynthesis evolved in cyanobacteria, primary producers of striking ecological importance. Like plants, cyanobacteria use the Calvin-Benson-Bassham cycle for CO2 fixation, fuelled by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). In a competitive reaction this enzyme also fixes O2 which makes it rather ineffective. To mitigate this problem, cyanobacteria evolved a CO2 concentrating mechanism (CCM) to pool CO2 in the vicinity of RuBisCO. However, the regulation of these carbon (C) assimilatory systems is understood only partially. Using the model Synechocystis sp. PCC 6803 we characterized an essential LysR-type transcriptional regulator encoded by gene sll0998. Transcript profiling of a knockdown mutant revealed diminished expression of several genes involved in C acquisition, including rbcLXS, sbtA and ccmKL encoding RuBisCO and parts of the CCM, respectively. We demonstrate that the Sll0998 protein binds the rbcL promoter and acts as a RuBisCO regulator (RbcR). We propose ATTA(G/A)-N5 -(C/T)TAAT as the binding motif consensus. Our data validate RbcR as a regulator of inorganic C assimilation and define the regulon controlled by it. Biological CO2 fixation can sustain efforts to reduce its atmospheric concentrations and is fundamental for the light-driven production of chemicals directly from CO2 . Information about the involved regulatory and physiological processes is crucial to engineer cyanobacterial cell factories.

The sRNA NsiR4 fine-tunes arginine synthesis in the cyanobacterium Synechocystis sp. PCC 6803 by post-transcriptional regulation of PirA.

As the only oxygenic phototrophs among prokaryotes, cyanobacteria employ intricate mechanisms to regulate common metabolic pathways. These mechanisms include small protein inhibitors exerting their function by protein-protein interaction with key metabolic enzymes and regulatory small RNAs (sRNAs). Here we show that the sRNA NsiR4, which is highly expressed under nitrogen limiting conditions, interacts with the mRNA of the recently described small protein PirA in the model strain Synechocystis sp. PCC 6803. In particular, NsiR4 targets the pirA 5'UTR close to the ribosome binding site. Heterologous reporter assays confirmed that this interaction interferes with pirA translation. PirA negatively impacts arginine synthesis under ammonium excess by competing with the central carbon/nitrogen regulator PII that binds to and thereby activates the key enzyme of arginine synthesis, N-acetyl-L-glutamate-kinase (NAGK). Consistently, ectopic nsiR4 expression in Synechocystis resulted in lowered PirA accumulation in response to ammonium upshifts, which also affected intracellular arginine pools. As NsiR4 and PirA are inversely regulated by the global nitrogen transcriptional regulator NtcA, this regulatory axis enables fine tuning of arginine synthesis and conveys additional metabolic flexibility under highly fluctuating nitrogen regimes. Pairs of small protein inhibitors and of sRNAs that control the abundance of these enzyme effectors at the post-transcriptional level appear as fundamental building blocks in the regulation of primary metabolism in cyanobacteria.

Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase.

Molecular hydrogen (H2) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H2 production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H2 syntheses rely mainly on light, water, and CO2 and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H2-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O2) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O2-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O2-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O2 sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O2 tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH+ to utilize H2 as sole electron source to fix CO2. Syn_ReSH+ also was able to produce H2 under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H2 production.

A glutamine riboswitch is a key element for the regulation of glutamine synthetase in cyanobacteria.

As the key enzyme of bacterial nitrogen assimilation, glutamine synthetase (GS) is tightly regulated. In cyanobacteria, GS activity is controlled by the interaction with inactivating protein factors IF7 and IF17 encoded by the genes gifA and gifB, respectively. We show that a glutamine-binding aptamer within the gifB 5' UTR of Synechocystis sp. PCC 6803 is critical for the expression of IF17. Binding of glutamine induced structural re-arrangements in this RNA element leading to enhanced protein synthesis in vivo and characterizing it as a riboswitch. Mutagenesis showed the riboswitch mechanism to contribute at least as much to the control of gene expression as the promoter-mediated transcriptional regulation. We suggest this and a structurally related but distinct element, to be designated type 1 and type 2 glutamine riboswitches. Extended biocomputational searches revealed that glutamine riboswitches are exclusively but frequently found in cyanobacterial genomes, where they are primarily associated with gifB homologs. Hence, this RNA-based sensing mechanism is common in cyanobacteria and establishes a regulatory feedback loop that couples the IF17-mediated GS inactivation to the intracellular glutamine levels. Together with the previously described sRNA NsiR4, these results show that non-coding RNA is an indispensable component in the control of nitrogen assimilation in cyanobacteria.

