Contaminant removal performance and lipid productivity of a cyanobacteria-bacteria consortium containing exogenous phytohormones during the treatment of antibiotic-polluted wastewater.

In this study, a cyanobacteria-bacteria consortium containing native wastewater bacteria and immobilized Synechococcus sp. was constructed. The cyanobacterial cellular responses (including growth, biomass and lipid productivity) and contaminant removal ability (for TN, TP, COD and antibiotics) in the consortium were evaluated during the advanced treatment of wastewater containing 10-50 µg/L of mixed antibiotics (amoxicillin, tetracycline, erythromycin, sulfadiazine and ciprofloxacin) with the addition of a certain phytohormone (indole-3-acetic acid, gibberellin A3 or 6-benzylaminopurine) at trace level within a period of four days. Each phytohormone promoted the growth of Synechococcus sp. and increased the tolerance of Synechococcus sp. to mixed antibiotics. Indole-3-acetic acid coupled to moderate antibiotic stress could elevate lipid productivity and lipid content of Synechococcus sp. to 33.50 mg/L/day and 43.75%, respectively. Phytohormones increased the pollutant removal performance of the cyanobacteria-bacteria consortium through the stimulation of cyanobacterial growth and the regulation of cyanobacteria-bacteria interaction, which increased the abundances of microalgae-associated bacteria including Flavobacterium, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Bosea, Sphingomonas and Emticicia. Up to 80.83%, 98.06%, 83.26%, 99.84%, 99.50%, 89.41%, 65.61% and 60.65% of TN, TP, COD, amoxicillin, tetracycline, erythromycin, sulfadiazine and ciprofloxacin were removed by the consortium with the addition of phytohormones. In general, indole-3-acetic acid was the optimal phytohormone for enhancing lipid production and contaminant removal performance of the cyanobacteria-bacteria consortium.

Microcystin-LR, a cyanotoxin, modulates division of higher plant chloroplasts through protein phosphatase inhibition and affects cyanobacterial division.

Microcystin-LR (MC-LR) is a harmful cyanotoxin that inhibits 1 and 2A serine-threonine protein phosphatases. This study examines the influence of MC-LR on chloroplast division and the underlying mechanisms and consequences in Arabidopsis. MC-LR increased the frequency of dividing chloroplasts in hypocotyls in a time range of 1-96 h. At short-term exposures to MC-LR, small-sized chloroplasts (longitudinal diameters ≤6 µm) were more sensitive to these stimulatory effects, while both small and large chloroplasts showed stimulations at long-term exposure. After 48 h, the cyanotoxin increased the frequency of small-sized chloroplasts, indicating the stimulation of division. MC-LR inhibited protein phosphatases in whole hypocotyls and isolated chloroplasts, while it did not induce oxidative stress. We show for the first time that total cellular phosphatases play important roles in chloroplast division and that particular chloroplast phosphatases may be involved in these processes. Interestingly, MC-LR has a protective effect on cyanobacterial division during methyl-viologen (MV) treatments in Synechococcus PCC6301. MC-LR production has harmful effects on ecosystems and it may have an ancient cell division regulatory role in stressed cyanobacterial cells, the evolutionary ancestors of chloroplasts. We propose that cytoplasmic (eukaryotic) factors also contribute to the relevant effects of MC-LR in plants.

Salt and heat stress enhances hydrogen production in cyanobacteria.

Cyanobacteria are among the most suitable organisms for the capture of excessive amounts of CO2 and can be grown in extreme environments. In our research we use the single-celled freshwater cyanobacteria Synechococcus elongatus PCC7942 PAMCOD strain and Synechocystis sp. PCC6714 for the production of carbohydrates and hydrogen. PAMCOD strain and Synechocystis sp. PCC6714 synthesize sucrose when exposed to salinity stress, as their main compatible osmolyte. We examined the cell proliferation rate and the sucrose accumulation in those two different strains of cyanobacteria under salt (0.4 M NaCl) and heat stress (35 0C) conditions. The intracellular sucrose (mol sucrose content per Chl a) was found to increase by 50% and 108% in PAMCOD strain and Synechocystis sp. PCC6714 cells, respectively. As previously reported, PAMCOD strain has the ability to produce hydrogen through the process of dark anaerobic fermentation (Vayenos D, Romanos GE, Papageorgiou GC, Stamatakis K (2020) Photosynth Res 146, 235-245). In the present study, we demonstrate that Synechocystis sp. PCC6714 has also this ability. We further examined the optimal conditions during the dark fermentation of PAMCOD and Synechocystis sp. PCC6714 regarding H2 formation, increasing the PAMCOD H2 productivity from 2 nmol H2 h- 1 mol Chl a- 1 to 23 nmol H2 h- 1 mol Chl a- 1. Moreover, after the dark fermentation, the cells demonstrated proliferation in both double BG-11 and BG-11 medium enriched in NaNO3, thus showing the sustainability of the procedure.

