Synechocystis Strain Research Articles

Escherichia coli AraJ boosts utilization of arabinose in metabolically engineered cyanobacterium Synechocystis sp. PCC 6803

Lignocellulosic biomass can serve as an inexpensive and renewable source of carbon for the biosynthesis of commercially important compounds. L-arabinose is the second most abundant pentose sugar present in the plant materials. Model cyanobacterium Synechocystis sp. PCC 6803 is incapable of catabolism of L-arabinose as a source of carbon and energy. In this study, all the heterologous genes expressed in Synechocystis were derived from Escherichia coli K-12. Initially we constructed four Synechocystis strains that expressed AraBAD enzymes involved in L-arabinose catabolism, either in combination with or without one of the three arabinose transporters, AraE, AraFGH or AraJ. Among the recombinants, the strain possessing AraJ transporter was observed to be the most efficient in terms of dry biomass production and L-arabinose consumption. Later, an additional strain was generated by the expression of AraJ in the AraE-possessing strain. The resultant strain was shown to be advantageous over its parent. This study demonstrates that AraJ, a protein with hitherto unknown function plays a role in the uptake of L-arabinose to boost its catabolism in the transgenic Synechocystis strains. The work also contributes to the current knowledge regarding metabolic engineering of cyanobacteria for the utilization of pentose sugars.

Differential Labeling of Chemically Modified Peptides and Lipids among Cyanobacteria Planktothrix and Microcystis.

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labeling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labeled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labeling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed increased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labeling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labeled peptides was not increased suggesting a reduced spatial overlap.

Introduction of the Carotenoid Biosynthesis α-Branch Into Synechocystis sp. PCC 6803 for Lutein Production.

Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous β-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD730 (1.03 ± 0.47 mmol mol–1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system.

Homologs of Circadian Clock Proteins Impact the Metabolic Switch Between Light and Dark Growth in the Cyanobacterium Synechocystis sp. PCC 6803.

The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and non-targeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaA and, to a lesser extent, in the ΔkaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.

Production of thermostable phycocyanin in a mesophilic cyanobacterium

Phycocyanin (PC) is a soluble phycobiliprotein found within the light-harvesting phycobilisome complex of cyanobacteria and red algae, and is considered a high-value product due to its brilliant blue colour and fluorescent properties. However, commercially available PC has a relatively low temperature stability. Thermophilic species produce more thermostable variants of PC, but are challenging and energetically expensive to cultivate. Here, we show that the PC operon from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (cpcBACD) is functional in the mesophile Synechocystis sp. PCC 6803. Expression of cpcBACD in an ‘Olive’ mutant strain of Synechocystis lacking endogenous PC resulted in high yields of thermostable PC (112 ± 1 mg g−1 DW) comparable to that of endogenous PC in wild-type cells. Heterologous PC also improved the growth of the Olive mutant, which was further supported by evidence of a functional interaction with the endogenous allophycocyanin core of the phycobilisome complex. The thermostability properties of the heterologous PC were comparable to those of PC from T. elongatus, and could be purified from the Olive mutant using a low-cost heat treatment method. Finally, we developed a scalable model to calculate the energetic benefits of producing PC from T. elongatus in Synechocystis cultures. Our model showed that the higher yields and lower cultivation temperatures of Synechocystis resulted in a 3.5-fold increase in energy efficiency compared to T. elongatus, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.

Impairing photorespiration increases photosynthetic conversion of CO2 to isoprene in engineered cyanobacteria

Photorespiration consumes fixed carbon and energy generated from photosynthesis to recycle glycolate and dissipate excess energy. The aim of this study was to investigate whether we can use the energy that is otherwise consumed by photorespiration to improve the production of chemicals which requires energy input. To this end, we designed and introduced an isoprene synthetic pathway, which requires ATP and NADPH input, into the cyanobacterium Synechocystis sp. 6803. We then deleted the glcD1 and glcD2 genes which encode glycolate dehydrogenase to impair photorespiration in isoprene-producing strain of Synechocystis. Production of isoprene in glcD1/glcD2 disrupted strain doubled, and stoichiometric analysis indicated that the energy saved from the impaired photorespiration was redirected to increase production of isoprene. Thus, we demonstrate we can use the energy consumed by photorespiration of cyanobacteria to increase the energy-dependent production of chemicals from CO2.

Enhanced Production of Photosynthetic Pigments and Various Metabolites and Lipids in the Cyanobacteria Synechocystis sp. PCC 7338 Culture in the Presence of Exogenous Glucose.

