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....

Protocol

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 ....

Results

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 relative to the .......

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.......

Materials

List of materials used in this article

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