Investigating zinc toxicity responses in marine Prochlorococcus and Synechococcus.

Marine plastic pollution is a growing concern worldwide and has the potential to impact marine life via leaching of chemicals, with zinc (Zn), a common plastic additive, observed at particularly high levels in plastic leachates in previous studies. At this time, however, little is known regarding how elevated Zn affects key groups of marine primary producers. Marine cyanobacterial genera Prochlorococcus and Synechococcus are considered to be some of the most abundant oxygenic phototrophs on earth, and together contribute significantly to oceanic primary productivity. Here we set out to investigate how two Prochlorococcus (MIT9312 and NATL2A) and two Synechococcus (CC9311 and WH8102) strains, representative of diverse ecological niches, respond to exposure to high Zn concentrations. The two genera showed differences in the timing and degree of growth and physiological responses to elevated Zn levels, with Prochlorococcus strains showing declines in their growth rate and photophysiology following exposure to 27 µg l-1 Zn, while Synechococcus CC9311 and WH8102 growth rates declined significantly on exposure to 52 and 152 µg l-1 Zn, respectively. Differences were also observed in each strain's capacity to maintain cell wall integrity on exposure to different levels of Zn. Our results indicate that excess Zn has the potential to pose a challenge to some marine picocyanobacteria and highlights the need to better understand how different marine Prochlorococcus and Synechococcus strains may respond to increasing concentrations of Zn in some marine regions.

Amino Acid Analog Induces Stress Response in Marine Synechococcus.

Characterizing the cell-level metabolic trade-offs that phytoplankton exhibit in response to changing environmental conditions is important for predicting the impact of these changes on marine food web dynamics and biogeochemical cycling. The time-selective proteome-labeling approach, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to provide insight into differential allocation of resources at the cellular level, especially when coupled with proteomics. However, the application of this technique in marine phytoplankton remains limited. We demonstrate that the marine cyanobacteria Synechococcus sp. and two groups of eukaryotic algae take up the modified amino acid l-homopropargylglycine (HPG), suggesting that BONCAT can be used to detect translationally active phytoplankton. However, the impact of HPG addition on growth dynamics varied between groups of phytoplankton. In addition, proteomic analysis of Synechococcus cells grown with HPG revealed a physiological shift in nitrogen metabolism, general protein stress, and energy production, indicating a potential limitation for the use of BONCAT in understanding the cell-level response of Synechococcus sp. to environmental change. Variability in HPG sensitivity between algal groups and the impact of HPG on Synechococcus physiology indicates that particular considerations should be taken when applying this technique to other marine taxa or mixed marine microbial communities. IMPORTANCE Phytoplankton form the base of the marine food web and substantially impact global energy and nutrient flow. Marine picocyanobacteria of the genus Synechococcus comprise a large portion of phytoplankton biomass in the ocean and therefore are important model organisms. The technical challenges of environmental proteomics in mixed microbial communities have limited our ability to detect the cell-level adaptations of phytoplankton communities to a changing environment. The proteome labeling technique, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to address some of these challenges by simplifying proteomic analyses. This study explores the ability of marine phytoplankton to take up the modified amino acid, l-homopropargylglycine (HPG), required for BONCAT, and investigates the proteomic response of Synechococcus to HPG. We not only demonstrate that cyanobacteria can take up HPG but also highlight the physiological impact of HPG on Synechococcus, which has implications for future applications of this technique in the marine environment.

The Influence of Metabolic Inhibitors, Antibiotics, and Microgravity on Intact Cell MALDI-TOF Mass Spectra of the Cyanobacterium Synechococcus Sp. UPOC S4.

The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 µM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).

Dissection of the Mechanisms of Growth Inhibition Resulting from Loss of the PII Protein in the Cyanobacterium Synechococcus elongatus PCC 7942.

