Increased Biomass and Polyhydroxybutyrate Production by Synechocystis sp. PCC 6803 Overexpressing RuBisCO Genes.

The overexpression of the RuBisCO (rbc) gene has recently become an achievable strategy for increasing cyanobacterial biomass and overcoming the biocompound production restriction. We successfully constructed two rbc-overexpressing Synechocystis sp. PCC 6803 strains (OX), including a strain overexpressing a large subunit of RuBisCO (OXrbcL) and another strain overexpressing all large, chaperone, and small subunits of RuBisCO (OXrbcLXS), resulting in higher and faster growth than wild type under sodium bicarbonate supplementation. This increased biomass of OX strains significantly contributed to the higher polyhydroxybutyrate (PHB) production induced by nutrient-deprived conditions, in particular nitrogen (N) and phosphorus (P). As a result of higher PHB contents in OX strains occurring at days 7 and 9 of nutrient deprivation, this enhancement was apparently made possible by cells preferentially maintaining their internal lipids while accumulating less glycogen. The OXrbcLXS strain, with the highest level of PHB at about 39 %w/dry cell weight (DCW) during 7 days of BG11-NP treatment, contained a lower glycogen level (31.9 %w/DCW) than wild type control (40 %w/DCW). In contrast, the wild type control strain exposed to N- and NP-stresses tended to retain lipid levels and store more glycogen than PHB. In this model, we, for the first time, implemented a RuBisCO-overexpressing cyanobacterial factory for overproducing PHB, destined for biofuel and biomaterial biotechnology.

Overexpressing Carotenoid Biosynthetic Genes in Synechocystis sp. PCC 6803 Improved Intracellular Pigments and Antioxidant Activity, Which Can Decrease the Viability and Proliferation of Lung Cancer Cells In Vitro.

In the antioxidant system in cyanobacteria, non-enzymatic antioxidants, such as carotenoids, are considered good candidates for coping with oxidative stress, particularly light stress, and pharmaceutical therapeutic applications. A significant amount of carotenoid accumulation has been recently improved by genetic engineering. In this study, to achieve higher carotenoid production with higher antioxidant activity, we successfully constructed five Synechocystis sp. PCC 6803 strains overexpressing (OX) native genes related to the carotenoids biosynthetic pathway, including OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. All of the engineered strains maintained a significant quantity of myxoxanthophyll, while increasing zeaxanthin and echinenone accumulation. In addition, higher components of zeaxanthin and echinenone were noted in all OX strains, ranging from 14 to 19% and from 17 to 22%, respectively. It is worth noting that the enhanced echinenone component responded to low light conditions, while the increased ß-carotene component contributed to a high light stress response. According to the higher antioxidant activity of all OX strains, the carotenoid extracts presented lower IC50 in lung cancer cell lines H460 and A549, with values less than 157 and 139 µg/mL, respectively, when compared with those of WTc, particularly OX_CrtR and OX_CrtQ. A higher proportion of zeaxanthin and ß-carotene in OX_CrtR and OX_CrtQ, respectively, may considerably contribute to the ability to treat lung cancer cells with antiproliferative and cytotoxic effects.

Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation.

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG11-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth.

Crystal structure of dimeric Synechococcus spermidine synthase with bound polyamine substrate and product.

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

Response of Synechocystis sp. PCC 6803 to UV radiations by alteration of polyamines associated with thylakoid membrane proteins.

The responses of Synechocystis sp. PCC 6803 exposed to UVA, UVB and UVC for at least 3 h were investigated with the emphasis on the changes of polyamines (PAs) levels in whole cells, thylakoid membrane fraction, and thylakoid membrane-associated proteins fraction. All UV radiations caused a slight decrease on cell growth but a drastic reduction of photosynthetic efficiency of Synechocystis cells. UV radiations, especially UVB and UVC, severely decreased the levels of PAs associated with thylakoid membrane proteins. The decreased PAs levels as affected by UV radiation correlated well with the decrease of photosynthetic efficiency, suggesting the role of PAs for the maintenance of photosynthetic activity of Synechocystis. PAs, especially spermidine (Spd) and putrescine (Put), were found abundantly in the thylakoid membrane fraction, and these PAs were associated mainly with the PSI trimer complex. Importantly, the exposure of Synechocystis cells to all UV radiations for 3 h resulted in the increase of Spd associated with the PSII monomer and dimer complex, suggesting its protective role against UV radiations despite the overall decrease of PAs.