Synechocystis strains are cyanobacteria that can produce useful biomaterials for biofuel and pharmaceutical resources. In this study, the effects of exogenous glucose (5-mM) on cell growth, photosynthetic pigments, metabolites, and lipids in Synechocystis sp. PCC 7338 (referred to as Synechocystis 7338) were investigated. Exogenous glucose increased cell growth on days 9 and 18. The highest production (mg/L) of chlorophyll a (34.66), phycocyanin (84.94), allophycocyanin (34.28), and phycoerythrin (6.90) was observed on day 18 in Synechocystis 7338 culture under 5-mM glucose. Alterations in metabolic and lipidomic profiles under 5-mM glucose were investigated using gas chromatography-mass spectrometry (MS) and nanoelectrospray ionization-MS. The highest production (relative intensity/L) of aspartic acid, glutamic acid, glycerol-3-phosphate, linolenic acid, monogalactosyldiacylglycerol (MGDG) 16:0/18:1, MGDG 16:0/20:2, MGDG 18:1/18:2, neophytadiene, oleic acid, phosphatidylglycerol (PG) 16:0/16:0, and PG 16:0/17:2 was achieved on day 9. The highest production of pyroglutamic acid and sucrose was observed on day 18. We suggest that the addition of exogenous glucose to Synechocystis 7338 culture could be an efficient strategy for improving growth of cells and production of photosynthetic pigments, metabolites, and intact lipid species for industrial applications.

Isobutene production in Synechocystis sp. PCC 6803 by introducing α-ketoisocaproate dioxygenase from Rattus norvegicus

Cyanobacteria can be utilized as a platform for direct phototrophic conversion of CO2 to produce several types of carbon-neutral biofuels. One promising compound to be produced photobiologically in cyanobacteria is isobutene. As a volatile compound, isobutene will quickly escape the cells without building up to toxic levels in growth medium or get caught in the membranes. Unlike liquid biofuels, gaseous isobutene may be collected from the headspace and thus avoid the costly extraction of a chemical from culture medium or from cells. Here we investigate a putative synthetic pathway for isobutene production suitable for a photoautotrophic host. First, we expressed α-ketoisocaproate dioxygenase from Rattus norvegicus (RnKICD) in Escherichia coli. We discovered isobutene formation with the purified RnKICD with the rate of 104.6 ​± ​9 ​ng (mg protein)-1 min-1 using α-ketoisocaproate as a substrate. We further demonstrate isobutene production in the cyanobacterium Synechocystis sp. PCC 6803 by introducing the RnKICD enzyme. Synechocystis strain heterologously expressing the RnKICD produced 91 ​ng ​l−1 OD750−1 ​h−1. Thus, we demonstrate a novel sustainable platform for cyanobacterial production of an important building block chemical, isobutene. These results indicate that RnKICD can be used to further optimize the synthetic isobutene pathway by protein and metabolic engineering efforts.

Trans-4-hydroxy-L-proline production by the cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria play an important role in photobiotechnology. Yet, one of their key central metabolic pathways, the tricarboxylic acid (TCA) cycle, has a unique architecture compared to most heterotrophs and still remains largely unexploited. The conversion of 2-oxoglutarate to succinate via succinyl-CoA is absent but is by-passed by several other reactions. Overall, fluxes under photoautotrophic growth conditions through the TCA cycle are low, which has implications for the production of chemicals. In this study, we investigate the capacity of the TCA cycle of Synechocystis sp PCC 6803 for the production of trans-4-hydroxy-L-proline (Hyp), a valuable chiral building block for the pharmaceutical and cosmetic industries. For the first time, photoautotrophic Hyp production was achieved in a cyanobacterium expressing the gene for the L-proline-4-hydroxylase (P4H) from Dactylosporangium sp. strain RH1. Interestingly, while elevated intracellular Hyp concentrations could be detected in the recombinant Synechocystis strains under all tested conditions, detectable Hyp secretion into the medium was only observed when the pH of the medium exceeded 9.5 and mostly in the late phases of the cultivation. We compared the rates obtained for autotrophic Hyp production with published sugar-based production rates in E. coli. The land-use efficiency (space-time yield) of the phototrophic process is already in the same order of magnitude as the heterotrophic process considering sugar farming as well. But, the remarkable plasticity of the cyanobacterial TCA cycle promises the potential for a 23–55 fold increase in space-time yield when using Synechocystis. Altogether, these findings contribute to a better understanding of bioproduction from the TCA cycle in photoautotrophs and broaden the spectrum of chemicals produced in metabolically engineered cyanobacteria.

Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis.

The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c=92.02 Å, β=90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.