In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PIIper se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

A cyclic lipopeptide surfactin is a species-selective Hsp90 inhibitor that suppresses cyanobacterial growth.

Heat shock protein 90 (Hsp90) is essential for eukaryotic cells, whereas bacterial homologs play a role under stresses and in pathogenesis. Identifying species-specific Hsp90 inhibitors is challenging because Hsp90 is evolutionarily conserved. We found that a cyclic lipopeptide surfactin inhibits the ATPase activity of Hsp90 from the cyanobacterium Synechococcus elongatus (S.elongatus) PCC 7942 but does not inhibit Escherichia coli (E.coli), yeast and human Hsp90s. Molecular docking simulations indicated that surfactin could bind to the N-terminal dimerization interface of the cyanobacterial Hsp90 in the ATP- and ADP-bound states, which provided molecular insights into the species-selective inhibition. The data suggest that surfactin inhibits a rate-limiting conformational change of S.elongatus Hsp90 in the ATP hydrolysis. Surfactin also inhibited the interaction of the cyanobacterial Hsp90 with a model substrate, and suppressed S.elongatus growth under heat stress, but not that of E.coli. Surfactin did not show significant cellular toxicity towards mammalian cells. These results indicate that surfactin inhibits the cellular function of Hsp90 specifically in the cyanobacterium. The present study shows that a cyclic peptide has a great specificity to interact with a specific homolog of a highly conserved protein family.

Modulation of photosynthesis in Synechocystis and Synechococcus grown with chromium (VI).

Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 exhibit dissimilar tolerance to Cr(VI) with a tenfold difference in their EC50 value for Cr(VI). This contrasting tolerance was attributed to the difference in the ability to transport Cr(VI) and to detoxify ROS. The present study used biochemical assays and chlorophyll fluorescence to investigate the effect of growth with Cr(VI) on photosynthesis in the two cyanobacteria. In absence of Cr(VI), all the measured parameters viz., rates of CO2 fixation, PSII and PSI activities were higher in Synechocystis in comparison to Synechococcus, suggesting intrinsic differences in their photosynthesis. Growth in the presence of Cr(VI) reduced the pigment content and photosystems' activities in both cyanobacteria. It was further observed that photosynthetic functions were more adversely affected in Synechocystis in comparison to Synechococcus, in spite of exposure to tenfold lower Cr(VI) concentration. The effective quantumyield of PSII and PSI obtained by chlorophyll fluorescence measurements increased in the presence of Cr(VI) in Synechococcus whereas it decreased in Synechocystis. However, the overall CO2 fixation remained unchanged. These results indicated that, in addition to the intrinsic difference in photosynthetic rates, the two cyanobacteria exhibit differential modulation of photosynthetic machinery upon Cr(VI) exposure and Synechococcus could adapt better it's photosystems to counter the oxidative stress.

Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance.

Cyanobacteria hold promise as cell factories for the photoautotrophic conversion of carbon dioxide to useful chemicals. For the eventual commercial viability of such processes, cyanobacteria need to be engineered for (i) efficient channeling of carbon flux toward the product of interest and (ii) improved product tolerance, the latter being the focus of this study. We chose the recently reported, fast-growing, high light and CO2 tolerant cyanobacterium Synechococcus elongatus PCC 11801 for adaptive laboratory evolution. In two parallel experiments that lasted over 8400 h of culturing and 100 serial passages, S. elongatus PCC 11801 was evolved to tolerate 5 g/L n-butanol or 30 g/L 2,3-butanediol representing a 100% improvement in concentrations tolerated. The evolved strains retained alcohol tolerance even after being passaged several times without the alcohol stress suggesting that the changes were permanent. Whole genome sequencing of the n-butanol evolved strains revealed mutations in a number of stress responsive genes encoding translation initiation factors, RpoB and an ABC transporter. In 2,3-butanediol evolved strains, genes for ClpC, a different ABC transporter, glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase were found to be mutated. Furthermore, the evolved strains showed significant improvement in tolerance toward several other alcohols. Notably, the n-butanol evolved strain could tolerate up to 32 g/L ethanol, thereby making it a promising host for photosynthetic production of biofuels via metabolic engineering.

Sponge-derived Ageladine A affects the in vivo fluorescence emission spectra of microalgae.

