Inhibition of Polyphosphate Degradation in Synechocystis sp. PCC6803 through Inactivation of the phoU Gene.

Phosphorus is an essential but non-renewable nutrient resource critical for agriculture. Luxury phosphorus uptake allows microalgae to synthesize polyphosphate and accumulate phosphorus, but, depending on the strain of algae, polyphosphate may be degraded within 4 hours of accumulation. We studied the recovery of phosphorus from wastewater through luxury uptake by an engineered strain of Synechocystis sp. with inhibited polyphosphate degradation and the effect of this engineered Synechocystis biomass on lettuce growth. First, a strain (ΔphoU) lacking the phoU gene, which encodes a negative regulator of environmental phosphate concentrations, was generated to inhibit polyphosphate degradation in cells. Polyphosphate concentrations in the phoU knock-out strain were maintained for 24 h and then decreased slowly. In contrast, polyphosphate concentrations in the wild-type strain increased up to 4 h and then decreased rapidly. In addition, polyphosphate concentration in the phoU knockout strain cultured in semi-permeable membrane bioreactors with artificial wastewater medium was 2.5 times higher than that in the wild type and decreased to only 16% after 48 h. The biomass of lettuce treated with the phoU knockout strain (0.157 mg P/m2) was 38% higher than that of the lettuce treated with the control group. These results indicate that treating lettuce with this microalgal biomass can be beneficial to crop growth. These results suggest that the use of polyphosphate-accumulating microalgae as biofertilizers may alleviate the effects of a diminishing phosphorous supply. These findings can be used as a basis for additional genetic engineering to increase intracellular polyphosphate levels.

Improved lipid production and component of mycosporine-like amino acids by co-overexpression of amt1 and aroB genes in Synechocystis sp. PCC6803.

Implementing homologous overexpression of the amt1 (A) and aroB (B) genes involved in ammonium transporter and the synthesis of mycosporine-like amino acids (MAAs) and aromatic amino acids, respectively, we created three engineered Synechocystis sp. PCC6803 strains, including Ox-A, Ox-B, and Ox-AB, to study the utilization of carbon and nitrogen in cyanobacteria for the production of valuable products. With respect to amt1 overexpression, the Ox-A and Ox-AB strains had a greater growth rate under (NH4)2SO4 supplemented condition. Both the higher level of intracellular accumulation of lipids in Ox-A and Ox-AB as well as the increased secretion of free fatty acids from the Ox-A strain were impacted by the late-log phase of cell growth. It is noteworthy that among all strains, the Ox-B strain undoubtedly spotted a substantial accumulation of glycogen as a consequence of aroB overexpression. Additionally, the ammonium condition drove the potent antioxidant activity in Ox strains with a late-log phase, particularly in the Ox-B and Ox-AB strains. This was probably related to the altered MAA component inside the cells. The higher proportion of P4-fraction was induced by the ammonium condition in both Ox-B and Ox-AB, while the noted increase of the P1 component was found in the Ox-A strain.

Chlorophyll biosynthesis under the control of arginine metabolism.

In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesis of tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.

A Non-functional γ-Aminobutyric Acid Shunt Pathway in Cyanobacterium Synechocystis sp. PCC 6803 Enhances δ-Aminolevulinic Acid Accumulation under Modified Nutrient Conditions.

To redirect carbon flux from the γ-aminobutyric acid (GABA) shunt to the δ-aminolevulinic acid (ALA) biosynthetic pathway, we disrupted the GABA shunt route of the model cyanobacterium Synechocystis sp. PCC 6803 by inactivating Gdc, the gene-encoding glutamate decarboxylase. The generated ΔGdc strain exhibited lower intracellular GABA and higher ALA levels than the wild-type (WT) one. The ΔGdc strain's ALA levels were ~2.8 times higher than those of the WT one when grown with levulinic acid (LA), a competitive inhibitor of porphobilinogen synthase. Abiotic stress conditions including salinity induced by 10 mM NaCl and cold at 4 °C increased the ALA levels in ΔGdc up to ~2.5 and 5 ng g−1 cell DW, respectively. The highest ALA production in the ΔGdc cyanobacteria grown in BG11 medium was triggered by glucose induction, followed by glutamate supplementation with 60 mM of LA, thereby resulting in ~360 ng g−1 cell DW of ALA, that is >300-fold higher ALA accumulation than that observed in ΔGdc cyanobacteria grown in normal medium. Increased levels of the gdhA (involved in the interconversion of α-ketoglutarate to glutamate) and the hemA (a major regulatory target of the ALA biosynthetic pathway) transcripts occurred in ΔGdc cyanobacteria grown under modified growth conditions. Our study provides critical insight into the facilitation of ALA production in cyanobacteria.

