Pinpointing Synechococcus Rubisco large subunit sections involved in heterologous holoenzyme formation in Escherichia coli

Aims@#Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447).@*Methodology and results@#Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli.@*Conclusion, significance and impact of study@#GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.

Construction of squalene producing cell factories and screening, cloning and expression of key genes / 生物工程学报

Squalene is widely used in pharmaceutical, nutraceutical, cosmetics and other fields because of its strong antioxidative, antibacterial and anti-tumor activities. In order to produce squalene, a gene ispA encoding farnesyl pyrophosphate synthase was overexpressed in a previously engineered Escherichia coli strain capable of efficiently producing terpenoids, resulting in a chassis strain that efficiently synthesizes triterpenoids. Through phylogenetic analysis, screening, cloning and expression of squalene synthase derived from different prokaryotes, engineered E. coli strains capable of efficiently producing squalene were obtained. Among them, squalene produced by strains harboring squalene synthase derived from Thermosynechococcus elongatus and Synechococcus lividus reached (16.5±1.4) mg/g DCW ((167.1±14.3) mg/L broth) and (12.0±1.9) mg/g DCW ((121.8±19.5) mg/L broth), respectively. Compared with the first-generation strains harboring the human-derived squalene synthase, the squalene synthase derived from T. elongatus and S. lividus remarkably increased the squalene production by 3.3 times and 2.4 times, respectively, making progress toward the cost-effective heterologous production of squalene.

Light and carbon dioxide-driven synthesis of high-density fuel in Synechococcus elongates UTEX 2973 / 生物工程学报

Development of "liquid sunshine" could be a key technology to deal with the issue of fossil fuel depletion. β-caryophyllene is a terpene compound with high energy density and has attracted attention for its potential application as a jet fuel. The high temperature and high light-tolerant photosynthetic cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973), whose doubling time is as short as 1.5 h, has great potential for synthesizing β-caryophyllene using sunlight and CO₂. In this study, a production of ~121.22 μg/L β-caryophyllene was achieved at 96 h via a combined strategy of pathway construction, key enzyme optimization and precursor supply enhancement. In addition, a final production of ~212.37 μg/L at 96 h was realized in a high-density cultivation. To our knowledge, this is the highest production reported for β-caryophyllene using cyanobacterial chassis and our study provide important basis for high-density fuel synthesis in cyanobacteria.

Pentoses Used in Cultures of Synechococcus nidulans and Spirulina paracas: Evaluation of Effects in Growth and in Content of Proteins and Carbohydrates

Abstract The biological assimilation of the sugars present in lignocellulosic residues has gained prominence since these residues are the most abundant and economic residues in nature. Thus, the objective of this work was to determine whether the use of D-xylose and L-arabinose as sources of carbon in Synechococcus nidulans and Spirulina paracas cultures affects the growth and production of proteins and carbohydrates. Kinetic growth parameters, pentose consumption, protein content and carbohydrates were evaluated. Synechococcus nidulans and Spirulina paracas consumed all concentrations of pentose used. The highest cellular concentration (1.37 g.L-1) and the highest protein productivity (54 mg.L-1.d-1) were obtained for Spirulina paracas, which was submitted to the addition of 38.33 mg.L-1 D-xylose and 1.79 mg.L-1 L-arabinose. The use of pentose promoted the accumulation of proteins for the studied microalgae. This is one of the first works to report protein bioaccumulation as a result of pentose addition.

Effects of light quality on cell growth and psbA promoter of engineered Synechococcus sp. PCC7002 / 生物工程学报

Light quality can regulate both psbA genes and vector promoter psbA of the engineered Synechococcus. Through light regulation, we tried to improve yield of the recombinant protein for vp28 gene-expressed Synechococcus sp. PCC7002. To drive photon-capturing efficiently, three limiting factors (irradiance, temperature and pH) were optimized by measuring net photosynthesis. High cell density cultures were performed with variant ratios of white, red and blue light in a 5-L photo-bioreactor. Yields of biomass, expressions of vp28 and transcription levels of psbA were compared. High ratio blue light-induced vp28 transcription had tripled and the relative accumulation of VP28 protein was doubled. The relative expressions of psbAII and psbAIII had positive correlations with higher ratio of blue light, not the red light. With high ratio red light inducing, dry biomass reached 1.5 g/L in three days. Therefore, we speculated that red light accelerated biomass accumulation of the transgenic strain and blue light promoted transcription for PpsbA and psbA. These results provided useful information for mass production of cyanobacteria and its secondary metabolites.