Molecular characterization and homology modeling of spermidine synthase from Synechococcus sp. PCC 7942.

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37 °C. The enzyme had higher affinity for dcSAM (K m, 20 µM) than for putrescine (K m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.

Spermidine Synthase is Required for Growth of Synechococcus sp. PCC 7942 Under Osmotic Stress.

The Synechococcus sp. PCC 7942 spermidine synthase encoded by spds gene (Synpcc7942_0628) is responsible for spermidine biosynthesis. Two Synechococcus strains, the overexpressing spds (OX-spds) and the spds knockout (Δspds), were constructed and characterized for their growth and photosynthetic efficiency under osmotic stress imposed by sorbitol. The growth of Δspds was completely inhibited when cells were grown in the presence of 400 mM sorbitol. Under the same condition, the OX-spds showed a slightly higher growth than the wild type. The OX-spds under osmotic stress also had a significant increase of spermidine level in conjunction with the up-regulation of the genes involved in spermidine biosynthesis. A higher ratio of spermidine to putrescine, an index for stress tolerance, under osmotic stress was found in the OX-spds strain than in the wild type. Overall results indicated that the spermidine synthase enzyme plays an essential role in the survival of Synechococcus sp. PCC 7942 under osmotic stress.

The adc1 knockout with proC overexpression in Synechocystis sp. PCC 6803 induces a diversion of acetyl-CoA to produce more polyhydroxybutyrate.

BACKGROUND: Lack of nutrients, in particular nitrogen and phosphorus, has been known in the field to sense glutamate production via 2-oxoglutarate and subsequently accelerate carbon storage, including glycogen and polyhydroxybutyrate (PHB), in cyanobacteria, but a few studies have focused on arginine catabolism. In this study, we first time demonstrated that gene manipulation on proC and adc1, related to proline and polyamine syntheses in arginine catabolism, had a significant impact on enhanced PHB production during late growth phase and nutrient-modified conditions. We constructed Synechocystis sp. PCC 6803 with an overexpressing proC gene, encoding Δ1pyrroline-5-carboxylate reductase in proline production, and adc1 disruption resulted in lower polyamine synthesis. RESULTS: Three engineered Synechocystis sp. PCC 6803 strains, including a ProC-overexpressing strain (OXP), adc1 mutant, and an OXP strain lacking the adc1 gene (OXP/Δadc1), certainly increased the PHB accumulation under nitrogen and phosphorus deficiency. The possible advantages of single proC overexpression include improved PHB and glycogen storage in late phase of growth and long-term stress situations. However, on day 7 of treatment, the synergistic impact created by OXP/Δadc1 increased PHB synthesis by approximately 48.9% of dry cell weight, resulting in a shorter response to nutrient stress than the OXP strain. Notably, changes in proline and glutamate contents in engineered strains, in particular OXP and OXP/Δadc1, not only partially balanced the intracellular C/N metabolism but also helped cells acclimate under nitrogen (N) and phosphorus (P) stress with higher chlorophyll a content in comparison with wild-type control. CONCLUSIONS: In Synechocystis sp. PCC 6803, overexpression of proC resulted in a striking signal to PHB and glycogen accumulation after prolonged nutrient deprivation. When combined with the adc1 disruption, there was a notable increase in PHB production, particularly in situations where there was a strong C supply and a lack of N and P.

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.

Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803.

BACKGROUND: Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl-acyl carrier protein synthetase), and surface layer protein (encoded by sll1951). RESULTS: Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG11-half N and BG11-N conditions, with the exception of strain KS. Under BG11-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG11-N growth medium. This strain KS significantly acclimated to the BG11-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion. CONCLUSIONS: Mutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation.

Native SodB Overexpression of Synechocystis sp. PCC 6803 Improves Cell Growth Under Alcohol Stresses Whereas Its Gpx2 Overexpression Impacts on Growth Recovery from Alcohol Stressors.