The iron-stress activated RNA 1 (IsaR1) coordinates osmotic acclimation and iron starvation responses in the cyanobacterium Synechocystis sp. PCC 6803.

In nature, microorganisms are exposed to multiple stress factors in parallel. Here, we investigated the response of the model cyanobacterium Synechocystis sp. PCC 6803 to simultaneous iron limitation and osmotic stresses. Iron is a major limiting factor for bacterial and phytoplankton growth in most environments. Thus, bacterial iron homeostasis is tightly regulated. In Synechocystis, it is mediated mainly by the transcriptional regulator FurA and the iron-stress activated RNA 1 (IsaR1). IsaR1 is an important riboregulator that affects the acclimation of the photosynthetic apparatus to iron starvation in multiple ways. Upon increases in salinity, Synechocystis responds by accumulating the compatible solute glucosylglycerol (GG). We show that IsaR1 overexpression causes a reduction in the de novo GG synthesis rate upon salt shock. We verified the direct interaction between IsaR1 and the 5'UTR of the ggpS mRNA, which in turn drastically reduced the de novo synthesis of the key enzyme for GG synthesis, glucosylglycerol phosphate synthase (GgpS). Thus, IsaR1 specifically interferes with the salt acclimation process in Synechocystis, in addition to its primary regulatory function. Moreover, the salt-stimulated GgpS production became reduced under parallel iron limitation in WT - an effect which is, however, attenuated in an isaR1 deletion strain. Hence, IsaR1 is involved in the integration of the responses to different environmental perturbations and slows the osmotic adaptation process in cells suffering from parallel iron starvation.

The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7.

Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5'UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus [Formula: see text] assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.

The glucosylglycerol-degrading enzyme GghA is involved in acclimation to fluctuating salinities by the cyanobacterium Synechocystis sp. strain PCC 6803.

The ggpS gene, which encodes the key enzyme for the synthesis of the compatible solute glucosylglycerol (GG), has a promoter region that overlaps with the upstream-located gene slr1670 in the cyanobacterium Synechocystissp. PCC 6803. Like ggpS, the slr1670 gene is salt-induced and encodes a putative glucosylhydrolase. A mutant strain with a slr1670 deletion was generated and found to be unable to adapt the internal GG concentrations in response to changes in external salinities. Whereas cells of the wild-type reduced the internal pool of GG when exposed to gradual and abrupt hypo-osmotic treatments, or when the compatible solute trehalose was added to the growth medium, the internal GG pool of ∆slr1670 mutant cells remained unchanged. These findings indicated that the protein Slr1670 is involved in GG breakdown. The biochemical activity of this GG-hydrolase enzyme was verified using recombinant Slr1670 protein, which split GG into glucose and glycerol. These results validate that Slr1670, which was named GghA, acts as a GG hydrolase. GghA is involved in GG turnover in fluctuating salinities, and similar proteins are found in the genomes of other GG-synthesizing cyanobacteria.

Ethylene production in Synechocystis sp. PCC 6803 promotes phototactic movement.

Ethylene is a gaseous signal sensed by plants and bacteria. Heterologous expression of the ethylene-forming enzyme (EFE) from Pseudomonas syringae in cyanobacteria leads to the production of ethylene under photoautotrophic conditions. The recent characterization of an ethylene-responsive signalling pathway affecting phototaxis in the cyanobacterium Synechocystis sp. PCC 6803 implied that biotechnologically relevant ethylene synthesis may induce regulatory processes that are not related to changes in metabolism. Here, we provide data that indicate that endogenously produced ethylene accelerates the movement of cells towards light. Microarray analysis demonstrates that ethylene mainly deactivates transcription from the csiR1/lsiR promoter, which is under the control of the two-component system consisting of the ethylene- and UV-A-sensing histidine kinase UirS and the DNA-binding response regulator UirR. Surprisingly, ethylene production triggers a very specific transcriptional response and only a few other smaller transcriptional changes are detected in the microarray analysis.