Effects of ggpS over-expression on glycosylglycerol and glycerol biosynthesis of Synechocystis sp. PCC 6803 / 生物工程学报

To study the roles of glucosylglycerol phosphate synthase (Ggps) in glucosylglycerol (GG) and glycerol biosynthesis, we over-expressed Ggps from either Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 in a Synechocystis strain with a high GG titer, and determined the GG and glycerol accumulation in the resultant mutants grown under different NaCl-stress conditions. Ion chromatography results revealed that GG yield was not improved, but glycerol production was significantly enhanced by over-expression of Ggps from Synechocystis sp. PCC 6803 (6803ggpS). In addition, increasing the NaCl concentration of medium from 600 to 900 mmol/L led to a further 75% increase of glycerol accumulation in the mutant strain with 6803ggpS over-expression. These findings show the role of ggpS in driving the carbon flux to the glycerol biosynthesis pathway, and will be helpful for further improvement of GG and glycerol production in Synechocystis.

Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases

The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of Cn fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco

Photosynthetic CO(2) fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of photosynthetic CO(2) fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving photosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G substitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxylation activity and a 45% improvement in catalytic efficiency towards CO(2). The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

Biodiesel production from marine cyanobacteria cultured in plate and tubular photobioreactors.

Carbon (neutral) based renewable liquid biofuels are alternative to petroleum derived transport fuels that contribute to global warming and are of a limited availability. Microalgae based biofuels are considered as promising source of energy. Lyngbya sp. and Synechococcus sp. were studied for the possibility of biodiesel production in different media such as ASNIII, sea water enrichment medium and BG11. The sea water enrichment medium was found superior in enhancing the growth rate of these microalgae. Nitrogen depletion has less effect in total chlorophyll a content, at the same time the lipid content was increased in both Lyngbya sp. and Synechococcus sp. by 1.4 and 1.2 % respectively. Increase in salinity from 0.5-1.0 M also showed an increase in the lipid content to 2.0 and 0.8 % in these strains; but a salinity of 1.5 M has a total inhibitory effect in the growth. The total biomass yield was comparatively higher in tubular LED photobioreactor than the fluorescent flat plated photobioreactor. Lipid extraction was obtained maximum at 60 ºC in 1:10 sample: solvent ratio. GC-MS analysis of biodiesel showed high content of polyunsaturated fatty acids (PUFA; 4.86 %) than saturated fatty acid (SFA; 4.10 %). Biodiesel production was found maximum in Synechococcus sp. than Lyngbya sp. The viscosity of the biodiesel was closely related to conventional diesel. The results strongly suggest that marine microalgae could be used as a renewable energy source for biodiesel production.

Carbon metabolism and energetic utilization of Synechococcus sp. PCC7942 under mixotrophic condition / 生物工程学报

To investigate the energy utilization efficiency of Synechococcus sp. PCC7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and acetic acid respectively and discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could better enhance the growth of Synechococcus sp. PCC7942, and the latter was more effective. The metabolic flux through glycolytic pathway in mixotrophic cultures was stimulated by glucose whereas depressed by acetate, while the flux through the tricarboxylic acid cycle increased in both cases. Under mixotrophic conditions, glucose makes more significant impact on the diminishment of photochemical efficiency of Synechococcus sp. PCC7942. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher comparing with photoautotrophic culture. The energy conversion efficiencies based on ATP synthesis in photoautotrophic culture, mixotrophic cultures with glucose and with acetate were evaluated to be 6.81%, 7.43% and 8.77%, respectively.

