Investigation of singlet-oxygen-responsive genes in the cyanobacterium Synechocystis PCC 6803.

Singlet oxygen (1O2) is an important reactive oxygen species whose formation by the type-II, light-dependent, photodynamic reaction is inevitable during photosynthetic processes. In the last decades, the recognition that 1O2 is not only a damaging agent, but can also affect gene expression and participates in signal transduction pathways has received increasing attention. However, contrary to several other taxa, 1O2-responsive genes have not been identified in the important cyanobacterial model organism Synechocystis PCC 6803. By using global transcript analysis we have identified a large set of Synechocystis genes, whose transcript levels were either enhanced or repressed in the presence of 1O2. Characteristic 1O2 responses were observed in several light-inducible genes of Synechocystis, especially in the hli (or scp) family encoding HLIP/SCP proteins involved in photoprotection. Other important 1O2-induced genes include components of the Photosystem II repair machinery (psbA2 and ftsH2, ftsH3), iron homeostasis genes isiA and idiA, the group 2 sigma factor sigD, some components of the transcriptomes induced by salt-, hyperosmotic and cold-stress, as well as several genes of unknown function. The most pronounced 1O2-induced upregulation was observed for the hliB and the co-transcribed lilA genes, whose deletion induced enhanced sensitivity against 1O2-mediated light damage. A bioreporter Synechocystis strain was created by fusing the hliB promoter to the bacterial luciferase (lux), which showed its utility for continuous monitoring of 1O2 concentrations inside the cell.

Development of a base editor for convenient and multiplex genome editing in cyanobacteria.

Cyanobacteria are important primary producers, contributing to 25% of the global carbon fixation through photosynthesis. They serve as model organisms to study the photosynthesis, and are important cell factories for synthetic biology. To enable efficient genetic dissection and metabolic engineering in cyanobacteria, effective and accurate genetic manipulation tools are required. However, genetic manipulation in cyanobacteria by the conventional homologous recombination-based method and the recently developed CRISPR-Cas gene editing system require complicated cloning steps, especially during multi-site editing and single base mutation. This restricts the extensive research on cyanobacteria and reduces its application potential. In this study, a highly efficient and convenient cytosine base editing system was developed which allows rapid and precise C → T point mutation and gene inactivation in the genomes of Synechocystis and Anabaena. This base editing system also enables efficient multiplex editing and can be easily cured after editing by sucrose counter-selection. This work will expand the knowledge base regarding the engineering of cyanobacteria. The findings of this study will encourage the biotechnological applications of cyanobacteria.

Fine-Tuning Gene Expression in Bacteria by Synthetic Promoters.

Promoters are key genetic elements in the initiation and regulation of gene expression. A limited number of natural promoters has been described for the control of gene expression in synthetic biology applications. Therefore, synthetic promoters have been developed to fine-tune the transcription for the desired amount of gene product. Mostly, synthetic promoters are characterized using promoter libraries that are constructed via mutagenesis of promoter sequences. The strength of promoters in the library is determined according to the expression of a reporter gene such as gfp encoding green fluorescent protein. Gene expression can be controlled using inducers. The majority of the studies on gram-negative bacteria are conducted using the expression system of the model organism Escherichia coli while that of the model organism Bacillus subtilis is mostly used in the studies on gram-positive bacteria. Additionally, synthetic promoters for the cyanobacteria, which are phototrophic microorganisms, are evaluated, especially using the model cyanobacterium Synechocystis sp. PCC 6803. Moreover, a variety of algorithms based on machine learning methods were developed to characterize the features of promoter elements. Some of these in silico models were verified using in vitro or in vivo experiments. Identification of novel synthetic promoters with improved features compared to natural ones contributes much to the synthetic biology approaches in terms of fine-tuning gene expression.

A global transcriptional activator involved in the iron homeostasis in cyanobacteria.