The small regulatory RNA SyR1/PsrR1 controls photosynthetic functions in cyanobacteria.

Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.

The Non-Coding RNA Ncr0700/PmgR1 is Required for Photomixotrophic Growth and the Regulation of Glycogen Accumulation in the Cyanobacterium Synechocystis sp. PCC 6803.

Carbohydrate metabolism is a tightly regulated process in photosynthetic organisms. In the cyanobacterium Synechocystis sp. PCC 6803, the photomixotrophic growth protein A (PmgA) is involved in the regulation of glucose and storage carbohydrate (i.e. glycogen) metabolism, while its biochemical activity and possible factors acting downstream of PmgA are unknown. Here, a genome-wide microarray analysis of a ΔpmgA strain identified the expression of 36 protein-coding genes and 42 non-coding transcripts as significantly altered. From these, the non-coding RNA Ncr0700 was identified as the transcript most strongly reduced in abundance. Ncr0700 is widely conserved among cyanobacteria. In Synechocystis its expression is inversely correlated with light intensity. Similarly to a ΔpmgA mutant, a Δncr0700 deletion strain showed an approximately 2-fold increase in glycogen content under photoautotrophic conditions and wild-type-like growth. Moreover, its growth was arrested by 38 h after a shift to photomixotrophic conditions. Ectopic expression of Ncr0700 in Δncr0700 and ΔpmgA restored the glycogen content and photomixotrophic growth to wild-type levels. These results indicate that Ncr0700 is required for photomixotrophic growth and the regulation of glycogen accumulation, and acts downstream of PmgA. Hence Ncr0700 is renamed here as PmgR1 for photomixotrophic growth RNA 1.

CyAbrB2 Contributes to the Transcriptional Regulation of Low CO2 Acclimation in Synechocystis sp. PCC 6803.

Acclimation to low CO2 conditions in cyanobacteria involves the co-ordinated regulation of genes mainly encoding components of the carbon-concentrating mechanism (CCM). Making use of several independent microarray data sets, a core set of CO2-regulated genes was defined for the model strain Synechocystis sp. PCC 6803. On the transcriptional level, the CCM is mainly regulated by the well-characterized transcriptional regulators NdhR (= CcmR) and CmpR. However, the role of an additional regulatory protein, namely cyAbrB2 belonging to the widely distributed AbrB regulator family that was originally characterized in the genus Bacillus, is less defined. Here we present results of transcriptomic and metabolic profiling of the wild type and a ΔcyabrB2 mutant of Synechocystis sp. PCC 6803 after shifts from high CO2 (5% in air, HC) to low CO2 (0.04%, LC). Evaluation of the transcriptomic data revealed that cyAbrB2 is involved in the regulation of several CCM-related genes such as sbtA/B, ndhF3/ndhD3/cupA and cmpABCD under LC conditions, but apparently acts supplementary to NdhR and CmpR. Under HC conditions, cyAbrB2 deletion affects the transcript abundance of PSII subunits, light-harvesting components and Calvin-Benson-Bassham cycle enzymes. These changes are also reflected by down-regulation of primary metabolite pools. The data suggest a role for cyAbrB2 in adjusting primary carbon and nitrogen metabolism to photosynthetic activity under fluctuating environmental conditions. The findings were integrated into the current knowledge about the acquisition of inorganic carbon (Ci), the CCM and parts of its regulation on the transcriptional level.

Small proteins in cyanobacteria provide a paradigm for the functional analysis of the bacterial micro-proteome.

