Determination of the Glycogen Content in Cyanobacteria
Summary
Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.
Abstract
Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.
Introduction
Cyanobacteria accumulate glycogen as the major carbohydrate store of carbon from CO2 fixed in light through photosynthesis. Glycogen is a glycan consisting of linear α-1,4-linked glucan with branches created by α-1,6-linked glucosyl linkages. Glycogen biosynthesis in cyanobacteria starts with the conversion of glucose-6-phosphate into ADP-glucose through the sequential action of phosphoglucomutase and ADP-glucose pyrophosphorylase. The glucose moiety in ADP-glucose is transferred to the non-reducing end of the α-1,4-glucan backbone of glycogen by one or more glycogen synthases (GlgA). Subsequently, a branching enzymes introduce the α-1,6-linked glucosyl linkage, which is further extended to generate the glycogen particle. In the dark, glycogen is broken down by glycogen phosphorylase, glycogen debranching enzymes, α-glucanotransferase, and malto-dextrin phosphorylase into phosphorylated glucose and free glucose. These feed into catabolic pathways, including the oxidative pentose phosphate pathway, the Embden-Meyerhof-Parnas pathway (glycolysis), and the Entner-Doudoroff pathway1,2,3,4.
Glycogen metabolism in cyanobacteria has garnered increasing interest in recent years because of the potential for cyanobacteria to develop into microbial cell factories driven by sunlight to produce chemicals and fuels. Glycogen metabolism could be modified to increase the yield of the products, because glycogen is the largest flexible carbon pool in these bacteria. An example is the cyanobacterium Synechococcus sp. PCC 7002, which has been genetically engineered to produce mannitol; the genetic disruption of glycogen synthesis increases the mannitol yield 3-fold5. Another example is the production of bioethanol from glycogen-loaded wildtype Synechococcus sp. PCC 70026. The wildtype cell glycogen content may be up to 60% of the dry weight of the cell during nitrogen starvation6.
Our understanding of glycogen metabolism and regulation has also expanded in recent years. While glycogen is known to accumulate in the light and to be catabolized in the dark, detailed kinetics of glycogen metabolism during the diel cycle was only recently revealed in Synechocystis sp. PCC 68037. Moreover, several genes affecting the accumulation of glycogen have been identified. A notable example is the discovery that the putative histidine kinase PmgA and the non-coding RNA PmgR1 form a regulatory cascade and control the accumulation of glycogen. Interestingly, the pmgA and pmgR1 deletion mutants accumulate twice as much glycogen as the wildtype strain of Synechocystis sp. PCC 68038,9. Other regulatory elements are also known to affect the accumulation of glycogen, including the alternative sigma factor E and the transcriptional factor CyAbrB210,11.
As interest in glycogen regulation and metabolism grow, a detailed protocol describing the determination of the glycogen content is warranted. Several methods are used in the literature. Acid hydrolysis followed by the determination of the monosaccharide content through high-pressure anion exchange liquid chromatography coupled with a pulsed amperometric detector or spectrometric determination following treatments with acid and phenol are widely used methods to approximate the glycogen content9,10,12,13. However, a high-pressure anion exchange liquid chromatographic instrument is very expensive and does not discriminate glucose derived from glycogen from that derived from other glucose-containing glycoconjugates, such as sucrose14, glucosylglycerol15, and cellulose16,17,18, which are known to accumulate in some cyanobacterial species. The acid-phenol method can be performed using standard laboratory equipment. However, it uses highly toxic reagents and does not distinguish glucose derived from different glycoconjugates, nor does it distinguish glucose from other monosaccharides that constitute cellular materials, such as glycolipids, lipopolysaccharides, and extracellular matrices12. Notably, the hot acid-phenol assay is often used for the determination of total carbohydrate content rather than for the specific determination of glucose content12. Enzymatic hydrolysis of glycogen to glucose by α-amyloglucosidase followed by the detection of glucose through an enzyme-coupled assay generates a colorimetric readout that is highly sensitive and specific to glucose derived from glycogen. The specificity can be enhanced further with the preferential precipitation of glycogen from cell lysates by ethanol5,8,19.
Here, we describe a detailed protocol for an enzyme-based assay of the glycogen content in two of the most widely studied cyanobacterial species, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, in the wildtype and mutant strains. In order to ensure efficient hydrolysis, a cocktail of α-amylase and α-amyloglucosidase is used8. The endo-acting α-amylase hydrolyzes the α-1,4-linkages in various glucans into dextrins, which are further hydrolyzed to glucose by exo-acting α-amyloglucosidase20. The synergistic effects of these enzymes are well known, and these enzymes are routinely used for the selective hydrolysis of starch, which is an α-linked glucan like glycogen, without affecting other glycoconjugants, such as cellulose, in the plant biomass21. The released glucose is quantitatively detected following an enzyme-coupled assay consisting of glucose oxidase-which catalyzes the reduction of oxygen to hydrogen peroxide and the oxidation of glucose to a lactone-and peroxidase-which produces a pink-colored quinoneimine dye from hydrogen peroxide, a phenolic compound, and 4-aminoantipyrine22.
Protocol
Representative Results
Discussion
Critical steps within the protocol are glycogen precipitation and resuspension. After centrifugation following ethanol precipitation, glycogen forms a translucent pellet that loosely adheres to the walls of the microcentrifuge tubes. Therefore, when removing the supernatant, special attention needs to be given so as not to remove the pellet. The glycogen pellet is sticky, and solubilization can be difficult if it dries out. Note that the complete solubilization of the glycogen pellet is important because incomplete solub…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge Nordic Energy Research (AquaFEED, project no. 24), Innovationfonden Denmark (Pant Power, project no. 12-131844), and Villum Fonden (project no. 13363)
Materials
| QSonica Sonicators Q700 | Qsonica, LLC | NA | QSonica |
| SpectraMax 190 Microplate Reader | Molecular Devices | NA | Eliza plate reader |
| Bullet Blender Storm | Next Advance | BBY24M-CE | Beads beater |
| Ultrospec 3100 pro UV/Visible Spectrophotometer | Amersham Biosciences | NA | Spectrophotometer |
| Tris | Sigma-Aldrich | T1503 | Buffer |
| HCl | Merck | 1-00317 | pH adjutment |
| Sodium acetate | Sigma-Aldrich | 32319 | Buffer |
| Amyloglycosidase (Rhizopus sp.) | Megazyme | E-AMGPU | Enzyme for glycogen depolymerization |
| α-Amylase, thermostable (Bacillus licheniformis) | Sigma-Aldrich | A3176 | Enzyme for glycogen depolymerization |
| D-Glucose | Merch | 8337 | Standard for the glucose assay |
| Pierce BCA Protein assay kit | Thermo Fisher scientific | 23225 | For determination of protein concentrations |
| Aluminum drying trays, disposable | VWR | 611-1362 | For determination of cell dry weights |
| D-Glucose assay kit (GODPOD format) | Megazyme | K-GLUC | For determination of glucose concentrations |
| Zirconium oxide breads, 0.15 mm | Next Advance | ZrOB015 | Beads for cell lysis in a Bullet Blendar Storm |
| RINO tubes | Next Advance | NA | Tubes for cell lysis in a Bullet Blendar Storm |
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