Altering the Structure of Carbohydrate Storage Granules in the Cyanobacterium Synechocystis sp Strain PCC 6803 through Branching-Enzyme Truncations

Abstract

Carbohydrate storage is an important element of metabolism in cyanobacteria and in the chloroplasts of plants. Understanding how to manipulate the metabolism and storage of carbohydrate is also an important factor toward harnessing cyanobacteria for energy production. While most cyanobacteria produce glycogen, some have been found to accumulate polysaccharides in the form of water-insoluble alpha-glucan similar to amylopectin. Notably, this alternative form, termed "semi-amylopectin," forms in cyanobacterial species harboring three branching-enzyme (BE) homologs, designated BE1, BE2, and BE3. In this study, mutagenesis of the branching genes found in Synechocystis sp. strain PCC 6803 was performed in order to characterize their possible impact on polysaccharide storage granule morphology. N-terminal truncations were made to the native BE gene of Synechocystis sp. PCC 6803. In addition, one of the two native debranching enzyme genes was replaced with a heterologous debranching enzyme gene from a semi-amylopectin-forming strain. Growth and glycogen content of mutant strains did not significantly differ from those of the wild type, and ultrastructure analysis revealed only slight changes to granule morphology. However, analysis of chain length distribution by anion-exchange chromatography revealed modest changes to the branchedchain length profile. The resulting glycogen shared structure characteristics similar to that of granules isolated from semi-amylopectin- producing strains. IMPORTANCE This study is the first to investigate the impact of branching-enzyme truncations on the structure of storage carbohydrates in cyanobacteria. The results of this study are an important contribution toward understanding the relationship between the enzymatic repertoire of a cyanobacterial species and the morphology of its storage carbohydrates.