Rerouting of Metabolism into Desired Cellular Products by Nutrient Stress: Fluxes Reveal the Selected Pathways in Cyanobacterial Photosynthesis

Boosting cellular
growth rates while redirecting metabolism to
make desired products are the preeminent goals of gene engineering
of photoautotrophs, yet so far these goals have been hardly achieved
owing to lack of understanding of the functional pathways and their
choke points. Here we apply a 13C mass isotopic method
(INST-MFA) to quantify instantaneous fluxes of metabolites during
photoautotrophic growth. INST-MFA determines the globally most accurate
set of absolute fluxes for each metabolite from a finite set of measured 13C-isotopomer fluxes by minimizing the sum of squared residuals
between experimental and predicted mass isotopomers. We show that
the widely observed shift in biomass composition in cyanobacteria,
demonstrated here with Synechococcus sp. PCC 7002,
favoring glycogen synthesis during nitrogen starvation is caused by
(1) increased flux through a bottleneck step in gluconeogenesis (3PG
→ GAP/DHAP), and (2) flux overflow through a previously unrecognized
hybrid gluconeogenesis–pentose phosphate (hGPP) pathway. Our
data suggest the slower growth rate and biomass accumulation under
N starvation is due to a reduced carbon fixation rate and a reduced
flux of carbon into amino acid precursors. Additionally, 13C flux from α-ketoglutarate to succinate is demonstrated to
occur via succinic semialdehyde, an alternative to
the conventional TCA cycle, in Synechococcus 7002
under photoautotrophic conditions. We found that pyruvate and oxaloacetate
are synthesized mainly by malate dehydrogenase with minimal flux into
acetyl coenzyme-A via pyruvate dehydrogenase. Nutrient
stress induces major shifts in fluxes into new pathways that deviate
from historical metabolic pathways derived from model bacteria.