Determination of the Glycogen Content in Cyanobacteria

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 pathway^1,2,3,4.

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 6803^8,9. Other regulatory elements are also known to affect the accumulation of glycogen, including the alternative sigma factor E and the transcriptional factor CyAbrB2^10,11.

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 content^9,10,12,13. 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 cellulose^16,17,18, 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 ethanol^5,8,19.

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.

Protokół

1. Preparation

1. Cyanobacterial cultures
   1. Grow Synechocystis sp. PCC 6803 at 30 °C in liquid BG11 medium⁸, with a constant supply of air supplemented with 1% (v/v) CO₂. Illuminate the cultures continuously with light at a photosynthetic photon flux density of 50 µmol photon/m²/s.
   2. Grow Synechococcus sp. PCC 7002 in liquid A+ medium²³ (BG11 medium can also be used), with a constant supply of air supplemented with 1% (v/v) CO₂. The temperature should be 37 °C. Illuminate the cultures continuously with light at a photosynthetic photon flux density of 150 µmol photon/m²/s.
   3. Measure the optical density (OD) of the culture at 730 nm using a cuvette with a light path of 1 cm. If the OD value is above 0.8, make appropriate dilutions to obtain OD measurements that are proportional to the cell concentration.
      NOTE: The protocols presented below are suitable for liquid cultures, with a cell density corresponding to an OD_730nm value of 2 or higher. When cultures in the exponential growth phase are desired, which typically have an OD_730nm value below 1, concentrate the cell density by centrifugation and resuspension in a buffer or medium to achieve an OD_730nm value of 2 or higher.
2. Buffers and reagents
   1. Make 50 mM Tris-HCl buffer at pH 8.
   2. Make 50 mM sodium acetate buffer at pH 5.
   3. Make a stock solution of 8 U/mL amyloglucosidase in 50 mM sodium acetate buffer, pH 5.
   4. Make a stock solution of 2 U/mL α-amylase in 50 mM sodium acetate buffer, pH 5.
   5. Using distilled water, makeglucose standard solutions at concentrations ranging between 0 and 100 µg/mL,.
   6. Prepare GOD-POD reagent from the D-Glucose Assay Kit (GODPOD Format), following the manufacturer´s instruction.

2. Determination of the Cell Dry Weight (Optional)

1. Transfer 2 mL of a culture or a cell resuspension (see step 1.1) to a 2.0 mL tube and centrifuge at 6,000 x g and 4 °C for 5 min. Discard the supernatant.
2. Resuspend the pellet in 1 mL of water and centrifuge at 6,000 x g and 4 °C for 5 min. Discard the supernatant and resuspend the cell pellet in 0.5 mL of water.
3. Transfer the suspension to a pre-weighed aluminum tray. Transfer the tray to a drying oven at 105 °C for overnight drying (approximately 18 h).
   CRITICAL: It is important that the tray is handled with forceps to avoid the transfer of material from the fingers. Dry an empty, pre-weighed aluminum tray under the same conditions to determine any weight loss from the trays during drying.
4. After drying, remove the tray from the oven and allow it to equilibrate at ambient conditions for 5 min before weighing it with an accuracy of 0.0001 g.
   NOTE: The value can be used to normalize the glycogen content on a cell-dry-weight basis.