Metabolic and Microbial Community Engineering for Four-Carbon Dicarboxylic Acid Production from CO2-Derived Glycogen in the Cyanobacterium Synechocystis sp. PCC6803.

The four-carbon (C4) dicarboxylic acids, fumarate, malate, and succinate, are the most valuable targets that must be exploited for CO2-based chemical production in the move to a sustainable low-carbon future. Cyanobacteria excrete high amounts of C4 dicarboxylic acids through glycogen fermentation in a dark anoxic environment. The enhancement of metabolic flux in the reductive TCA branch in the Cyanobacterium Synechocystis sp. PCC6803 is a key issue in the C4 dicarboxylic acid production. To improve metabolic flux through the anaplerotic pathway, we have created the recombinant strain PCCK, which expresses foreign ATP-forming phosphoenolpyruvate carboxykinase (PEPck) concurrent with intrinsic phosphoenolpyruvate carboxylase (Ppc) overexpression. Expression of PEPck concurrent with Ppc led to an increase in C4 dicarboxylic acids by autofermentation. Metabolome analysis revealed that PEPck contributed to an increase in carbon flux from hexose and pentose phosphates into the TCA reductive branch. To enhance the metabolic flux in the reductive TCA branch, we examined the effect of corn-steep liquor (CSL) as a nutritional supplement on C4 dicarboxylic acid production. Surprisingly, the addition of sterilized CSL enhanced the malate production in the PCCK strain. Thereafter, the malate and fumarate excreted by the PCCK strain are converted into succinate by the CSL-settling microorganisms. Finally, high-density cultivation of cells lacking the acetate kinase gene showed the highest production of malate and fumarate (3.2 and 2.4 g/L with sterilized CSL) and succinate (5.7 g/L with non-sterile CSL) after 72 h cultivation. The present microbial community engineering is useful for succinate production by one-pot fermentation under dark anoxic conditions.

Specific Incorporation of Polyunsaturated Fatty Acids into the sn-2 Position of Phosphatidylglycerol Accelerates Photodamage to Photosystem II under Strong Light.

Free fatty acids (FFAs) are generated by the reaction of lipases with membrane lipids. Generated polyunsaturated fatty acids (PUFAs) containing more than two double bonds have toxic effects in photosynthetic organisms. In the present study, we examined the effect of exogenous FFAs in the growth medium on the activity of photosystem II (PSII) under strong light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PUFAs but not monounsaturated fatty acids accelerated the rate of photodamage to PSII by inactivating electron transfer at the oxygen-evolving complex. Moreover, supplemented PUFAs were specifically incorporated into the sn-2 position of phosphatidylglycerol (PG), which usually contains C16 fatty acids at the sn-2 position in Synechocystis cells. The disruption of the gene for an acyl-ACP synthetase reduced the effect of PUFAs on the photoinhibition of PSII. Thus, the specific incorporation of PUFAs into PG molecules requires acyl-ACP synthetase and leads to an unstable PSII, thereby accelerating photodamage to PSII. Our results are a breakthrough into elucidating the molecular mechanism of the toxicity of PUFAs to photosynthetic organisms.

Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt.

BACKGROUND: Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. RESULTS: We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. CONCLUSIONS: Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Requirement of the exopolyphosphatase gene for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803.

Polyphosphate, which is ubiquitous in cells in nature, is involved in a myriad of cellular functions, and has been recently focused on its metabolism related with microbial acclimation to phosphorus-source fluctuation. In view of the ecological importance of cyanobacteria as the primary producers, this study investigated the responsibility of polyphosphate metabolism for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803, with the use of a disruptant (Δppx) as to the gene of exopolyphosphatase that is responsible for polyphosphate degradation. Δppx was similar to the wild type in the cellular content of polyphosphate to show no defect in cell growth under phosphorus-replete conditions. However, under phosphorus-starved conditions, Δppx cells were defective in a phosphorus-starvation dependent decrease of polyphosphate to show deleterious phenotypes as to their survival and the stabilization of the photosystem complexes. These results demonstrated some crucial role of exopolyphosphatase to degrade polyP in the acclimation of cyanobacterial cells to phosphorus-starved conditions. Besides, it was found that ppx expression is induced in Synechocystis cells in response to phosphorus starvation through the action of the two-component system, SphS and SphR, in the phosphate regulon. The information will be a foundation for a fuller understanding of the process of cyanobacterial acclimation to phosphorus fluctuation.

A unique porin meditates iron-selective transport through cyanobacterial outer membranes.