BACKGROUND: Despite their versatile functions in multimeric protein complexes, in the modification of enzymatic activities, intercellular communication or regulatory processes, proteins shorter than 80 amino acids (µ-proteins) are a systematically underestimated class of gene products in bacteria. Photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of 19 small proteins of less than 50 amino acids. In analogy, previously unstudied small ORFs with similar degrees of conservation might encode small proteins of high relevance also in other functional contexts. RESULTS: Here we used comparative transcriptomic information available for two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechocystis sp. PCC 6714 for the prediction of small ORFs. We found 293 transcriptional units containing candidate small ORFs ≤80 codons in Synechocystis sp. PCC 6803, also including the known mRNAs encoding small proteins of the photosynthetic apparatus. From these transcriptional units, 146 are shared between the two strains, 42 are shared with the higher plant Arabidopsis thaliana and 25 with E. coli. To verify the existence of the respective µ-proteins in vivo, we selected five genes as examples to which a FLAG tag sequence was added and re-introduced them into Synechocystis sp. PCC 6803. These were the previously annotated gene ssr1169, two newly defined genes norf1 and norf4, as well as nsiR6 (nitrogen stress-induced RNA 6) and hliR1(high light-inducible RNA 1) , which originally were considered non-coding. Upon activation of expression via the Cu2+.responsive petE promoter or from the native promoters, all five proteins were detected in Western blot experiments. CONCLUSIONS: The distribution and conservation of these five genes as well as their regulation of expression and the physico-chemical properties of the encoded proteins underline the likely great bandwidth of small protein functions in bacteria and makes them attractive candidates for functional studies.

Integrated Analysis of Engineered Carbon Limitation in a Quadruple CO2/HCO3- Uptake Mutant of Synechocystis sp. PCC 6803.

Cyanobacteria have efficient carbon concentration mechanisms and suppress photorespiration in response to inorganic carbon (Ci) limitation. We studied intracellular Ci limitation in the slow-growing CO2/HCO3 (-)-uptake mutant ΔndhD3 (for NADH dehydrogenase subunit D3)/ndhD4 (for NADH dehydrogenase subunit D4)/cmpA (for bicarbonate transport system substrate-binding protein A)/sbtA (for sodium-dependent bicarbonate transporter A): Δ4 mutant of Synechocystis sp. PCC 6803. When cultivated under high-CO2 conditions, ∆4 phenocopies wild-type metabolic and transcriptomic acclimation responses after the shift from high to low CO2 supply. The ∆4 phenocopy reveals multiple compensation mechanisms and differs from the preacclimation of the transcriptional Ci regulator mutant ∆ndhR (for ndhF3 operon transcriptional regulator). Contrary to the carboxysomeless ∆ccmM (for carbon dioxide concentrating mechanism protein M) mutant, the metabolic photorespiratory burst triggered by shifting to low CO2 is not enhanced in ∆4. However, levels of the photorespiratory intermediates 2-phosphoglycolate and glycine are increased under high CO2. The number of carboxysomes is increased in ∆4 under high-CO2 conditions and appears to be the major contributing factor for the avoidance of photorespiration under intracellular Ci limitation. The ∆4 phenocopy is associated with the deregulation of Ci control, an overreduced cellular state, and limited photooxidative stress. Our data suggest multiple layers of Ci regulation, including inversely regulated modules of antisense RNAs and cognate target messenger RNAs and specific trans-acting small RNAs, such as the posttranscriptional PHOTOSYNTHESIS REGULATORY RNA1 (PsrR1), which shows increased expression in ∆4 and is involved in repressing many photosynthesis genes at the posttranscriptional level. In conclusion, our insights extend the knowledge on the range of compensatory responses of Synechocystis sp. PCC 6803 to intracellular Ci limitation and may become a valuable reference for improving biofuel production in cyanobacteria, in which Ci is channeled off from central metabolism and may thus become a limiting factor.

Integrated Transcriptomic and Metabolomic Characterization of the Low-Carbon Response Using an ndhR Mutant of Synechocystis sp. PCC 6803.

