| 1. |
- Durall, Claudia, et al.
(författare)
-
Robust QCM-Based Sensing and Assay Formats in Commercialized Systems
- 2023
-
Ingår i: Springer Series on Chemical Sensors and Biosensors. - : Springer.
-
Bokkapitel (refereegranskat)abstract
- Attana’s Quartz Crystal Microbalance (QCM) analytical instruments have been developed to study in vitro biological interactions, mimicking the in vivo conditions. Attana’s superior technology for kinetic interaction studies allows to perform different assays, including biochemical, crude, sera, cell, and tissue-based, in vitro diagnostic and material chemistry assays, in real time and label free. With the focus to validate, select, and optimize drug candidates prior to clinical trials, Attana has helped pharmaceutical companies to increase their efficiency and profitability. In addition, the Attana instruments and services have been used in many other applications and research as described in this chapter.
|
|
| 2. |
- Durall de la Fuente, Claudia, et al.
(författare)
-
Enhanced growth at low light intensity in the cyanobacterium Synechocystis PCC 6803 by overexpressing phosphoenolpyruvate carboxylase
- 2016
-
Ingår i: Algal Research. - : Elsevier BV. - 2211-9264. ; 16, s. 275-281
-
Tidskriftsartikel (refereegranskat)abstract
- Synechocystis PCC 6803 strains overexpressing pepc, gene encoding the carbon fixing enzyme phosphoenolpyruvate carboxylase (PEPc), were constructed and characterized for growth, PEPc protein content and in vitro PEPc activities. Synechocystis strains WT + Km(r) - one (native) copy of pepc (control), WT + 2xPEPc - native copy of pepc and two additional native copies of pepc (in total three copies of pepc), and WT + PPM - native copies of ppsa (encoding phosphoenolpyruvate synthase), pepc and mdh (encoding malate dehydrogenase) and one additional copy of each gene (in total two copies each of ppsa, pepc and mdh) were analyzed for growth under normal and low light intensities, and in darkness (no growth). No significant differences in the growth rates were observed when the cells were grown under normal light intensity. However, growth under low light intensity (3 mu mol photons.m(-2).sec(-1)) resulted in increased growth rate, in particular in the strain with 3 copies of pepc. SDS-PAGE/Western immunoblots using antibodies directed against PEPc demonstrated an increased level of PEPc protein with increasing number of copies of pepc. This was followed by increased levels of in vitro PEPc activities. A less efficient ribulose 1,5-bisphosphate carboxylase/oxygenase in combination with reduced levels of NADPH and ATP under low light condition may make the relatively more efficient carbon fixing enzyme PEPc the limiting step for growth under this condition.
|
|
| 3. |
- Durall de la Fuente, Claudia
(författare)
-
Increased Carbon Fixation for Chemical Production in Cyanobacteria
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The combustion of fossil fuels has created many environmental problems, the major one, the greenhouse effect. Thus, we need solutions in order to replace fossil fuels and recycle the CO2 in the atmosphere. Renewable energies have created attention the last decades but electricity is the main energy form obtained. Photosynthetic organisms (including cyanobacteria) can be used as cell factories since they can convert solar energy to chemical energy. In addition, the requisites to grow them are few; light water, CO2 and inorganic nutrients. Cyanobacteria have been genetically engineered in order to produce numerous chemicals and fuels of human interest in direct processes. However, the amount of product obtained is still low. Increased carbon fixation in cyanobacteria results in higher production of carbon-based substances. This thesis focuses on the effects of overexpressing the native phosphoenolpyruvate carboxylase (PEPc) in the model cyanobacterium Synechocystis PCC 6803. PEPc is an essential enzyme and provides oxaloacetate, an intermediate of the tricarboxylic acid cycle (TCA cycle). The TCA cycle is involved in connecting the carbon and nitrogen metabolism in cyanobacteria. The strains were further engineered to produce ethylene and succinate, two examples of interests for the chemical and fuel industry. Strains with additional PEPc produced significantly more ethylene and succinate. Moreover, an in vitro characterization of PEPc from the cyanobacterium Synechococcus PCC 7002 was performed. The focus was on oligomerization state, kinetics and the structure of the carboxylase. This thesis demonstrates that increasing carbon fixation and discovering the bottlenecks in chemical production can lead to higher yields and gives us hope that cyanobacteria can be commercialized.
|
|
| 4. |
- Durall de la Fuente, Claudia, et al.