Cyanobacteria are globally important primary producers and nitrogen fixers with high iron demands. Low ambient dissolved iron concentrations in many aquatic environments mean that these organisms must maintain sufficient and selective transport of iron into the cell. However, the nature of iron transport pathways through the cyanobacterial outer membrane remains obscure. Here we present multiple lines of experimental evidence that collectively support the existence of a novel class of substrate-selective iron porin, Slr1908, in the outer membrane of the cyanobacterium Synechocystis sp. PCC 6803. Elemental composition analysis and short-term iron uptake assays with mutants in Slr1908 reveal that this protein is primarily involved in inorganic iron uptake and contributes less to the accumulation of other metals. Homologues of Slr1908 are widely distributed in both freshwater and marine cyanobacteria, most notably in unicellular marine diazotrophs. Complementary experiments with a homologue of Slr1908 in Synechococcus sp. PCC 7002 restored the phenotype of Synechocystis knockdown mutants, showing that this siderophore producing species also possesses a porin with a similar function in Fe transport. The involvement of a substrate-selective porins in iron uptake may allow cyanobacteria to tightly control iron flux into the cell, particularly in environments where iron concentrations fluctuate.

The global explosion of eukaryotic algae: The potential role of phosphorus?

There arose one of the most important ecological transitions in Earth's history approximately 750 million years ago during the middle Neoproterozoic Era (1000 to 541 million years ago, Ma). Biomarker evidence suggests that around this time there was a rapid shift from a predominantly bacterial-dominated world to more complex ecosystems governed by eukaryotic primary productivity. The resulting 'Rise of the algae' led to dramatically altered food webs that were much more efficient in terms of nutrient and energy transfer. Yet, what triggered this ecological shift? In this study we examined the theory that it was the alleviation of phosphorus (P) deficiency that gave eukaryotic alga the prime opportunity to flourish. We performed laboratory experiments on the cyanobacterium Synechocystis salina and the eukaryotic algae Tetraselmis suecica and examined their ability to compete for phosphorus. Both these organisms co-occur in modern European coastal waters and are not known to have any allelopathic capabilities. The strains were cultured in mono and mixed cultures in chemostats across a range of dissolved inorganic phosphorus (DIP) concentrations to reflect modern and ancient oceanic conditions of 2 µM P and 0.2 µM P, respectively. Our results show that the cyanobacteria outcompete the algae at the low input (0.2 µM P) treatment, yet the eukaryotic algae were not completely excluded and remained a constant background component in the mixed-culture experiments. Also, despite their relatively large cell size, the algae T. suecica had a high affinity for DIP. With DIP input concentrations resembling modern-day levels (2 µM), the eukaryotic algae could effectively compete against the cyanobacteria in terms of total biomass production. These results suggest that the availability of phosphorus could have influenced the global expansion of eukaryotic algae. However, P limitation does not seem to explain the complete absence of eukaryotic algae in the biomarker record before ca. 750 Ma.

Contribution of protein synthesis depression to poly-ß-hydroxybutyrate accumulation in Synechocystis sp. PCC 6803 under nutrient-starved conditions.

Poly-ß-hydroxybutyrate (PHB) in cyanobacteria, which accumulates as energy and carbon sources through the action of photosynthesis, is expected to substitute for petroleum-based plastics. This study first demonstrated that PHB accumulation was induced, with the appearance of lipid droplets, in sulfur (S)-starved cells of a cyanobacterium, Synechocystis sp. PCC 6803, however, to a lower level than in nitrogen (N)- or phosphorus (P)-starved cells. Concomitantly found was repression of the accumulation of total cellular proteins in the S-starved cells to a similar level to that in N-starved cells, and a severer level than in P-starved cells. Intriguingly, PHB accumulation was induced in Synechocystis even under nutrient-replete conditions, upon repression of the accumulation of total cellular proteins through treatment of the wild type cells with a protein synthesis inhibitor, chloramphenicol, or through disruption of the argD gene for Arg synthesis. Meanwhile, the expression of the genes for PHB synthesis was hardly induced in S-starved cells, in contrast to their definite up-regulation in N- or P-starved cells. It therefore seemed that PHB accumulation in S-starved cells is achieved through severe repression of protein synthesis, but is smaller than in N- or P-starved cells, owing to little induction of the expression of PHB synthesis genes.

Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

Heme ligation in hemoglobin is typically assumed by the "proximal" histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two α-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a "distal" histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has been replaced with a leucine, though expected to bind a water molecule and yield a high-spin iron complex at neutral pH, has low-spin spectral properties. Here, we applied nuclear magnetic resonance and electronic absorption spectroscopic methods to GlbN modified with heme and amino acid replacements to identify the distal ligand in H46L GlbN. We found that His117, a residue located in the C-terminal portion of the protein and on the proximal side of the heme, is responsible for the formation of an alternative bis-histidine complex. Simultaneous coordination by His70 and His117 situates the heme in a binding site different from the canonical site. This new holoprotein form is achieved with only local conformational changes. Heme affinity in the alternative site is weaker than in the normal site, likely because of strained coordination and a reduced number of specific heme-protein interactions. The observation of an unconventional heme binding site has important implications for the interpretation of mutagenesis results and globin homology modeling.

Single Amino Acid Change in 6-Phosphogluconate Dehydrogenase from Synechocystis Conveys Higher Affinity for NADP+ and Altered Mode of Inhibition by NADPH.

In the oxidative pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) is one of the enzymes that catalyzes reactions generating NADPH. The model cyanobacterium Synechocystis sp. PCC 6803 is widely studied for numerous applications; however, biochemical knowledge of the NADPH production pathway in Synechocystis sp. PCC 6803 is limited. In this study, we conducted biochemical analysis of a 6-phosphogluconate dehydrogenase from Synechocystis sp. PCC 6803 (Sy6PGDH). We found that Sy6PGDH has unconventional characteristics, i.e. the highest kcat value and non-competitive inhibition by NADPH. Additionally, phylogenetic analysis of cyanobacterial 6PGDHs revealed that an amino acid residue at position 42 in Sy6PGDH is highly conserved for each order of cyanobacteria, but Sy6PGDH is phylogenetically unique. In Sy6PGDH, a single amino acid substitution at position 42 from serine to threonine enhanced the affinity for NADP+ and altered the mode of inhibition by NADPH. The amino acid substitution equivalent to Ser42 also altered the affinity for NADP+ and mode of inhibition by NADPH in Arthrospira platensis. These data suggested that an amino acid residue corresponding to position 42 in Sy6PGDH is one of the important residues that possibly determines the function of cyanobacterial 6PGDHs.

Cyanobacterial production of plant essential oils.

MAIN CONCLUSION: Synechocystis (a cyanobacterium) was employed as an alternative host for the production of plant essential oil constituents. ß-Phellandrene synthase (PHLS) genes from different plants, when expressed in Synechocystis, enabled synthesis of variable monoterpene hydrocarbon blends, converting Synechocystis into a cell factory that photosynthesized and released useful products. Monoterpene synthases are secondary metabolism enzymes that catalyze the generation of essential oil constituents in terrestrial plants. Essential oils, including monoterpene hydrocarbons, are of interest for their commercial application and value. Therefore, heterologous expression of monoterpene synthases for high-capacity essential oil production in photosynthetic microorganism transformants is of current interest. In the present work, the cyanobacterium Synechocystsis PCC 6803 was employed as an alternative host for the production of plant essential oil constituents. As a case study, ß-phellandrene synthase (PHLS) genes from different plants were heterologously expressed in Synechocystis. Genomic integration of individual PHLS-encoding sequences endowed Synechocystis with constitutive monoterpene hydrocarbons generation, occurring concomitant with photosynthesis and cell growth. Specifically, the ß-phellandrene synthase from Lavandula angustifolia (lavender), Solanum lycopersicum (tomato), Pinus banksiana (pine), Picea sitchensis (Sitka spruce) and Abies grandis (grand fir) were active in Synechocystis transformants but, instead of a single product, they generated a blend of terpene hydrocarbons comprising ß-phellandrene, α-phellandrene, ß-myrcene, ß-pinene, and δ-carene with variable percentage ratios ranging from < 10 to > 90% in different product combinations and proportions. Our results suggested that PHLS enzyme conformation and function depends on the cytosolic environment in which they reside, with the biochemical properties of the latter causing catalytic deviations from the products naturally observed in the corresponding gene-encoding plants, giving rise to the terpene hydrocarbon blends described in this work. These findings may have commercial application in the generation of designer essential oil blends and will further assist the development of heterologous cyanobacterial platforms for the generation of desired monoterpene hydrocarbon products.

Deletion of sll1541 in Synechocystis sp. Strain PCC 6803 Allows Formation of a Far-Red-Shifted holo-Proteorhodopsin In Vivo.

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of ß-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal.IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis.

Processing recommendations for using low-solids digestate as nutrient solution for poly-ß-hydroxybutyrate production with Synechocystis salina.