Comparative Proteomic Profiling of Marine and Freshwater Synechocystis Strains Using Liquid Chromatography-Tandem Mass Spectrometry

Freshwater Synechocystis sp. PCC 6803 has been considered to be a platform for the production of the next generation of biofuels and is used as a model organism in various fields. Various genomics, transcriptomics, metabolomics, and proteomics studies have been performed on this strain, whereas marine Synechocystis sp. PCC 7338 has not been widely studied despite its wide distribution. This study analyzed the proteome profiles of two Synechocystis strains using a liquid chromatography–tandem mass spectrometry-based bottom-up proteomic approach. Proteomic profiling of Synechocystis sp. PCC 7338 was performed for the first time with a data-dependent acquisition method, revealing 18,779 unique peptides and 1794 protein groups. A data-independent acquisition method was carried out for the comparative quantitation of Synechocystis sp. PCC 6803 and 7338. Among 2049 quantified proteins, 185 up- and 211 down-regulated proteins were defined in Synechocystis sp. PCC 7338. Some characteristics in the proteome of Synechocystis sp. PCC 7338 were revealed, such as its adaptation to living conditions, including the down-regulation of some photosynthesis proteins, the up-regulation of kdpB, and the use of osmolyte glycine as a substrate in C1 metabolism for the regulation of carbon flow. This study will facilitate further studies on Synechocystis 7338 to define in depth the proteomic differences between it and other Synechocystis strains.

Heterologous expression and purification of the bicarbonate transporter BicA from Synechocystis sp. PCC 6803

The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA6803) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA6803 relative to other strains screened, and membrane solubilization with n-dodecyl-β-d-maltopyranoside facilitated the purification of high levels of stable and homogenous BicA6803. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture.

Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803

BackgroundMannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p, in short: a ‘mannitol cassette’), proved to be genetically unstable. In this study, we aim to overcome this genetic instability by conceiving a strategy to stabilize mannitol production using Synechocystis sp. PCC6803 as a model cyanobacterium.ResultsHere, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild-type Synechocystis, complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production.ConclusionsMannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.

Identification of the electron donor to flavodiiron proteins in Synechocystis sp. PCC 6803 by in vivo spectroscopy

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO2, both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δflv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (FAFB), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δflv1/3 (13 vs 2 s−1). These data unambiguously show that reduced ferredoxin, or possibly reduced FAFB, is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (FAFB) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.

Comparative Primary Metabolic and Lipidomic Profiling of Freshwater and Marine Synechocystis Strains Using by GC-MS and NanoESI-MS Analyses

Synechocystis strains are considered as valuable resources for production of useful metabolites. Comparative primary metabolic and lipidomic profiling of freshwater and marine Synechocystis strains is needed to understand the physiological characteristics of each strain at molecular level. In this study, we performed comparative primary metabolic and lipidomic profiling of freshwater Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) and marine Synechocystis sp. PCC 7338 (hereafter Synechocystis 7338) by using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry. Neophytadiene, phytol, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) were significantly higher in Synechocystis 6803. Levels of alanine, aspartic acid, valine, linoleic acid, linolenic acid, oleic acid, plamitic acid, stearic acid, glucosylglycerol, sucrose, phosphatidylglycerol (PG), and diacylglyceryltrimethylhomoserine (DGTS) (only detected in Synechocystis 7338) were significantly higher in Synechocystis 7338. Our results provide basic information to choose Synechocystis strains for various industrial usages, and also provide fundamental information on the future development of novel Synechocystis strains to produce high levels of specific metabolites by metabolic pathway modulation.

Assembly properties of the bacterial tubulin homolog FtsZ from the cyanobacterium Synechocystis sp. PCC 6803

The tubulin homolog FtsZ is the major cytoskeletal protein in the bacterial cell division machinery, conserved in almost all bacteria, archaea, and chloroplasts. Bacterial FtsZ assembles spontaneously into single protofilaments, sheets, and bundles in vitro, and it also accumulates at the site of division early during cell division, where it forms a dynamic protein complex, the contractile ring or Z-ring. The biochemical properties of FtsZ proteins from many bacteria have been studied, but comparable insights into FtsZs from cyanobacteria are limited. Here, using EM and light-scattering assays, we studied the biochemical and assembly properties of SyFtsZ, the FtsZ protein from the cyanobacterial strain Synechocystis sp. PCC 6803. SyFtsZ had a slow GTPase activity of ∼0.4 GTP/FtsZ molecule/min and assembled into thick, straight protofilament bundles and curved bundles, designated toroids. The assembly of SyFtsZ in the presence of GTP occurred in two stages. The first stage consisted of the assembly of single-stranded straight protofilaments and opened circles; in the second stage, the protofilaments associated into straight protofilament bundles and toroids. In addition to these assemblies, we also observed highly curved oligomers and minirings after GTP hydrolysis or in the presence of excess GDP. The three types of protofilaments of SyFtsZ observed here provide support for the hypothesis that a constriction force due to curved protofilaments bends the membrane. In summary, our findings indicate that, unlike other bacterial FtsZ, SyFtsZ assembles into thick protofilament bundles. This bundling is similar to that of chloroplast FtsZ, consistent with its origin in cyanobacteria.