Carbon and energetic metabolism of Synechococcus sp. PCC7942 under photoautotrophic conditions / 生物工程学报

Metabolic flux analysis is a very powerful tool to understand CO2 fixation and light energy utilization of microalgae during photoautotrophic cultivation. A comprehensive network structure for the autotrophic growth of Synechococcus sp. PCC7942 was proposed, and the carbon and energetic metabolism under different incident light intensity was investigated based on metabolic flux analysis in this paper. These results showed that CO2 fixation was the main energy and reducing potential trap which accounted for 85% and 70% of the total energy and reducing potential consumption respectively. We also found that the cell yield and the maximum cell yield based on ATP synthesis were maintained 2.80 g/mol and 2.97 g/mol respectively under the appointed incident intensity. But the cell yield on absorbed light energy their corresponding energy conversion efficiency were descended with the increasing of incident intensity.

influence of picocyanobacterial photosynthesis on calcite precipitation

This study assessed the role of picocyanobacterial photosynthesis in the induction of calcite precipitation. It aimed at establishing whether photosynthetic uptake of bicarbonate by Synechoccoccus cells leads to calcite nucleation. The precipitation of calcite was initiated by addition of previously washed cyanobacterial cells of Svnechococcus strain PCC 7942 to solutions of calcium carbonate at different saturation levels with respect to calcite. Precipitation experiments were performed under controlled laboratory conditions in two set-ups: one in which photosynthesis was inhibited using a herbicide called Diuron and the other one in which photosynthesis was taking place. During the experiments, a pH meter monitored the pH and ion selective electrodes monitored concentrations of carbonate and calcium ions. The morphology of the precipitated crystals was analysed using Scanning Electron Microscopy. When the kinetics of calcium carbonate nucleation by the Synechococcus cells were compared for the two sets of experiments, there were very little differences. In fact, the induction times for precipitation reactions with photosynthesis were shorter due to the uptake of carbon dioxide. It is therefore, concluded that photosynthesis does not directly influence the nucleation of calcite at the surface of Synechococcus cells with sufficient supply of carbon dioxide, i.e. cells took up carbon dioxide and not bicarbonate. The microscopic observations, however, provided some evidence that picocyanobacterial cell walls act as a template for calcite nucleation

Crecimiento y producción de metabolitos de la cianobacteria marina Synechococcus sp. (Chroococcales) en función de la irradiancia

Growth and metabolite production of the marine cyanobacterium Synechococcus sp. (Chroococcales) in function to irradiance. Changes in salinity, temperature and irradiance during wet and dry seasons have induced metabolic versatility in cyanobacteria from saline environments. Cyanobacteria from these environments have biotechnological potential for the production of metabolites with pharmaceutical and industrial interest. We studied the growth, dry mass and metabolite production of the cyanobacterium Synechococcus sp. MOF-03 in function of irradiance (78, 156 and 234 µmol q m-2 s-1). All batch cultures were maintained by triplicate in constant aeration, 12:12 h photoperiod, 30 ±2ºC and 35‰. Maximum values of protein, carbohydrates and lipids, of 530.19 ±11.16, 408.94 ±4.27 and 56.20 ±1.17 µg ml-1, respectively, were achieved at 78 µmol q m-2 s-1. Pigments, analyzed by HPLC, showed maximum values at 78 µmol q m-2 s-1 for chlorophyll a with 7.72 ±0.16 µg ml-1, and at 234 µmol q m-2 s-1 for ß-carotene and zeaxanthin with 0.70 ±0.01 and 0.67 ±0.05 µg ml-1. Chlorophyll a:ß-carotene ratio decreased from 17.15 to 6.91 at 78 and 234 µmol q m-2 s-1; whereas ß-carotene:zeaxanthin ratio showed no changes between 78 and 156 µmol q m-2 s-1, around 1.21, and decreased at 234 µmol q m-2 s-1, to 1.04. Also, this cyanobacterium produced the greatest cell density and dry mass at 156 µmol q m-2 s-1, with 406.13 ±21.74 x106 cell ml-1 and 1.49 ±0.11 mg ml-1, respectively. Exopolysaccharide production was stable between 156 y 234 µmol q m-2 s-1, around 110 µg ml-1. This Synechococcus strain shows a great potential for the production of enriched biomass with high commercial value metabolites. Rev. Biol. Trop. 56 (2): 421-429. Epub 2008 June 30.

