A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms.
Metabolic flux analysis is increasingly recognized as an integral component of systems biology. However, techniques for experimental measurement of system-wide metabolic fluxes in purely photoautotrophic systems (growing on CO(2) as the sole carbon source) have not yet been developed due to the unique problems posed by such systems. In this paper, we demonstrate that an approach that balances positional isotopic distributions transiently is the only route to obtaining system-wide metabolic flux maps for purely autotrophic metabolism. The outlined transient (13)C-MFA methodology enables measurement of fluxes at a metabolic steady-state, while following changes in (13)C-labeling patterns of metabolic intermediates as a function of time, in response to a step-change in (13)C-label input. We use mathematical modeling of the transient isotopic labeling patterns of central intermediates to assess various experimental requirements for photoautotrophic MFA. This includes the need for intracellular metabolite concentration measurements and isotopic labeling measurements as a function of time. We also discuss photobioreactor design and operation in order to measure fluxes under precise environmental conditions. The transient MFA technique can be used to measure and compare fluxes under different conditions of light intensity, nitrogen sources or compare strains with various mutations or gene deletions and additions.
Shastri
Avantika A
AA
School of Chemical Engineering, Purdue University, 480 Stadium Mall Dr., West Lafayette, IN 47907, USA.
Morgan
John A
JA
D019149:Bioreactors; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism*; D002247:Carbon Isotopes; D007553:Isotope Labeling / Q000379:methods*; D010788:Photosynthesis; D046939:Synechocystis / Q000378:metabolism*; D013997:Time Factors
D002247:Carbon Isotopes; D002245:Carbon Dioxide