In several marine hosts of microalgae, fluorescent natural products may play an important role. While the ecological function of these compounds is not well understood, an interaction of these molecules with the photosynthesis of the symbionts has been suggested. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on microalgal fluorescence was examined. The spectra showed an accumulation of Ag A within the cells, but with variable impacts on fluorescence. While in two Synechococcus strains, fluorescence of phycoerythrin increased significantly, the fluorescence of other Synechococcus strains was not affected. In four out of the five eukaryote species examined, chlorophyll a (Chl a) fluorescence intensity was modulated. In Tisochrysis lutea, for example, the position of the fluorescence emission maximum of Chl a was shifted. The variety of these effects of Ag A on microalgal fluorescence suggests that fluorophores derived from animals could play a crucial role in shaping the composition of marine host/symbiont systems.

Assessment of transcriptomic constraint-based methods for central carbon flux inference.

MOTIVATION: Determining intracellular metabolic flux through isotope labeling techniques such as 13C metabolic flux analysis (13C-MFA) incurs significant cost and effort. Previous studies have shown transcriptomic data coupled with constraint-based metabolic modeling can determine intracellular fluxes that correlate highly with 13C-MFA measured fluxes and can achieve higher accuracy than constraint-based metabolic modeling alone. These studies, however, used validation data limited to E. coli and S. cerevisiae grown on glucose, with significantly similar flux distribution for central metabolism. It is unclear whether those results apply to more diverse metabolisms, and therefore further, extensive validation is needed. RESULTS: In this paper, we formed a dataset of transcriptomic data coupled with corresponding 13C-MFA flux data for 21 experimental conditions in different unicellular organisms grown on varying carbon substrates and conditions. Three computational flux-balance analysis (FBA) methods were comparatively assessed. The results show when uptake rates of carbon sources and key metabolites are known, transcriptomic data provides no significant advantage over constraint-based metabolic modeling (average correlation coefficients, transcriptomic E-Flux2 0.725 and SPOT 0.650 vs non-transcriptomic pFBA 0.768). When uptake rates are unknown, however, predictions obtained utilizing transcriptomic data are generally good and significantly better than those obtained using constraint-based metabolic modeling alone (E-Flux2 0.385 and SPOT 0.583 vs pFBA 0.237). Thus, transcriptomic data coupled with constraint-based metabolic modeling is a promising method to obtain intracellular flux estimates in microorganisms, particularly in cases where uptake rates of key metabolites cannot be easily determined, such as for growth in complex media or in vivo conditions.

The response of Synechococcus sp. PCC 7002 to micro-/nano polyethylene particles - Investigation of a key anthropogenic stressor.

Microplastics or plastic particles less than 5 mm in size are a ubiquitous and damaging pollutant in the marine environment. However, the interactions between these plastic particles and marine microorganisms are just starting to be understood. The objective of this study was to measure the responses of a characteristic marine organism (Synechococcus sp. PCC 7002) to an anthropogenic stressor (polyethelene nanoparticles and microparticles) using molecular techniques. This investigation showed that polyethylene microparticles and nanoparticles have genetic, enzymatic and morphological effects on Synechococcus sp. PCC 7002. An RT-PCR analysis showed increases in the expression of esterase and hydrolase genes at 5 days of exposure to polyethylene nanoparticles and at 10 days of exposure to polyethylene microparticles. A qualitative enzymatic assay also showed esterase activity in nanoparticle exposed samples. Cryo-scanning electron microscopy was used to assess morphological changes in exopolymer formation resulting from exposure to polyethylene microparticles and nanoparticles. The data from this paper suggests that microplastic and nanoplastics could be key microbial stressors and should be investigated in further detail.

Bayesian modeling reveals metabolite-dependent ultrasensitivity in the cyanobacterial circadian clock.

Mathematical models can enable a predictive understanding of mechanism in cell biology by quantitatively describing complex networks of interactions, but such models are often poorly constrained by available data. Owing to its relative biochemical simplicity, the core circadian oscillator in Synechococcus elongatus has become a prototypical system for studying how collective dynamics emerge from molecular interactions. The oscillator consists of only three proteins, KaiA, KaiB, and KaiC, and near-24-h cycles of KaiC phosphorylation can be reconstituted in vitro. Here, we formulate a molecularly detailed but mechanistically naive model of the KaiA-KaiC subsystem and fit it directly to experimental data within a Bayesian parameter estimation framework. Analysis of the fits consistently reveals an ultrasensitive response for KaiC phosphorylation as a function of KaiA concentration, which we confirm experimentally. This ultrasensitivity primarily results from the differential affinity of KaiA for competing nucleotide-bound states of KaiC. We argue that the ultrasensitive stimulus-response relation likely plays an important role in metabolic compensation by suppressing premature phosphorylation at nighttime.