The acquisition and assimilation of inorganic carbon (Ci) represents the largest flux of inorganic matter in photosynthetic organisms; hence, this process is tightly regulated. We examined the Ci-dependent transcriptional and metabolic regulation in wild-type Synechocystis sp. PCC 6803 compared with a mutant defective in the main transcriptional repressor for Ci acquisition genes, the NAD(P)H dehydrogenase transcriptional regulator NdhR. The analysis revealed that many protein-coding transcripts that are normally repressed in the presence of high CO2 (HC) concentrations were strongly expressed in ∆ndhR, whereas other messenger RNAs were strongly down-regulated in mutant cells, suggesting a potential activating role for NdhR. A conserved NdhR-binding motif was identified in the promoters of derepressed genes. Interestingly, the expression of some NdhR-regulated genes remained further inducible under low-CO2 conditions, indicating the involvement of additional NdhR-independent Ci-regulatory mechanisms. Intriguingly, we also observed that the abundance of 52 antisense RNAs and 34 potential noncoding RNAs was affected by Ci supply, although most of these molecules were not regulated through NdhR. Thus, antisense and noncoding RNAs could contribute to NdhR-independent carbon regulation. In contrast to the transcriptome, the metabolome in ∆ndhR cells was similar to that of wild-type cells under HC conditions. This observation and the delayed metabolic responses to the low-CO2 shift in ∆ndhR, specifically the lack of transient increases in the photorespiratory pathway intermediates 2-phosphoglycolate, glycolate, and glycine, suggest that the deregulation of gene expression in the ΔndhR mutant successfully preacclimates cyanobacterial cells to lowered Ci supply under HC conditions.

The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply.

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.

The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories.

Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis, a solar-driven process which allows them to obtain electrons from water to reduce and finally assimilate carbon dioxide. Consequently, they are in the spotlight of biotechnology as photoautotrophic cell factories to generate a large variety of chemicals and biofuels in a sustainable way. Recent progress in synthetic biology has enlarged the molecular toolset to genetically engineer the metabolism of cyanobacteria, mainly targeting common model strains, such as Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, Synechococcus sp. PCC 7002, or Anabaena sp. PCC 7120. Nevertheless, the accessibility and flexibility of engineering cyanobacteria is still somewhat limited and less predictable compared to other biotechnologically employed microorganisms.This chapter gives a broad overview of currently available methods for the genetic modification of cyanobacterial model strains as well as more recently discovered and promising species, such as Synechococcus elongatus PCC 11801. It comprises approaches based on homologous recombination, replicative broad-host-range or strain-specific plasmids, CRISPR/Cas, as well as markerless selection. Furthermore, common and newly introduced molecular tools for gene expression regulation are presented, comprising promoters, regulatory RNAs, genetic insulators like transcription terminators, ribosome binding sites, CRISPR interference, and the utilization of heterologous RNA polymerases. Additionally, potential DNA assembly strategies, like modular cloning, are described. Finally, considerations about post-translational control via protein degradation tags and heterologous proteases, as well as small proteins working as enzyme effectors are briefly discussed.

Cyanobacteria as whole-cell factories: current status and future prospectives.

Cyanobacteria as phototrophic microorganisms bear great potential to produce chemicals from sustainable resources such as light and CO2. Most studies focus on either strain engineering or tackling metabolic constraints. Recently gained knowledge on internal electron and carbon fluxes and their regulation provides new opportunities to efficiently channel cellular resources toward product formation. Concomitantly, novel photobioreactor concepts are developed to ensure sufficient light supply. This review summarizes the newest developments in the field of cyanobacterial engineering to finally establish photosynthesis-based production processes. A holistic approach tackling genetic, metabolic, and biochemical engineering in parallel is considered essential to turn their application into an ecoefficient and economically feasible option for a future green bioeconomy.

Toward a synthetic hydrogen sensor in cyanobacteria: Functional production of an oxygen-tolerant regulatory hydrogenase in Synechocystis sp. PCC 6803.