To overcome the limited resistance to alcohol stress, genetically engineered Synechocystis sp. PCC 6803 strains with overexpressions of genes related with the ROS detoxification system (sodB and gpx2, which encode superoxide dismutase and glutathione peroxidase, respectively) were developed. Three engineered strains including a sodB-overexpressing strain (OE + S), a gpx2-overexpressing strain (OE + G), and a sodB/gpx2-overexpressing strain (OE + SG) grew similarly as wild-type control under normal condition. When compared to wild-type control, OE + S and OE + SG strains grew faster for 4 days under 2.0% (v/v) ethanol and 0.3% (v/v) n-butanol conditions, as well as having higher chlorophyll a levels. On the other hand, the prominent growth recovery of OE + G and OE + SG was noted within 4 days in normal BG11 medium after treating cells with high alcohol stresses for 1 h, in particular 15% ethanol and 2.5% n-butanol. Under 4 days of recovery from butanol stress, specific levels of intracellular pigments including chlorophyll a and carotenoids were dramatically increased in all modified strains. The overexpression of antioxidant genes then revealed a significant improvement of alcohol tolerance in Synechocystis sp. PCC 6803.

Increased H2 production in the cyanobacterium Synechocystis sp. strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway.

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 contains a single bidirectional NiFe-Hox-hydrogenase, which evolves hydrogen under certain environmental conditions. The nitrate assimilation pathway is a potential competing pathway that may reduce the electron flow to the hydrogenase and thereby limit hydrogen production. To improve H(2) production, the nitrate assimilation pathway was disrupted by genetic engineering to redirect the electron flow towards the Hox-hydrogenase. Mutant strains disrupted in either nitrate reductase (ΔnarB) or nitrite reductase (ΔnirA) or both nitrate reductase and nitrite reductase (ΔnarB:ΔnirA) were constructed and tested for their ability to produce hydrogen. H(2) production and Hox-hydrogenase activities in all the mutant strains were higher than those in wild-type. Highest H(2) production was observed in the ΔnarB:ΔnirA strain. Small changes were observed for Hox-hydrogenase enzyme activities and only minor changes in transcript levels of hoxH and hoxY were not correlated with H(2) production. The results suggest that the high rate of H(2) production observed in the ΔnarB:ΔnirA strain of the cyanobacterium Synechocystis sp. strain PCC 6803 is the result of redirecting the electron supply from the nitrate assimilation pathway, through genetic engineering, towards the Hox-hydrogenase.

Short-term UV-B and UV-C radiations preferentially decrease spermidine contents and arginine decarboxylase transcript levels of Synechocystis sp. PCC 6803.

To investigate the short term effect of ultraviolet (UV) radiations on changes in pigments and polyamine contents, Synechocystis sp. PCC 6803 cells after exposure to UV-radiation were extracted by dimethylformamide and perchloric acid for pigments and polyamines determination, respectively. Cell growth was slightly decreased after 1 h exposure to UV-A and UV-B radiations. UV-C had little effect on cell growth despite the decrease of photosynthetic rate by about 18%. UV-A and UV-B decreased the contents of chlorophyll a and carotenoids whereas UV-C decreased chlorophyll a but had no effect on carotenoids. Spermidine contents were unaffected by UV-A, in contrast to the reduction of 25 and 50% by UV-B and UV-C, respectively. All three types of UV-radiation particularly reduced perchloric acid-insoluble spermidine. Importantly, putrescine and spermine which accounted for less than 1% of intracellular polyamines were increased by about three- to eight-fold by UV-B and UV-C, respectively. The changes in polyamines contents by UV-B and UV-C were consistent with the changes in transcript levels of arginine decarboxylase mRNA, but not with the protein levels. The decrease in the transcripts of adc2 but not adc1 was observed with UV-B and UV-C treatments.

Cyanobacterium Synechocystis sp. PCC 6803 lacking adc1 gene produces higher polyhydroxybutyrate accumulation under modified nutrients of acetate supplementation and nitrogen-phosphorus starvation.