Cyanobacteria use a series of adaptation strategies and a complicated regulatory network to maintain intracellular iron (Fe) homeostasis. Here, a global activator named IutR has been identified through three-dimensional chromosome organization and transcriptome analysis in a model cyanobacterium Synechocystis sp. PCC 6803. Inactivation of all three homologous IutR-encoding genes resulted in an impaired tolerance of Synechocystis to Fe deficiency and loss of the responses of Fe uptake-related genes to Fe-deplete conditions. Protein-promoter interaction assays confirmed the direct binding of IutR with the promoters of genes related to Fe uptake, and chromatin immunoprecipitation sequencing analysis further revealed that in addition to Fe uptake, IutR could regulate many other physiological processes involved in intracellular Fe homeostasis. These results proved that IutR is an important transcriptional activator, which is essential for cyanobacteria to induce Fe-deficiency response genes. This study provides in-depth insights into the complicated Fe-deficient signaling network and the molecular mechanism of cyanobacteria adaptation to Fe-deficient environments.

A type III-Dv CRISPR-Cas system is controlled by the transcription factor RpaB and interacts with the DEAD-box RNA helicase CrhR.

How CRISPR-Cas systems defend bacteria and archaea against invading genetic elements is well understood, but less is known about their regulation. In the cyanobacterium Synechocystis sp. PCC 6803, the expression of one of the three different CRISPR-Cas systems responds to changes in environmental conditions. The cas operon promoter of this system is controlled by the light- and redox-responsive transcription factor RpaB binding to an HLR1 motif, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, the leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled with mass spectrometry analysis and site-directed mutagenesis revealed six residues involved in the CrhR-RNA interaction, with C371 being critically important. Thus, the expression of a type III-Dv CRISPR-Cas system is linked to the redox status of the photosynthetic cell at the transcriptional and post-transcriptional levels.

Disrupted H2 synthesis combined with methyl viologen treatment inhibits photosynthetic electron flow to synergistically enhance glycogen accumulation in the cyanobacterium Synechocystis sp. PCC 6803.

Under nitrogen deprivation (-N), cyanobacterium Synechocystis sp. PCC 6803 exhibits growth arrest, reduced protein content, and remarkably increased glycogen accumulation. However, producing glycogen under this condition requires a two-step process with cell transfer from normal to -N medium. Metabolic engineering and chemical treatment for rapid glycogen accumulation can bypass the need for two-step cultivation. For example, recent studies indicate that individually disrupting hydrogen (H2) or poly(3-hydroxybutyrate) (PHB) synthesis, or treatment with methyl viologen (MV), effectively increases glycogen accumulation in Synechocystis. Here we explore the effects of disrupted H2 or poly(3-hydroxybutyrate) synthesis, together with MV treatment to on enhanced glycogen accumulation in Synechocystis grown in normal medium. Wild-type cells without MV treatment exhibited low glycogen content of less than 6% w/w dry weight (DW). Compared with wild type, disrupting PHB synthesis combined with MV treatment did not increase glycogen content. Disrupted H2 production without MV treatment yielded up to 11% w/w DW glycogen content. Interestingly, when combined, disrupted H2 production with MV treatment synergistically enhanced glycogen accumulation to 51% and 59% w/w DW within 3 and 7 days, respectively. Metabolomic analysis suggests that MV treatment mediated the conversion of proteins into glycogen. Metabolomic and transcriptional-expression analysis suggests that disrupted H2 synthesis under MV treatment positively influenced glycogen synthesis. Disrupted H2 synthesis under MV treatment significantly increased NADPH levels. This increased NADPH content potentially contributed to the observed enhancements in antioxidant activity against MV-induced oxidants, O2 evolution, and metabolite substrates levels for glycogen synthesis in normal medium, ultimately leading to enhanced glycogen accumulation in Synechocystis. KEY MESSAGE: Combining disrupted hydrogen-gas synthesis and the treatment by photosynthesis electron-transport inhibitor significantly enhance glycogen production in cyanobacteria.