Cyanobacteria have raised great interest in biotechnology, e.g., for the sustainable production of molecular hydrogen (H2) using electrons from water oxidation. However, this is hampered by various constraints. For example, H2-producing enzymes compete with primary metabolism for electrons and are usually inhibited by molecular oxygen (O2). In addition, there are a number of other constraints, some of which are unknown, requiring unbiased screening and systematic engineering approaches to improve the H2 yield. Here, we introduced the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator (formerly Ralstonia eutropha) H16 into the cyanobacterial model strain Synechocystis sp. PCC 6803. In its natural host, the RH serves as a molecular H2 sensor initiating a signal cascade to express hydrogenase-related genes when no additional energy source other than H2 is available. Unlike most hydrogenases, the C. necator enzymes are O2-tolerant, allowing their efficient utilization in an oxygenic phototroph. Similar to C. necator, the RH produced in Synechocystis showed distinct H2 oxidation activity, confirming that it can be properly matured and assembled under photoautotrophic, i.e., oxygen-evolving conditions. Although the functional H2-sensing cascade has not yet been established in Synechocystis yet, we utilized the associated two-component system consisting of a histidine kinase and a response regulator to drive and modulate the expression of a superfolder gfp gene in Escherichia coli. This demonstrates that all components of the H2-dependent signal cascade can be functionally implemented in heterologous hosts. Thus, this work provides the basis for the development of an intrinsic H2 biosensor within a cyanobacterial cell that could be used to probe the effects of random mutagenesis and systematically identify promising genetic configurations to enable continuous and high-yield production of H2 via oxygenic photosynthesis.

The marine cyanobacterium Crocosphaera watsonii WH8501 synthesizes the compatible solute trehalose by a laterally acquired OtsAB fusion protein.

Compatible solutes are small organic molecules that are involved in the acclimation to various stresses such as temperature and salinity. Marine or moderate halotolerant cyanobacteria accumulate glucosylglycerol, while cyanobacteria with low salt tolerance (freshwater strains) usually accumulate sucrose or trehalose as the main compatible solutes. The screening of the genome of the marine, unicellular N(2) -fixing cyanobacterium Crocosphaera watsonii WH8501 revealed that instead of genes for glucosylglycerol biosynthesis, a fusion protein for the synthesis of trehalose was found that displayed similarities to trehalose-phosphate-synthase and -phosphatase (OtsAB pathway) from enterobacteria. Accordingly, cells of Crocosphaera showed salt-stimulated expression of the otsAB gene as well as a salt-dependent trehalose accumulation. The biochemical characterization of recombinant full-length OtsAB and truncated OtsB versions revealed that the otsAB gene in Crocosphaera encodes for an active trehalose-phosphate-synthase/phosphatase fusion protein. Genes coding for such proteins were not found in the genomes of other cyanobacteria but were present in many other, non-related marine bacteria, suggesting that otsAB might have been acquired by lateral gene transfer into the Crocosphaera genome.

Whole-genome sequence of the filamentous diazotrophic cyanobacterium Tolypothrix sp. PCC 7712 and its comparison with non-diazotrophic Tolypothrix sp. PCC 7601.

Cyanobacteria are highly promising microorganisms in forthcoming biotechnologies. Besides the systematic development of molecular tools for genetic engineering, the design of chassis strains and novel reactor concepts are in focus. The latter includes capillary biofilm reactors (CBR), which offer a high surface area-to-volume ratio and very high cell densities. In this context, Tolypothrix sp. PCC 7712 was found to be highly suited for this reactor system due to maximal surface coverage, extraordinarily strong biofilm attachment, and high biomass formation. Here, we provide the genome sequence of Tolypothrix sp. PCC 7712 to potentially allow targeted strain engineering. Surprisingly, it was almost identical to an available incomplete genome draft of Tolypothrix sp. PCC 7601. Thus, we completely sequenced this strain as well and compared it in detail to strain PCC 7712. Comparative genome analysis revealed 257 and 80 unique protein-coding sequences for strains PCC 7601 and PCC 7712, respectively. Clustering genomes based on average nucleotide identity (ANI) and 16S rRNA homology showed 99.98% similarity and only minor distance, respectively, between the two strains in contrast to 21 other cyanobacterial genomes. Despite these high similarities, both strains differ in the ability to fix atmospheric nitrogen and show specific sequence variations, which are discussed in the paper.