Increased polyhydroxybutyrate production in cyanobacterium Synechocystis sp. PCC 6803 lacking adc1 gene (Δadc1) is first-timely reported in this study. We constructed the mutant by disrupting adc1 gene encoding arginine decarboxylase, thereby exhibiting a partial blockade of polyamine synthesis. This Δadc1 mutant had a proliferative growth and certain contents of intracellular pigments including chlorophyll a and carotenoids as similar as those of wild type (WT). Highest PHB production was certainly induced by BG11-N-P+A condition in both WT and Δadc1 mutant of about 24.9 %w/DCW at day 9 and 36.1 %w/DCW at day 7 of adaptation time, respectively. Abundant PHB granules were also visualized under both BG11-N-P and BG11-N-P+A conditions. All pha transcript amounts of Δadc1 mutant grown at 7 days-adaptation time were clearly upregulated corresponding to its PHB content under BG11-N-P+A condition. Our finding indicated that this adc1 perturbation is alternatively achieved for PHB production in Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 overexpressing genes involved in CBB cycle and free fatty acid cycling enhances the significant levels of intracellular lipids and secreted free fatty acids.

The integrative aspect on carbon fixation and lipid production is firstly implemented in cyanobacterium Synechocystis sp. PCC 6803 using metabolic engineering approach. Genes related to Calvin-Benson-Bassham (CBB) cycle including rbcLXS and glpD and free fatty acid recycling including aas encoding acyl-ACP synthetase were practically manipulated in single, double and triple overexpressions via single homologous recombination. The significantly increased growth rate and intracellular pigment contents were evident in glpD-overexpressing (OG) strain among all strains studied under normal growth condition. The triple aas_glpD_rbcLXS-overexpressing (OAGR) strain notably gave the highest contents of both intracellular lipids and extracellular free fatty acids (FFAs) of about 35.9 and 9.6% w/DCW, respectively, when compared to other strains at day 5 of cultivation. However, the highest intracellular lipid titer and production rate were observed in OA strain at day 5 (228.7 mg/L and 45.7 mg/L/day, respectively) and OG strain at day 10 (358.3 mg/L and 35.8 mg/L/day, respectively) due to their higher growth. For fatty acid (FA) compositions, the main saturated fatty acid of palmitic acid (C16:0) was dominantly found in both intracellular lipid and secreted FFAs fractions. Notably, intracellular FA proportion of myristic acid (C14:0) was induced in all engineered strains whereas the increase of stearic acid (C18:0) composition was found in extracellular FFAs fraction. Altogether, these overexpressing strains efficiently produced higher lipid production via homeostasis balance on both its lipid synthesis and FFAs secretion.

Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803.

BACKGROUND: Cyanobacteria are potential sources for third generation biofuels. Their capacity for biofuel production has been widely improved using metabolically engineered strains. In this study, we employed metabolic engineering design with target genes involved in selected processes including the fatty acid synthesis (a cassette of accD, accA, accC and accB encoding acetyl-CoA carboxylase, ACC), phospholipid hydrolysis (lipA encoding lipase A), alkane synthesis (aar encoding acyl-ACP reductase, AAR), and recycling of free fatty acid (FFA) (aas encoding acyl-acyl carrier protein synthetase, AAS) in the unicellular cyanobacterium Synechocystis sp. PCC 6803. RESULTS: To enhance lipid production, engineered strains were successfully obtained including an aas-overexpressing strain (OXAas), an aas-overexpressing strain with aar knockout (OXAas/KOAar), and an accDACB-overexpressing strain with lipA knockout (OXAccDACB/KOLipA). All engineered strains grew slightly slower than wild-type (WT), as well as with reduced levels of intracellular pigment levels of chlorophyll a and carotenoids. A higher lipid content was noted in all the engineered strains compared to WT cells, especially in OXAas, with maximal content and production rate of 34.5% w/DCW and 41.4 mg/L/day, respectively, during growth phase at day 4. The OXAccDACB/KOLipA strain, with an impediment of phospholipid hydrolysis to FFA, also showed a similarly high content of total lipid of about 32.5% w/DCW but a lower production rate of 31.5 mg/L/day due to a reduced cell growth. The knockout interruptions generated, upon a downstream flow from intermediate fatty acyl-ACP, an induced unsaturated lipid production as observed in OXAas/KOAar and OXAccDACB/KOLipA strains with 5.4% and 3.1% w/DCW, respectively. CONCLUSIONS: Among the three metabolically engineered Synechocystis strains, the OXAas with enhanced free fatty acid recycling had the highest efficiency to increase lipid production.

Co-overexpression of native phospholipid-biosynthetic genes plsX and plsC enhances lipid production in Synechocystis sp. PCC 6803.