Interplay between photosynthetic electron flux and organic carbon sinks in sucrose-excreting Synechocystis sp. PCC 6803 revealed by omics approaches.

BACKGROUND: Advancing the engineering of photosynthesis-based prokaryotic cell factories is important for sustainable chemical production and requires a deep understanding of the interplay between bioenergetic and metabolic pathways. Rearrangements in photosynthetic electron flow to increase the efficient use of the light energy for carbon fixation must be balanced with a strong carbon sink to avoid photoinhibition. In the cyanobacterium Synechocystis sp. PCC 6803, the flavodiiron protein Flv3 functions as an alternative electron acceptor of photosystem I and represents an interesting engineering target for reorganizing electron flow in attempts to enhance photosynthetic CO2 fixation and increase production yield. RESULTS: We have shown that inactivation of Flv3 in engineered sucrose-excreting Synechocystis (S02:Δflv3) induces a transition from photoautotrophic sucrose production to mixotrophic growth sustained by sucrose re-uptake and the formation of intracellular carbon sinks such as glycogen and polyhydroxybutyrate. The growth of S02:Δflv3 exceeds that of the sucrose-producing strain (S02) and demonstrates unforeseen proteomic and metabolomic changes over the course of the nine-day cultivation. In the absence of Flv3, a down-regulation of proteins related to photosynthetic light reactions and CO2 assimilation occurred concomitantly with up-regulation of those related to glycolytic pathways, before any differences in sucrose production between S02 and S02:Δflv3 strains were observed. Over time, increased sucrose degradation in S02:Δflv3 led to the upregulation of respiratory pathway components, such as the plastoquinone reductase complexes NDH-11 and NDH-2 and the terminal respiratory oxidases Cyd and Cox, which transfer electrons to O2. While glycolytic metabolism is significantly up-regulated in S02:Δflv3 to provide energy for the cell, the accumulation of intracellular storage compounds and the increase in respiration serve as indirect sinks for photosynthetic electrons. CONCLUSIONS: Our results show that the presence of strong carbon sink in the engineered sucrose-producing Synechocystis S02 strain, operating under high light, high CO2 and salt stress, cannot compensate for the lack of Flv3 by directly balancing the light transducing source and carbon fixing sink reactions. Instead, the cells immediately sense the imbalance, leading to extensive reprogramming of cellular bioenergetic, metabolic and ion transport pathways that favor mixotrophic growth rather than enhancing photoautotrophic sucrose production.

Engineered Cyanobacteria-Based Living Materials for Bioremediation of Heavy Metals Both In Vitro and In Vivo.

The pollution caused by heavy metals (HMs) represents a global concern due to their serious environmental threat. Photosynthetic cyanobacteria have a natural niche and the ability to remediate HMs such as cadmium. However, their practical application is hindered by a low tolerance to HMs and issues related to recycling. In response to these challenges, this study focuses on the development and evaluation of engineered cyanobacteria-based living materials for HMs bioremediation. Genes encoding phytochelatins (PCSs) and metallothioneins (MTs) were introduced into the model cyanobacterium Synechocystis sp. PCC 6803, creating PM/6803. The strain exhibited improved tolerance to multiple HMs and effectively removed a combination of Cd2+, Zn2+, and Cu2+. Using Cd2+ as a representative, PM/6803 achieved a bioremediation rate of approximately 21 µg of Cd2+/OD750 under the given test conditions. To facilitate its controllable application, PM/6803 was encapsulated using sodium alginate-based hydrogels (PM/6803@SA) to create "living materials" with different shapes. This system was feasible, biocompatible, and effective for removing Cd2+ under simulated conditions of zebrafish and mice models. Briefly, in vitro application of PM/6803@SA efficiently rescued zebrafish from polluted water containing Cd2+, while in vivo use of PM/6803@SA significantly decreased the Cd2+ content in mice bodies and restored their active behavior. The study offers feasible strategies for HMs bioremediation using the interesting biomaterials of engineered cyanobacteria both in vitro and in vivo.

