Imaging and modelling of poly(3-hydroxybutyrate) synthesis in Paracoccus denitrificans

Abstract

Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).

Keywords: Acetyl-CoA; LSCM; Metabolic modelling; Nile Red; Poly-3-hydroxybutyrate (PHB); Polyhydroxyalkanoates (PHA); Storage inclusions.

Fig. 1

Staining of P. denitrificans cells with different fluorescent dyes. In all cases, cells were grown to an OD₆₆₀ of about 0.75–1 and stained with: A MitoTracker™ Green FM (MTG) (1 µM); B FM4-64 (1 µM); C Nile Red (1 µM); D Double staining with MTG (1 µM) and Syto80 (500 nM). E–G Details of daughter cells from a recently divided mother cell. MTG image (E), Syto80 image (F), overlay (G). Cells were imaged using two different lasers (a blue-argon Laser, 488 nm, for excitation of MTG and a green-helium/neon laser, 543 nm, for excitation of Syto80)

Fig. 2

The PHB granules appear before the first cellular division. A Mid-exponential phase cells. B Late stationary phase cells, used as inoculum (T₀) for the second time-series experiment. T₁ Cells after 30 min of shaking at 34 °C and 300 rpm. The microscopy images of T2, T3 and T4 samples are not shown in Fig. 2 because they are very similar to the T1 sample. The scale bar is 1 µm

Fig. 3

Distributions of metabolic fluxes under PHB accumulation and exponential growth. The numbers are reaction rates in mmol g-DW⁻¹ h⁻¹. Only the main metabolic fluxes are shown