Within the last decades, environmental pollution with persistent plastics steadily increased; therefore the production of biodegradable materials like poly-ß-hydroxybutyrate (PHB) is essential. Currently, PHB is produced with heterotrophic bacteria from crops. This leads to competition with food and feed production, which can be avoided by using photoautotrophic cyanobacteria, as Synechocystis salina, synthesizing PHB from CO2 at nutrient limitation. This study aims to increase the economic efficiency of PHB production with cyanobacteria by using nutrients from anaerobic digestate. First, growth and PHB production of S. salina in digestate fractions (supernatant and permeate, with/without precipitating agents) and dilutions thereof and then the scale-up (photobioreactor, 200 L working volume) were evaluated. With precipitated and centrifuged digestate diluted 1/3 the highest biomass (1.55gL-1) and PHB concentrations (95.4mgL-1), being 78% of those in mineral media, were achieved. In the photobioreactor-experiments biomass (1.63gL-1) and PHB concentrations (88.7mgL-1), being 79% and 72% of those in mineral medium, were reached, but in a cultivation time 10days longer than in mineral medium. The possibility to use digestate as sustainable and low cost nutrient solution for microalgae cultivation and photoautotrophic PHB production, instead of applying it on fields or processing it to achieve discharge limits, makes this application a highly valid option.

Transcriptional regulator PrqR plays a negative role in glucose metabolism and oxidative stress acclimation in Synechocystis sp. PCC 6803.

Plant and cyanobacteria can perceive signals from soluble sugar and reactive oxygen species (ROS) and then coordinate gene expression under stress acclimation, but the underlying mechanism remains unclear. In this study, we found that the transcriptional factor PrqR (Slr0895) in Synechocystis can perceive signals from ROS generated after shifting from prolonged darkness with glucose into high-light. The deletion mutant (DprqR) showed increased growth rate and decreased ROS content, whereas the complementary strain (CprqR) restored the growth characteristics, phenotypes and ROS status of WT, thereby establishing PrqR as a negative regulator of ROS.LC/GC-MS-based metabolic profiling also showed active ROS mitigation in DprqR mutant. Further study by qRT-PCR, ChIP-PCR and deletion of both prqR and prqA (DprqR-DprqA mutant) revealed that PrqR exerts this negative regulation of ROS removal by controlling the expression of sodB and prqA (slr0896). Furthermore, PrqR also found to control glucose metabolism by regulating a positive regulator of glucose metabolism, sigE, and its regulons. Results suggest that PrqR was involved in perceiving signals from ROS under physiological condition, as well as in regulating stress removal and glucose metabolism.

Biotechnological production of limonene in microorganisms.

This mini review describes novel, biotechnology-based, ways of producing the monoterpene limonene. Limonene is applied in relatively highly priced products, such as fragrances, and also has applications with lower value but large production volume, such as biomaterials. Limonene is currently produced as a side product from the citrus juice industry, but the availability and quality are fluctuating and may be insufficient for novel bulk applications. Therefore, complementary microbial production of limonene would be interesting. Since limonene can be derivatized to high-value compounds, microbial platforms also have a great potential beyond just producing limonene. In this review, we discuss the ins and outs of microbial limonene production in comparison with plant-based and chemical production. Achievements and specific challenges for microbial production of limonene are discussed, especially in the light of bulk applications such as biomaterials.

Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production.

BACKGROUND: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. RESULTS: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. CONCLUSION: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

Biocatalytic role of potato starch synthase III for α-glucan biosynthesis in Synechocystis sp. PCC6803 mutants.

A potato starch synthase III (PSSIII) was expressed in the Synechocystis mutants deficient in either glycogen synthase I (M1) or II (M2) to replenish α-(1,4) linkage synthesizing activity, resulting in new mutants, PM1 and PM2, respectively. These mutants were applied to study the role of exogenous plant starch synthase for starch/glycogen biosynthesis mechanism established in the cyanobacteria. The remaining glycogen synthase genes in PM1 and PM2 were further disrupted to make the mutants PM12 and PM21 which contained PSSIII as the sole glycogen/starch synthase. Among wild type and mutants, there were no significant differences in the amount of α-glucan produced. All the mutants harboring active PSSIII produced α-glucans with relatively much shorter and less longer α-1,4 chains than wild-type glycogen, which was exactly in accordance with the increase in glycogen branching enzyme activity. In fact, α-glucan structure of PM1 was very similar to those of PM12 and PM21, and PM2 had more intermediate chains than M2. This result suggests PSSIII may have distributive elongation property during α-glucan synthesis. In conclusion, the Synechocystis as an expression model system of plant enzymes can be applied to determine the role of starch synthesizing enzymes and their association during α-glucan synthesis.