Glycosyltransferase Slr1064 regulates carbon metabolism by modulating the levels of UDP-GlcNAc in Synechocystis sp. PCC 6803.

Glycosyltransferases (GTs) are enzymes that transfer sugars to various targets. They play important roles in diverse biological processes, including photosynthesis, cell motility, exopolysaccharide biosynthesis, and lipid metabolism; however, their involvement in regulating carbon metabolism in Synechocystis sp. PCC 6803 has not been reported. We identified a novel GT protein, Slr1064, involved in carbon metabolism. The effect of slr1064 deletion on the growth of Synechocystis cells and functional mechanisms of Slr1064 on carbon metabolism were thoroughly investigated through physiological, biochemistry, proteomic, and metabolic analyses. We found that this GT, which is mainly distributed in the membrane compartment, is essential for the growth of Synechocystis under heterotrophic and mixotrophic conditions, but not under autotrophic conditions. The deletion of slr1064 hampers the turnover rate of Gap2 under mixotrophic conditions and disrupts the assembly of the PRK/GAPDH/CP12 complex under dark culture conditions. Additionally, UDP-GlcNAc, the pivotal metabolite responsible for the O-GlcNAc modification of GAPDH, is downregulated in the Δslr1064. Our work provides new insights into the role of GTs in carbon metabolism in Synechocystis and elucidate the mechanism by which carbon metabolism is regulated in this important model organism.

Functional consequences of modification of the photosystem I/photosystem II ratio in the cyanobacterium Synechocystis sp. PCC 6803.

The stoichiometry of photosystem II (PSII) and photosystem I (PSI) varies between photoautotrophic organisms. The cyanobacterium Synechocystis sp. PCC 6803 maintains two- to fivefold more PSI than PSII reaction center complexes, and we sought to modify this stoichiometry by changing the promoter region of the psaAB operon. We thus generated mutants with varied psaAB expression, ranging from ~3% to almost 200% of the wild-type transcript level, but all showing a reduction in PSI levels, relative to wild type, suggesting a role of the psaAB promoter region in translational regulation. Mutants with 25%-70% of wild-type PSI levels were photoautotrophic, with whole-chain oxygen evolution rates on a per-cell basis comparable to that of wild type. In contrast, mutant strains with <10% of the wild-type level of PSI were obligate photoheterotrophs. Variable fluorescence yields of all mutants were much higher than those of wild type, indicating that the PSI content is localized differently than in wild type, with less transfer of PSII-absorbed energy to PSI. Strains with less PSI saturate at a higher light intensity, enhancing productivity at higher light intensities. This is similar to what is found in mutants with reduced antennae. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea present, P700+ re-reduction kinetics in the mutants were slower than in wild type, consistent with the notion that there is less cyclic electron transport if less PSI is present. Overall, strains with a reduction in PSI content displayed surprisingly vigorous growth and linear electron transport. IMPORTANCE: Consequences of reduction in photosystem I content were investigated in the cyanobacterium Synechocystis sp. PCC 6803 where photosystem I far exceeds the number of photosystem II complexes. Strains with less photosystem I displayed less cyclic electron transport, grew more slowly at lower light intensity and needed more light for saturation but were surprisingly normal in their whole-chain electron transport rates, implying that a significant fraction of photosystem I is dispensable for linear electron transport in cyanobacteria. These strains with reduced photosystem I levels may have biotechnological relevance as they grow well at higher light intensities.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption.

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Is the A-1 Pigment in Photosystem I Part of P700? A (P700+-P700) FTIR Difference Spectroscopy Study of A-1 Mutants.

The involvement of the second pair of chlorophylls, termed A-1A and A-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A-1B and A-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700+-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700+-P700) FTIR DS cannot be due to only the PA and PB pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A-1A and A-1B pigments are modified upon P700+ formation. Previously, we showed that the A-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

Novel candidate genes for environmental stresses response in Synechocystis sp. PCC 6803 revealed by machine learning algorithms.