Las cianobacterias de ambientes salinos presentan una versatilidad metabólica inducida por los cambios de salinidad, temperatura e irradiancia, durante los períodos de sequía y lluvias. Por ello es importante la búsqueda en estos ambientes de cianobacterias con potencial biotecnológico para la producción de metabolitos de interés farmacéutico e industrial. Se reporta el crecimiento, masa seca y producción de metabolitos de la cianobacteria Synechococcus sp. MOF-03 en función de la irradiancia (78, 156 y 234 µmol q m-2 s-1). Los cultivos discontinuos por triplicado, fueron mantenidos con aireación constante, fotoperiodo 12:12 h, 30 ±2ºC y a 35‰. Los máximos valores de proteínas, carbohidratos y lípidos de 530.19 ±11.16, 408.94 ±4.27 y 56.20 ±1.17 µg ml-1 respectivamente, fueron obtenidos a 78 µmol q m-2 s-1. Los pigmentos, analizados por HPLC, mostraron los máximos a 78 µmol q m-2 s-1 para clorofila a con 7.72 ±0.16 µg ml-1; y a 234 µmol q m-2 s-1 para ß-caroteno y zeaxantina con 0.70 ±0.01 and 0.67 ±0.05 µg ml-1. La relación clorofila a:ß-caroteno disminuyó de 17.15 hasta 6.91 a 78 y 234 µmol q m-2 s-1; mientras que la relación ß-caroteno:zeaxantina se mantuvo sin cambios entre 78 y 156 µmol q m-2 s-1, con cerca de 1.21 y disminuyó a 234 µmol q m-2 s-1 a 1.04. La cianobacteria produjo la mayor densidad celular y masa seca a 156 µmol q m-2 s-1, con 406.13 ±21.74 x106 cel ml-1 y 1.49 ±0.11 mg ml-1 respectivamente. La producción de exopolisacáridos se mantuvo alrededor de 110 µg ml-1 entre 156 y 234 µmol q m-2 s-1. Así, esta cepa de Synechococcus muestra un gran potencial para la producción de biomasa enriquecida con metabolitos de alto valor comercial.

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942.

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.

Single gene retrieval from thermally degraded DNA.

To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both crucial factors influencing DNA degradation. The heat influence, mainly represented by temperature and heating time, affects the DNA degradation via DNA depurination followed by cleavage of nearby phosphodiesters. The heating time influence is temperature-dependent. By reactive oxygen species (ROS) scavenging and 1,3-diphenyl-isobenzofuran (DPBF) bleaching experiments the influence of oxygen on DNA thermal degradation was shown to occur via a singlet oxygen pathway. A comparative study of the thermal degradation of cellular DNA and isolated DNA showed that cellular lipids can aggravate DNA thermal degradation. These results confirm the possibility of gene amplification from thermally degraded DNA. They can be used to evaluate the feasibility of the retrieval of single gene from ancient remains.

The upstream sequence of the phycocyanin b subunit gene from Arthrospira platensis regulates expression of gfp gene in response to light intensity

In cyanobacteria, few details are known of the mechanisms through which the expression of the light-harvesting pigment c-phycocyanin is regulated. In the present study, a 419 bp upstream sequence of the phycocyanin b subunit (cpcB) gene from Arthrospira platensis FACHB341 was fused with green fluorescent protein (gfp) gene, and a heterologous reporting system was built up to investigate the influence of light intensity on the expression of gfp gene, and the regulation function of different region of the upstream sequence of cpcB gene. Results showed that the upstream sequence of cpcB gene could drive the expression of gfp gene in Synechococcus sp. strain PCC7942, and the expression was influenced by light intensity, the lower the light intensity, the higher the GFP level. Deletion analysis revealed that a light-responsive element was located in the region -276 to-218, a promoter sequence was in the region -85 to -1, and two positive cis elements were in the -419 to -276 and the -218 to -130 regions, respectively.