Metabolic Changes in Synechocystis PCC6803 upon Nitrogen-Starvation: Excess NADPH Sustains Polyhydroxybutyrate Accumulation

Abstract

Polyhydroxybutyrate (PHB) is a common carbon storage polymer among heterotrophic bacteria. It is also accumulated in some photoautotrophic cyanobacteria; however, the knowledge of how PHB accumulation is regulated in this group is limited. PHB synthesis in Synechocystis sp. PCC 6803 is initiated once macronutrients like phosphorus or nitrogen are limiting. We have previously reported a mutation in the gene sll0783 that impairs PHB accumulation in this cyanobacterium upon nitrogen starvation. In this study we present data which explain the observed phenotype. We investigated differences in intracellular localization of PHB synthase, metabolism, and the NADPH pool between wild type and mutant. Localization of PHB synthase was not impaired in the sll0783 mutant; however, metabolome analysis revealed a difference in sorbitol levels, indicating a more oxidizing intracellular environment than in the wild type. We confirmed this by directly measuring the NADPH/NADP ratio and by altering the intracellular redox state of wild type and sll0783 mutant. We were able to physiologically complement the mutant phenotype of diminished PHB synthase activity by making the intracellular environment more reducing. Our data illustrate that the NADPH pool is an important factor for regulation of PHB biosynthesis and metabolism, which is also of interest for potential biotechnological applications.

Integrated analysis of engineered carbon limitation in a quadruple CO2/HCO3--uptake mutant of Synechocystis sp. PCC 6803

“…After the LC shift, the carboxysome number increased in both strains, the wild type and the D4 mutant. Additionally, the D4 mutant also had more PHB bodies under HC conditions, which is characteristic for Synechocystis 6803 wild-type cells exposed to carbon excess under high-light conditions, leading to a reducing intracellular environment (Hauf et al, 2013). In addition to Ci-specific genes, a transcript subset indicated a general stress response of D4.…”

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate

“…6). Hauf et al (2013) have shown that the reduction state of the cofactor pool can influence PHB production. PHB production observed here could be the result of an increased level of NAD(P)H and indirectly be a way to deal with NAD + photoreduction (Herter et al, 1998), notably occurring at the beginning of the growth curve.…”

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

“…In the last step of biosynthesis, hydroxybutyryl-CoA is polymerized to polyhydroxybutyrate by a class III PHB synthase, which is a heterodimer of PhaE (slr1829) and PhaC (slr1830) that builds the catalytically active enzyme in cyanobacteria (11). The PhaEC heterodimer (12) (or tetramer [13]) synthesizes PHB, thereby building up PHB granules, to which it is attached (4). This marks the boundaries of our understanding of PHB accumulation in cyanobacteria on a molecular level.…”

“…This metabolite can be utilized for either gluconeogenesis or glycolysis, providing the necessary carbon skeletons for biosynthesis of amino acids and other metabolites required for cell growth (2), when growth conditions are suitable and nutrients are abundant. In fact, carbon flux is greatly affected by the availability of macronutrients like nitrogen (3)(4)(5) and phosphorus, which may limit growth (6). Under nutrient-limiting conditions, cyanobacteria undergo a stress adaptation process termed chlorosis (7).…”