Cyanobacteria have developed acclimation strategies to adapt to harsh environments, making them a model organism. Understanding the molecular mechanisms of tolerance to abiotic stresses can help elucidate how cells change their gene expression patterns in response to stress. Recent advances in sequencing techniques and bioinformatics analysis methods have led to the discovery of many genes involved in stress response in organisms. The Synechocystis sp. PCC 6803 is a suitable microorganism for studying transcriptome response under environmental stress. Therefore, for the first time, we employed two effective feature selection techniques namely and support vector machine recursive feature elimination (SVM-RFE) and LASSO (Least Absolute Shrinkage Selector Operator) to pinpoint the crucial genes responsive to environmental stresses in Synechocystis sp. PCC 6803. We applied these algorithms of machine learning to analyze the transcriptomic data of Synechocystis sp. PCC 6803 under distinct conditions, encompassing light, salt and iron stress conditions. Seven candidate genes namely sll1862, slr0650, sll0760, slr0091, ssl3044, slr1285, and slr1687 were selected by both LASSO and SVM-RFE algorithms. RNA-seq analysis was performed to validate the efficiency of our feature selection approach in selecting the most important genes. The RNA-seq analysis revealed significantly high expression for five genes namely sll1862, slr1687, ssl3044, slr1285, and slr0650 under ion stress condition. Among these five genes, ssl3044 and slr0650 could be introduced as new potential candidate genes for further confirmatory genetic studies, to determine their roles in their response to abiotic stresses.

Glycogen synthesis prevents metabolic imbalance and disruption of photosynthetic electron transport from photosystem II during transition to photomixotrophy in Synechocystis sp. PCC 6803.

Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO2 and glucose. The switch between these trophic modes and the role of glycogen, their main carbon storage macromolecule, was investigated. We analysed the effect of glucose addition on the physiology, metabolic and photosynthetic state of Synechocystis sp. PCC 6803 and mutants lacking phosphoglucomutase and ADP-glucose pyrophosphorylase, with limitations in glycogen synthesis. Glycogen acted as a metabolic buffer: glucose addition increased growth and glycogen reserves in the wild-type (WT), but arrested growth in the glycogen synthesis mutants. Already 30 min after glucose addition, metabolites from the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate shunt increased threefold more in the glycogen synthesis mutants than the WT. These alterations substantially affected the photosynthetic performance of the glycogen synthesis mutants, as O2 evolution and CO2 uptake were both impaired. We conclude that glycogen synthesis is essential during transitions to photomixotrophy to avoid metabolic imbalance that induces inhibition of electron transfer from PSII and subsequently accumulation of reactive oxygen species, loss of PSII core proteins, and cell death. Our study lays foundations for optimising photomixotrophy-based biotechnologies through understanding the coordination of the crosstalk between photosynthetic electron transport and metabolism.

Altered excitation energy transfer between phycobilisome and photosystems in the absence of ApcG, a small linker peptide, in Synechocystis sp. PCC 6803, a cyanobacterium.

Phycobilisome (PBS) is a large pigment-protein complex in cyanobacteria and red algae responsible for capturing sunlight and transferring its energy to photosystems (PS). Spectroscopic and structural properties of various PBSs have been widely studied, however, the nature of so-called complex-complex interactions between PBS and PSs remains much less explored. In this work, we have investigated the function of a newly identified PBS linker protein, ApcG, some domain of which, together with a loop region (PB-loop in ApcE), is possibly located near the PBS-PS interface. Using Synechocystis sp. PCC 6803, we generated an ApcG deletion mutant and probed its deletion effect on the energetic coupling between PBS and photosystems. Steady-state and time-resolved spectroscopic characterization of the purified ΔApcG-PBS demonstrated that ApcG removal weakly affects the photophysical properties of PBS for which the spectroscopic properties of terminal energy emitters are comparable to those of PBS from wild-type strain. However, analysis of fluorescence decay imaging datasets reveals that ApcG deletion induces disruptions within the allophycocyanin (APC) core, resulting in the emergence (splitting) of two spectrally diverse subgroups with some short-lived APC. Profound spectroscopic changes of the whole ΔApcG mutant cell, however, emerge during state transition, a dynamic process of light scheme adaptation. The mutant cells in State I show a substantial increase in PBS-related fluorescence. On the other hand, global analysis of time-resolved fluorescence demonstrates that in general ApcG deletion does not alter or inhibit state transitions interpreted in terms of the changes of the PSII and PSI fluorescence emission intensity. The results revealed yet-to-be discovered mechanism of ApcG-docking induced excitation energy transfer regulation within PBS or to Photosystems.