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Structure optimization and bioactivity evaluation of ThDP analogs targeting cyanobacterial pyruvate dehydrogenase E1

Harmful cyanobacteria bloom (HCB) has occurred frequently in recent years and it is urgent to develop novel algicides to deal with this problem. In this paper, a series of novel thiamin diphosphate (ThDP) analogs 5a−5g were designed and synthesized targeting cyanobacterial pyruvate dehydrogenase complex E1 (Cy-PDHc E1). Our results showed that compounds 5a−5g have higher inhibitory activities against Cy-PDHc E1 (IC50 9.56–3.48 µM) and higher inhibitory activities against two model cyanobacteria strains Synechocystis sp PCC6803 (EC50 2.03–1.58 µM) and Microcystis aeruginosa FACHB905 (EC50 1.86–0.95 µM). Especially, compound 5b displayed highest inhibitory activities (IC50 = 3.48 µM) against Cy-PDHc E1 and powerful inhibitory activities against cyanobacteria Synechocystis sp PCC6803 (EC50 = 1.58 µM) and Microcystis aeruginosa FACHB905 (EC50 = 1.04 µM). Moreover, the inhibitory activities of compound 5b were even higher than those of copper sulfate (EC50 = 2.02 and 1.71 µM separately) which has been widely used as algicide against cyanobacteria PCC6803 and FACHB905. The more important was that compound 5b display much higher inhibitory selectivity between Cy-PDHc E1 (Inhibitory rate 97.4%) and porcine PDHc E1 (Inhibitory rate 11.8%) under the same concentration (100 μM). The inhibition kinetic experiment and molecular docking research showed that compound 5b can inhibit Cy-PDHc E1 by occupying the ThDP-binding pocket and then blocking Cy-PDHc E1 bound to ThDP as competitive inhibitor. The imagines of SEM and TEM showed that cellular microstructures were heavily destroyed under compound 5b stress. Our results demonstrated compound 5b could be taken as a potential lead compound targeting Cy-PDHc E1 to obtain environment-friendly algicide for harmful cyanobacterial blooms control.

Combining isotopically non-stationary metabolic flux analysis with proteomics to unravel the regulation of the Calvin-Benson-Bassham cycle in Synechocystis sp. PCC 6803

Photosynthetic microorganisms are increasingly being investigated as a sustainable alternative to existing bio-industrial processes, converting CO2 into desirable end products without the use of carbohydrate feedstock. The Calvin-Benson-Bassham (CBB) cycle is the main pathway of carbon fixation metabolism in photosynthetic organisms. In this study, we analyzed the metabolic fluxes in two strains of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) that overexpressed fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) and transketolase (TK), respectively. These two potential carbon flux control enzymes in the CBB cycle had previously been shown to improve biomass accumulation when overexpressed under air and low light (15 μmol m−2 s−1) conditions (Liang and Lindblad, 2016). We measured the growth rates of Synechocystis under atmospheric and high (3% v/v) CO2 conditions at 80 μmol m−2 s−1. Surprisingly, the cells overexpressing transketolase (tktA) demonstrated no significant increase in growth rates when CO2 was increased, suggesting an altered carbon flux distribution and a potential metabolic bottleneck in carbon fixation. Moreover, the tktA strain had an increased susceptibility to oxidative stress under high light as revealed by its chlorotic phenotype under high light conditions. In contrast, the fructose-1,6/sedoheptulose-1,7-bisphosphatase (70glpX) and wild-type cells demonstrated increases in growth rates as expected.To investigate the disparate phenotypical responses of these different Synechocystis strains, isotopically non-stationary metabolic flux analysis (INST-MFA) was used to estimate the carbon flux distribution of tktA, 70glpX, and a kanamycin-resistant control (Km), under atmospheric conditions. In addition, untargeted label-free proteomics, which can detect changes in relative enzymatic abundance, was employed to study the possible effects caused by overexpressing each enzyme. Fluxomic and proteomic results indicated a decrease in oxidative pentose phosphate pathway activity when either FBP/SBPase or TK were overexpressed, resulting in increased carbon fixation efficiency. These results are an example of the integration of multiple omic-level experimental techniques and can be used to guide future metabolic engineering efforts to improve performances and efficiencies.