(författare)
-
Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803
- 2020
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Cyanobacteria can be metabolically engineered to convert CO2 to fuels and chemicals such as ethylene. A major challenge in such efforts is to optimize carbon fixation and partition towards target molecules.Results: The efe gene encoding an ethylene-forming enzyme was introduced into a strain of the cyanobacterium Synechocystis PCC 6803 with increased phosphoenolpyruvate carboxylase (PEPc) levels. The resulting engineered strain (CD-P) showed significantly increased ethylene production (10.5 +/- 3.1 mu g mL(-1) OD-1 day(-1)) compared to the control strain (6.4 +/- 1.4 mu g mL(-1) OD-1 day(-1)). Interestingly, extra copies of the native pepc or the heterologous expression of PEPc from the cyanobacterium Synechococcus PCC 7002 (Synechococcus) in the CD-P, increased ethylene production (19.2 +/- 1.3 and 18.3 +/- 3.3 mu g mL(-1) OD-1 day(-1), respectively) when the cells were treated with the acetyl-CoA carboxylase inhibitor, cycloxydim. A heterologous expression of phosphoenolpyruvate synthase (PPSA) from Synechococcus in the CD-P also increased ethylene production (16.77 +/- 4.48 mu g mL(-1) OD-1 day(-1)) showing differences in the regulation of the native and the PPSA from Synechococcus in Synechocystis.Conclusions: This work demonstrates that genetic rewiring of cyanobacterial central carbon metabolism can enhance carbon supply to the TCA cycle and thereby further increase ethylene production.
|
|
| 5. |
|
|
| 6. |
- Durall de la Fuente, Claudia, et al.
(författare)
-
Mechanisms of carbon fixation and engineering for increased carbon fixation in cyanobacteria
- 2015
-
Ingår i: Algal Research. - : Elsevier BV. - 2211-9264. ; 11, s. 263-270
-
Forskningsöversikt (refereegranskat)abstract
- Cyanobacteria, gram-negative prokaryotic microorganisms, perform oxygenic photosynthesis with a photosynthetic machinery similar to higher plants which includes ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) as the main CO2-fixing enzyme. Currently, there is a growing interest to use cyanobacteria as photosynthetic microbial cell factories for the direct production of solar fuels or other compounds of human interest. However, rates and efficiencies to produce e.g. biofuels are still very low. The amount of available fixed carbon for the synthesis of desired product(s) may be one of the limiting steps. This contribution reviews CO2-fixation in cyanobacteria with focus on CO2-concentrating mechanisms, RuBisCO, phosphoenolpyruvate carboxylase and other carboxylases, engineering approaches for increased carbon fixation, and finally the synthetic malonyl-CoA-oxaloacetate-glyoxylate pathways.
|
|
| 7. |
- Durall de la Fuente, Claudia, et al.