Phosphorus deficiency alleviates iron limitation in Synechocystis cyanobacteria through direct PhoB-mediated gene regulation.

Iron and phosphorus are essential nutrients that exist at low concentrations in surface waters and may be co-limiting resources for phytoplankton growth. Here, we show that phosphorus deficiency increases the growth of iron-limited cyanobacteria (Synechocystis sp. PCC 6803) through a PhoB-mediated regulatory network. We find that PhoB, in addition to its well-recognized role in controlling phosphate homeostasis, also regulates key metabolic processes crucial for iron-limited cyanobacteria, including ROS detoxification and iron uptake. Transcript abundances of PhoB-targeted genes are enriched in samples from phosphorus-depleted seawater, and a conserved PhoB-binding site is widely present in the promoters of the target genes, suggesting that the PhoB-mediated regulation may be highly conserved. Our findings provide molecular insights into the responses of cyanobacteria to simultaneous iron/phosphorus nutrient limitation.

Structure and genome editing of type I-B CRISPR-Cas.

Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3+ T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

Erythromycin mediates co-flocculation between cyanobacterium Synechocystis sp. PCC 6803 and filamentous fungi in liquid cultivation without organic compounds.

Photoautotrophic cyanobacteria assimilate the greenhouse gas carbon dioxide as their sole carbon source for producing useful bioproducts. However, harvesting the cells from their liquid media is a major bottleneck in the process. Thus, an easy-to-harvest method, such as auto-flocculation, is desirable. Here, we found that cyanobacterium Synechocystis sp. PCC 6803 co-flocculated with a natural fungal contamination in the presence of the antibiotic erythromycin (EM) but not without EM. The fungi in the co-flocculated biomass were isolated and found to consist of five species with the filamentous Purpureocillium lilacinum and Aspergillus protuberus making up 71% of the overall fungal population. The optimal co-cultivation for flocculation was an initial 5 mg (fresh weight) of fungi, an initial cell density of Synechocystis of 0.2 OD730, 10 µM EM, and 14 days of cultivation in 100 mL of BG11 medium with no organic compound. This yielded 248 ± 28 mg/L of the Synechocystis-fungi flocculated biomass from 560 ± 35 mg/L of total biomass, a 44 ± 2% biomass flocculation efficiency. Furthermore, the EM treated Synechocystis cells in the Synechocystis-fungi flocculate had a normal cell color and morphology, while those in the axenic suspension exhibited strong chlorosis. Thus, the occurrence of the Synechocystis-fungi flocculation was mediated by EM, and the co-flocculation with the fungi protected Synechocystis against the development of chlorosis. Transcriptomic analysis suggested that the EM-mediated co-flocculation was a result of down-regulation of the minor pilin genes and up-regulation of several genes including the chaperone gene for pilin regulation, the S-layer protein genes, the exopolysaccharide-polymerization gene, and the genes for signaling proteins involved in cell attachment and abiotic-stress responses. The CuSO4 stress can also mediate Synechocystis-fungi flocculation but at a lower flocculation efficiency than that caused by EM. The EM treatment may be applied in the co-culture between other cyanobacteria and fungi to mediate cell bio-flocculation.