3. Lysis of Cyanobacterial Cells

1. Transfer 1 mL of a culture or a cell resuspension (see step 1.1) to a 1.5 mL tube and centrifuge at 6,000 x g and 4 °C for 10 min. Discard the supernatant.
2. Resuspend the pellet in 1 mL of 50 mM Tris-HCl, pH 8, and centrifuge at 6,000 x g and 4 °C for 10 min. Discard the supernatant and resuspend the cell pellet in 1 mL of the Tris-HCl buffer. Repeat the process.
3. Thoroughly resuspend the pellet in 500 µL of 50 mM Tris-HCl buffer, pH 8.
   CRITICAL: Resuspend the pellet well for an efficient cell lysis. Keep the resuspension in ice.
4. Lyse the resuspended cells at 4 °C by performing 30 cycles of ultrasonication, each cycle consisting of 30 s at a frequency of 20 kHz with the maximum amplitude, followed by 90 s without.
   NOTE: This method can efficiently lyse Synechococcus sp. PCC 7002.
   1. Alternatively, transfer the cell resuspension to a screw-cap tube and add zirconium oxide beads to the tube, following manufacturer's instructions. Set the tube in a tissue homogenizer and lyse the cells at 4 °C by performing 2 cycles of beating, each cycle consisting of 5 min at the frequency setting of 5.
      NOTE: This method can efficiently lyse Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002.
5. Centrifuge the tube containing the lysate for 10 min at 6,000 x g and 4 °C.
   NOTE: The pellet should mainly consist of large cell debris. If there is a significant number of unbroken cells, repeat step 3.4. Keep the supernatant on ice.
6. Determine the protein concentration using a commercial BCA Protein assay kit.
   NOTE: The value can be used to normalize the glycogen content on a protein content basis.

4. Glycogen Precipitation

1. Remove chlorophyll a from the cell lysate by mixing 900 µL of 96% (v/v) ethanol and 100 µL of the supernatant from step 3.5 in a 1.5 mL screw-cap tube. After closing the cap, heat the tube at 90 °C for 10 min using a standard laboratory heating block.
2. Incubate the tube on ice for 30 min.
3. Centrifuge the tube at 20,000 x g and 4 °C for 30 min and carefully remove the supernatant; the pellet contains glycogen. Lightly dry the pellet in air to remove excess ethanol.
   CRITICAL:Excessive drying of the pellet must be avoided, otherwise it becomes difficult to solubilize in step 5.1.
4. OPTIONAL: Measure the absorbance of the supernatant obtained in step 4.3 at 664 nm to determine the chlorophyll a content. Use an absorption coefficient of 84.6 L/g/cm²⁴.
   NOTE: The value can be used to normalize the glycogen content.

5. Enzymatic Hydrolysis and Glycogen Determination

1. Solubilize the pellet obtained in step 4.3 in 100 µL of 50 mM sodium acetate, pH 5, and add 50 µL of 8 U/mL amyloglucosidase and 50 µL of 2 U/mL α-amylase. Mix these materials well using a vortex.
   NOTE: Mixing by pipetting is not recommended because the glycogen pellet is viscous.
2. Incubate the mixture at 60 °C on a heating block for 2 h to enable the digestion of glycogen into glucose molecules.
3. Centrifuge the sample at 10,000 x g for 5 min and transfer the supernatant to a new 1.5 mL tube.

6. Determination of the Total Glucose Content Using the GOD-POD Reagent

1. Measure the concentration of glucose in the supernatant obtained in step 5.3 using the GOD-POD reagent. Transfer 100 µL of the supernatant from step 5.3 to a well in a 96-well plate. As a negative control, use 100 µL of 50 mM sodium acetate, pH 5. For generating a calibration curve, also measure the glucose standard solutions.
2. Add 150 µL of GOD-POD reagent to each sample and quickly mix by pipetting.
3. Incubate the plate statically at 25 °C for 30 min. Record the absorbance value at 510 nm using a plate reader.
4. Calculate the concentration of glucose using a calibration curve obtained from the glucose standards.
   NOTE: The glycogen concentration in the cell lysate is expressed as the concentration of glucose (µg/mL).

Wyniki

10 mL of wildtype Synechocystis sp. PCC 6803 were grown under photoautotrophic conditions until the OD_730nm value reached approximately 0.8. The cells were harvested and resuspended in 50 mM Tris-HCl, pH 8. The OD_730nm value was adjusted to 2-3. The glycogen content was analyzed following the protocol described above. The glycogen content per the OD_730nm was 13 ± 1.8 µg/mL/OD_730nm (N = 12). The glycogen content relati...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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