Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence

Abstract

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner-Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me) corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593-1602. © 2017 Wiley Periodicals, Inc.

Glycolytic Shunts Replenish the Calvin–Benson–Bassham Cycle as Anaplerotic Reactions in Cyanobacteria

“…Our previous finding that deletion of the ED pathway (Deda) had the strongest effect on the ability to grow under photomixotrophic conditions was confirmed ( Figure 1B) (Chen et al, 2016). In darkness, i.e., under LAH and fermentative conditions, the OPP pathway (Dzwf, Dgnd) was most important ( Figure 1C and 1D), which is well in line with previous findings (Scanlan et al, 1995;Yang et al, 2002;Jansen et al, 2010;Wan et al, 2017). Deletion of the EMP pathway (Dpfk) led to diminished growth under LAH conditions but the effect was less severe than in Dzwf and Dgnd.…”

“…Deletion of the OPP pathway in cyanobacteria results in mutants that are unable to grow under light-activated heterotrophic (LAH) conditions (Scanlan et al, 1995;Jansen et al, 2010). This is well in line with flux analyses that found that glucose is predominantly metabolized by the OPP pathway in darkness (Yang et al, 2002;Wan et al, 2017). It has furthermore been reported that the PK pathway in cyanobacteria contributes to heterotrophic metabolism as well (Xiong et al, 2015).…”

“…In line with these results, CO 2 fixation was severely reduced in DedaDgnd in comparison with the WT and all other glycolytic mutants ( Figure 2E). Fluxes through the ED pathway were shown to be negligible under heterotrophic and photomixotrophic conditions in Synechocystis (Wan et al, 2017), whereas photoautotrophic conditions were not tested. A requirement for classical flux analyses are constant conditions that result in metabolic steady states.…”

Model metabolic strategy for heterotrophic bacteria in the cold ocean based on Colwellia psychrerythraea 34H

“…Pool Size and Dynamic Labeling of Central Metabolites. The metabolic pool size (i.e., concentration of intracellular metabolites) of 34H was compared with that of E. coli during exponential growth at their respective normal-growth temperatures (4°C and 37°C) using the isotopic ratio method previously described (37). The analysis (Fig.…”

Metabolic pathway rewiring in engineered cyanobacteria for solar-to-chemical and solar-to-fuel production from CO2

“…Metabolic plasticity is also associated with the metabolic capabilities under various environmental growth conditions (e.g., photoautotrophic, photomixotrophic, or heterotrophic growth), 29,30 and this has been demonstrated through the rewiring of metabolic pathways for the production of 2,3-BDO. 12 Thus, the rewiring of metabolic pathways for directing carbon fluxes toward the desired products in cyanobacteria could facilitate the development of feasible S2C and S2F platforms.…”