PHB is Produced from Glycogen Turn-over during Nitrogen Starvation in Synechocystis sp. PCC 6803

Abstract

Polyhydroxybutyrate (PHB) is a polymer of great interest as a substitute for conventional plastics, which are becoming an enormous environmental problem. PHB can be produced directly from CO₂ in photoautotrophic cyanobacteria. The model cyanobacterium Synechocystis sp. PCC 6803 produces PHB under conditions of nitrogen starvation. However, it is so far unclear which metabolic pathways provide the precursor molecules for PHB synthesis during nitrogen starvation. In this study, we investigated if PHB could be derived from the main intracellular carbon pool, glycogen. A mutant of the major glycogen phosphorylase, GlgP2 (slr1367 product), was almost completely impaired in PHB synthesis. Conversely, in the absence of glycogen synthase GlgA1 (sll0945 product), cells not only produced less PHB, but were also impaired in acclimation to nitrogen depletion. To analyze the role of the various carbon catabolic pathways (EMP, ED and OPP pathways) for PHB production, mutants of key enzymes of these pathways were analyzed, showing different impact on PHB synthesis. Together, this study clearly indicates that PHB in glycogen-producing Synechocystis sp. PCC 6803 cells is produced from this carbon-pool during nitrogen starvation periods. This knowledge can be used for metabolic engineering to get closer to the overall goal of a sustainable, carbon-neutral bioplastic production.

Keywords: PHB; Synechocystis; bioplastic; cyanobacteria; glycogen; metabolic engineering; sustainable.

Figure 1

Overview of central metabolism of Synechocystis. Genes which were deleted in this study are highlighted in with a red background. Dotted lines represent several enzymatic reactions. The EMP, ED and OPP (Embden-Meyerhof-Parnas, Entner-Doudoroff, Oxidative Pentose Phosphate) pathways are highlighted in green, blue and yellow, respectively.

Figure 2

Characterization of the glycogen synthase mutants, ∆glgA1 and ∆glgA2. (A) Cultures after five days of nitrogen starvation. (B) Recovery assay of chlorotic wild-type (WT) and mutants ∆glgA1 and ∆glgA2, using the drop agar method. Cultures that were nitrogen-starved for 14 days were serially diluted from 1 to 1:10,000 and from each dilution, a drop of 5 µL was plated on BG11 agar and grown for seven days.

Figure 3

Polyhydroxybutyrate (PHB) content in percentage of cell dry weight (CDW) (A) and cellular glycogen content (B) of mutants impaired in the glycogen synthesis. Cultures were shifted to nitrogen free medium at day 0 and were subsequently grown for 21 days. Each point represents a mean of three independent biological replicates.

Figure 4

PHB content in percentage of cell dry weight (CDW) (A) and glycogen content (B) of mutants impaired in the glycogen degradation. Cultures were shifted to nitrogen free medium at day 0 and were subsequently grown for 21 days. Each point represents a mean of three independent biological replicates.

Figure 5

Fluorescence microscopic picture of Nile-red stained PHB granules in chlorotic cells. Cultures where grown for 14 days in nitrogen depleted medium BG11⁰. Shown is an overlay of phase contrast with a CY3 channel of the WT (A), ∆glgP1 (B), ∆glgP2 (C) and ∆glgP1/2 (D). Scale bar corresponds to 7.5 µm.

Figure 6

PHB content in percentage of cell dry weight (CDW) (A) and glycogen content (B) of mutants with disrupted carbon pathway. Cultures were shifted to nitrogen free medium at day 0 and were subsequently grown for 21 days. Each point represents a mean of three independent biological replicates.

Figure 7

Glycogen content of wild-type and mutant lacking the PHB synthase genes (PhaEC). Cultures were shifted to nitrogen free medium at day 0 and were subsequently grown for 21 days. Each point represents a mean of three independent biological replicates.