Oxidative stress measurement in different morphological forms of wild-type and mutant cyanobacterial strains: Overcoming the limitation of fluorescence microscope-based method.

Monitoring of oxidative stress caused by a wide range of reactive oxygen species (ROS) is essential to have an idea about the fitness and growth of photosynthetic organisms. The imaging-based oxidative stress measurement in cyanobacteria using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye has the limitation of small sample size as the only selected number of cells are analyzed to measure the ROS levels. Here, we developed a method for oxidative stress measurement by DCFH-DA and flow cytometer (FCM) using unicellular Synechococcus elongatus PCC 7942 and filamentous Fremyella diplosiphon BK14 cyanobacteria. F. diplosiphon BK14 inherently possess high levels of ROS and showed higher sensitivity to hydrogen peroxide treatment in comparison to S. elongatus PCC 7942. We successfully measured oxidative stress in glutaredoxin lacking strain (Δgrx3) of S. elongatus PCC 7942, and wild-type Synechocystis sp. PCC 6803 using FCM based method. Importantly, ROS were not detected in these two strains of cyanobacteria by fluorescence microscope-based method due to their small spherical morphology. Δgrx3 strain showed high ROS levels in comparison to its wild-type strain. Treatment of abiotic factors such as high PAR in wild-type and Δgrx3 strains of S. elongatus PCC 7942, low PAR or low PAR + UVR in wild-type S. elongatus PCC 7942, and high PAR or high PAR + NaCl in Synechocystis sp. PCC 6803 increased oxidative stress. In summary, the FCM based method can measure ROS levels produced due to physiological conditions associated with genetic changes or abiotic stress in a large population of cells regardless of their morphology. Therefore, the present study shows the usefulness of the method in monitoring the health of organisms in a large scale cultivation system.

Accumulation of sugars and nucleosides in response to high salt and butanol stress in 1-butanol producing Synechococcus elongatus.

1-Butanol production using photosynthetic organisms such as cyanobacteria has garnered interest among researchers due to its high potential as a sustainable biofuel. Previously, the cyanobacterium Synechococcus elongatus PCC 7942 was engineered to produce 1-butanol through the introduction of a modified CoA-dependent pathway. S. elongatus strain DC11, a high producer of 1-butanol, was constructed based on metabolomics-assisted strain engineering. DC11 can reach a production titer of 418.7 mg/L in 6 days, cutting the production time in half compared to the previously constructed DC7. Regardless, the final 1-butanol titer of DC11 was still low compared to other microbial hosts. Sensitivity towards 1-butanol of the producing strain has been known as one of main hurdles for improving cyanobacterial production system. Thus, to improve cyanobacterial-based 1-butanol production in the future, we employed the metabolomics approach to study the intrinsic effect of improved 1-butanol productivity in DC11. This study focused on metabolite profiling of DC11 using LC/MS/MS. Results showed that there is an accumulation of disaccharide-P and sucrose/trehalose in DC11 compared to the DC7. These metabolites were previously reported to have a role in salt and alcohol stress response in cyanobacteria and therefore, DC11 was subjected to 0.2 M of NaCl and 1000 mg/L of 1-butanol for further investigation. DC11 with stress treatment showed a more prominent accumulation of sugars and nucleosides compared to control. The results obtained from this study may be beneficial for future strain improvement strategies in S. elongatus, particularly addressing the metabolic response of this strain upon 1-butanol stress.

Development of a New Biocontainment Strategy in Model Cyanobacterium Synechococcus Strains.