The overexpression of native plsX and plsC genes involving in fatty acid/phospholipid synthesis first timely-reported the significantly enhanced lipid contents in Synechocystis sp. PCC 6803. Growth rate, intracellular pigment contents including chlorophyll a and carotenoids, and oxygen evolution rate of all overexpressing (OX) strains were normally similar as wild type. For fatty acid compositions, saturated fatty acid, in particular palmitic acid (16:0) was dominantly increased in OX strains whereas slight increases of unsaturated fatty acids were observed, specifically linoleic acid (18:2) and alpha-linolenic acid (18:3). The plsC/plsX-overexpressing (OX + XC) strain produced high lipid content of about 24.3%w/dcw under normal condition and was further enhanced up to 39.1%w/dcw by acetate induction. This OX + XC engineered strain was capable of decreasing phaA transcript level which related to poly-3-hydroxybutyrate (PHB) synthesis under acetate treatment. Moreover, the expression level of gene transcripts revealed that the plsX- and plsC/plsX-overexpression strains had also increased accA transcript amounts which involved in the irreversible carboxylation of acetyl-CoA to malonyl-CoA. Altogether, these overexpressing strains significantly augmented higher lipid contents when compared to wild type by partly overcoming the limitation of lipid production.

Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp. PCC 6803 by overexpression of its native biosynthetic genes.

Synechocystis sp. PCC 6803 strains overexpressing pha genes were constructed and characterized for poly-3-hydroxybutyrate (PHB) production. These pha overexpressing strains showed slightly reduced growth rates. Under N-deprived condition, the strains overexpressing (OE) phaAB, phaEC and phaABEC showed significantly higher PHB contents than the wild type. The maximum PHB content, a 2.6-fold increase producing 26% PHB (dcw), was observed in OE phaAB cells grown for 9days in N-deprived medium. Under this condition, these OE phaAB cells increased PHB production to 35% PHB (dcw) upon addition of 0.4% (w/v) acetate. Higher PHB granules in OE phaAB cells were clearly visualized by both Nile red staining and TEM imaging. All OE strains under N-deficient condition had increased glgX transcript levels. Overall results demonstrate an enhanced PHB production in Synechocystis cells overexpressing pha genes, particularly phaA and phaB, when grown in N-deprived medium containing 0.4% (w/v) acetate.

A novel high light-inducible carotenoid-binding protein complex in the thylakoid membranes of Synechocystis PCC 6803.

Synechocystis sp. PCC 6803 is a model cyanobacterium extensively used to study photosynthesis. Here we reveal a novel high light-inducible carotenoid-binding protein complex (HLCC) in the thylakoid membranes of Synechocystis PCC 6803 cells exposed to high intensity light. Zeaxanthin and myxoxanthophyll accounted for 29.8% and 54.8%, respectively, of the carotenoids bound to the complex. Using Blue-Native PAGE followed by 2D SDS-PAGE and mass spectrometry, we showed that the HLCC consisted of Slr1128, IsiA, PsaD, and HliA/B. We confirmed these findings by SEAD fluorescence cross-linking and anti-PsaD immuno-coprecipitation analyses. The expression of genes encoding the protein components of the HLCC was enhanced by high light illumination and artificial oxidative stress. Deletion of these proteins resulted in impaired state transition and increased sensitivity to oxidative and/or high light stress, as indicated by increased membrane peroxidation. Therefore, the HLCC protects thylakoid membranes from extensive photooxidative damage, likely via a mechanism involving state transition.

Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp. PCC 6803.

The effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of the polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S-adenosyl methionine decarboxylase (SAMDC), were investigated in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis cells showed no difference in growth rate when the concentration of NaCl was raised up to 550 mM. The growth rate decreased at 300 mM sorbitol, and complete inhibition of growth occurred at concentrations of > or =700 mM sorbitol. Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular. Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol. Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp. PCC 6803. ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a six-fold increase in enzyme activity upon osmotic stress imposed by 700 mM sorbitol. SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to the pattern observed for ADC. An analysis by reverse transcription-polymerase chain reaction revealed an increase of ADC mRNA level in cells under salt and osmotic stresses. Most importantly, the increase of ADC mRNA was attributed to its slower turnover rate under both stress conditions. Interestingly, the samdc gene(s) of Synechocystis appear to be unique since comparisons with known gene sequences from other organisms resulted in no homologous sequences identified in the Synechocystis genome.