(författare)
-
Oligomerization and characteristics of phosphoenolpyruvate carboxylase in Synechococcus PCC 7002
- 2020
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Phosphoenolpyruvate carboxylase (PEPc) is an essential enzyme in plants. A photosynthetic form is present both as dimer and tetramer in C4 and CAM metabolism. Additionally, non-photosynthetic PEPcs are also present. The single, non-photosynthetic PEPc of the unicellular cyanobacterium Synechococcus PCC 7002 (Synechococcus), involved in the TCA cycle, was examined. Using size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS), we observed that PEPc in Synechococcus exists as both a dimer and a tetramer. This is the first demonstration of two different oligomerization states of a non-photosynthetic PEPc. High concentration of Mg2+, the substrate PEP and a combination of low concentration of Mg2+ and HCO3− induced the tetramer form of the carboxylase. Using SEC-SAXS analysis, we showed that the oligomerization state of the carboxylase is concentration dependent and that, among the available crystal structures of PEPc, the scattering profile of PEPc of Synechococcus agrees best with the structure of PEPc from Escherichia coli. In addition, the kinetics of the tetramer purified in presence of Mg2+ using SEC, and of the mixed population purified in presence of Mg2+ using a Strep-tagged column were examined. Moreover, the enzyme showed interesting allosteric regulation, being activated by succinate and inhibited by glutamine, and not affected by either malate, 2-oxoglutarate, aspartic acid or citric acid.
|
|
| 8. |
- Durall de la Fuente, Claudia, et al.
(författare)
-
Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt
- 2021
-
Ingår i: Microbial Cell Factories. - : BioMed Central (BMC). - 1475-2859. ; 20
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium.Results: We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium.Conclusions: Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.
|
|
| 9. |
|
|
| 10. |
- Persson Skare, Tor, et al.
(författare)
-
Quartz Crystal Microbalance Measurement of Histidine-Rich Glycoprotein and Stanniocalcin-2 Binding to Each Other and to Inflammatory Cells
- 2022
-
Ingår i: Cells. - : MDPI. - 2073-4409. ; 11:17
-
Tidskriftsartikel (refereegranskat)abstract
- The plasma protein histidine-rich glycoprotein (HRG) is implicated in the polarization of macrophages to an M1 antitumoral phenotype. The broadly expressed secreted protein stanniocalcin 2 (STC2), also implicated in tumor inflammation, is an HRG interaction partner. With the aim to biochemically characterize the HRG and STC2 complex, binding of recombinant HRG and STC2 preparations to each other and to cells was explored using the quartz crystal microbalance (QCM) methodology. The functionality of recombinant proteins was tested in a phagocytosis assay, where HRG increased phagocytosis by monocytic U937 cells while STC2 suppressed HRG-induced phagocytosis. The binding of HRG to STC2, measured using QCM, showed an affinity between the proteins in the nanomolar range, and both HRG and STC2 bound individually and in combination to vitamin D3-treated, differentiated U937 monocytes. HRG, but not STC2, also bound to formaldehyde-fixed U937 cells irrespective of their differentiation stage in part through the interaction with heparan sulfate. These data show that HRG and STC2 bind to each other as well as to U937 monocytes with high affinity, supporting the relevance of these interactions in monocyte/macrophage polarity.
|
|
| 11. |
- Tor, Persson Skare, et al.
(författare)
-
Histidine-rich glycoprotein and stanniocalcin-2 high affinity interactions with inflammatory cells
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- AbstractThe plasma protein Histidine-rich protein (HRG), is implicated in macrophage polarization to an M1 antitumoral phenotype. The broadly expressed, secreted protein Stanniocalcin 2 (STC2), also implicated in tumor inflammation, is an HRG interaction partner. To assess the effects of HRG and STC2 on inflammation in a biologically relevant model, binding of recombinant HRG and STC2 preparations to each other and to cells was explored using quartz crystal microbalance (QCM) methodology. Protein functionality was tested in a phagocytosis assay. Stimulation with HRG increased phagocytosis in U937 cells while STC2 suppressed HRG-induced phagocytosis. Binding of HRG to STC2 measured using QCM showed an affinity in the nanomolar range and occurred in a conformation-dependent manner. Both HRG and STC2 bound individually and in combination to monocytic U937 cells with higher efficiency after vitamin D3-induced differentiation. HRG, but not STC2, also bound to formaldehyde-fixed U937 cells irrespective of differentiation stage, potentially through heparan sulphate. These data show that binding of HRG to STC2 is specific and conformation-dependent, and HRG and STC2 bind to separate sites on U937 cells, suggesting that they exert their effects through distinct cell surface entities.
|
|
