282337 Isotopically Nonstationary 13C Flux Analysis of Isobutyraldehyde Production in S. Elongatus
The development of fuel substitutes from renewable resources has become significantly important as a result of current global energy and environmental problems. Recent studies have demonstrated the feasibility of converting energy from sunlight and carbon from CO2 directly into biofuels using photosynthetic microorganisms. Despite the advances made in cyanobacterial biofuels production, the productivities achieved by cyanobacterial fermentations are currently too low for industrial feasibility and few tools are available that specifically address the challenges of redirecting and enhancing metabolic flux in photosynthetic microbes.
The ability to perform quantitative studies using isotope tracers and metabolic flux analysis (MFA) is critical for accurately assessing in vivo regulation of photoautotrophic metabolism, as well as crucial to identifying pathways that will maximize carbon flux from Calvin cycle intermediates into biofuel-producing pathways. Although 13C is the preferred isotope tracer for mapping central carbon metabolism in heterotrophic organisms, photoautotrophs assimilate carbon solely from CO2 and therefore produce a uniform steady-state 13C-labeling pattern that is insensitive to fluxes. However, transient measurements of isotope incorporation following a step change from labeled to unlabeled CO2 can be used to map carbon fluxes under autotrophic growth conditions. This involves quantification of intracellular metabolic fluxes based upon computational analysis of dynamic isotope labeling trajectories, which applies the technique of isotopically nonstationary MFA (INST-MFA).
We have recently introduced an INST-MFA approach to quantify intracellular fluxes in the cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism, under photoautotrophic conditions (1). This involved using both GC/MS and LC/MS/MS to quantify the trajectories of metabolite labeling that result from introduction of 13C-labeled bicarbonate. Comparison of the INST-MFA flux map to theoretical values predicted by a linear programming (LP) method revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. Our ongoing work is focused on applying the INST-MFA approach to several engineered strains of Synechococcus elongatus PCC 7942 to enable the systematic improvement of cyanobacterial production of isobutyraldehyde (IBA, a direct precursor of isobutanol). Quantification of photosynthetic carbon fluxes in cyanobacteria will pinpoint pathway bottlenecks that can be subsequently removed in further rounds of metabolic engineering, thus leading to maximal IBA productivity by redirecting flux into IBA-producing pathways.
1. Young, J.D., Shastri, A. a, Stephanopoulos, G., et al. (2011) Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis. Metabolic engineering. 13, 656-665.
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