Recent synthetic biology efforts have raised biosafety concerns for possible release of engineered cyanobacteria into natural environments. To address the issues, we developed a controllable metal ion induced biocontainment system for two model cyanobacteria. First, six ion-inducible promoters were respectively evaluated in both Synechococcus elongatus PCC 7942 and the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, leading to the identification of an iron ion-repressed promoter PisiAB with low leakage and a reduction-fold of 5.4 and 7.9, respectively. Second, holin-endolysin and nuclease NucA systems were introduced, the inhibition rate of which against two Synechococcus strains varied from 61% to 86.4%. Third, two toxin/antitoxin modules were identified capable of inducing programmed suicide in both Synechococcus strains after induction. Furthermore, an escape experiment was conducted and the results showed that the system was able to achieve an escape frequency below the detection limit of 10-9 after 3 days' duration, demonstrating the strategy integrating iron ion-inducible promoter PisiAB and that toxin/antitoxin modules could be a useful tool for cyanobacterium biocontainment.

Enhanced stable production of ethylene in photosynthetic cyanobacterium Synechococcus elongatus PCC 7942.

Ethylene is a volatile alkene which is used in large commercial scale as a precursor in plastic industry, and is currently derived from petroleum refinement. As an alternative production strategy, photoautotrophic cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have been previously evaluated as potential biotechnological hosts for producing ethylene directly from CO2, by the over-expression of ethylene forming enzyme (efe) from Pseudomonas syringae. This work addresses various open questions related to the use of Synechococcus as the engineering target, and demonstrates long-term ethylene production at rates reaching 140 µL L-1 h-1 OD750-1 without loss of host vitality or capacity to produce ethylene. The results imply that the genetic instability observed earlier may be associated with the expression strategies, rather than efe over-expression, ethylene toxicity or the depletion of 2-oxoglutarate-derived cellular precursors in Synechococcus. In context with literature, this study underlines the critical differences in expression system design in the alternative hosts, and confirms Synechococcus as a suitable parallel host for further engineering.

Expression of a stress-responsive gene cluster for mycosporine-2-glycine confers oxidative stress tolerance in Synechococcus elongatus PCC7942.

Mycosporine-like amino acids (MAAs) are a class of well-documented UV-screening compounds produced by taxonomically diverse organisms. Extensive studies revealed that a rare MAA, mycosporine-2-glycine (M2G), possesses unique biological activities and functions. M2G is not only a potent antioxidant, but also suppresses protein glycation in vitro, and production of inflammatory mediators in RAW 264.7 macrophages. The present study evaluates vital functions of M2G in a heterologous expression system. The stress-sensitive fresh water cyanobacterium Synechococcus elongatus PCC7942, carrying a M2G biosynthetic gene cluster, was generated. The M2G-expressing cells were more tolerant to H2O2-induced oxidative stress than the wild type, with a half-maximal inhibitory concentration (IC50) value of 2.3 ± 0.06 mM. Transcriptional analysis revealed that all M2G biosynthetic genes were highly up-regulated under oxidative stress. Further, expression of vital genes in the cellular antioxidant defense system, including sodB, cat and tpxA were modulated and up-regulated. Elevated M2G was detected under oxidative stress as well as salt stress treatments. This study provides insight into the molecular and cellular effects of the M2G biosynthetic gene cluster, contributing to understanding of the mechanism behind physiological plasticity under this heterologous expression system.

Elevated carbon dioxide levels lead to proteome-wide alterations for optimal growth of a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801.

The environmental considerations attributing to the escalation of carbon dioxide emissions have raised alarmingly. Consequently, the concept of sequestration and biological conversion of CO2 by photosynthetic microorganisms is gaining enormous recognition. In this study, in an attempt to discern the synergistic CO2 tolerance mechanisms, metabolic responses to increasing CO2 concentrations were determined for Synechococcus elongatus PCC 11801, a fast-growing, novel freshwater strain, using quantitative proteomics. The protein expression data revealed that the organism responded to elevated CO2 by not only regulating the cellular transporters involved in carbon-nitrogen uptake and assimilation but also by inducing photosynthesis, carbon fixation and glycolysis. Several components of photosynthetic machinery like photosystem reaction centers, phycobilisomes, cytochromes, etc. showed a marked up-regulation with a concomitant downshift in proteins involved in photoprotection and redox maintenance. Additionally, enzymes belonging to the TCA cycle and oxidative pentose phosphate pathway exhibited a decline in their expression, further highlighting that the demand for reduced cofactors was fulfilled primarily through photosynthesis. The present study brings the first-ever comprehensive assessment of intricate molecular changes in this novel strain while shifting from carbon-limited to carbon-sufficient conditions and may pave the path for future host and pathway engineering for production of sustainable fuels through efficient CO2 capture.