(645c) A Nonstationary 13C Labeling Approach For Metabolic Flux Analysis In A Photoautotrophic System

Photoautotrophic metabolism involves the utilization of light energy to fix
freely available CO₂ into complex organic molecules. ¹³C-MFA
is widely used for quantification of flux-phenotypes under varying
environmental and genetic conditions. However, inherent limitations have
prevented the application of steady state ¹³C-MFA to purely
autotrophic metabolism, wherein CO₂ is the sole carbon source. This
is due to the fact that, in autotrophic systems under conditions of isotopic
steady state, every single carbon atom in every downstream molecule has the
same relative labeling as the single input carbon of CO₂, irrespective
of flux distribution. However, the trajectory of label incorporation during the
period preceding isotopic steady state is sensitive to fluxes. This transient
labeling information is utilized in recently developed techniques of
nonstationary ¹³C-MFA, which enable estimation of metabolic fluxes
from measurements of dynamic changes in ¹³C labeling patterns of
intracellular metabolite pools in response to a step change in CO₂
labeling.

In this work, we utilize the nonstationary ¹³C- MFA technique with
the elementary metabolite unit (EMU) formulation to estimate central carbon
fluxes under photoautotrophic conditions for the first time. The technique is
applied to a prokaryotic cyanobacterium, Synechocystis sp. PCC 6803.
Reactions of the central carbon network consisting of
glycolysis/gluconeogenesis, pentose phosphate/Calvin cycle, and the
TCA/glyoxylate shunt, are modeled. Tandem mass spectrometry based techniques
are used to measure mass isotopomer distributions as well as concentrations
(pool sizes) of intracellular metabolites. Metabolite pool size measurements
are subject to several inaccuracies during the quenching and extraction
procedure. Therefore, the effect of number and accuracy of pool size
measurements on flux identifiability will also be discussed.