Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants.

Photorespiration is a central component of photosynthesis; however to better understand its role it should be viewed in the context of an integrated metabolic network rather than a series of individual reactions that operate independently. Isotopically nonstationary C metabolic flux analysis (INST-MFA), which is based on transient labeling studies at metabolic steady state, offers a comprehensive platform to quantify plant central metabolism. In this chapter, we describe the application of INST-MFA to investigate metabolism in leaves.

Isotopically nonstationary C flux analysis of cyanobacterial isobutyraldehyde production.

We applied isotopically nonstationary C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity.

Application of isotope labeling experiments and (13)C flux analysis to enable rational pathway engineering.

Isotope labeling experiments (ILEs) and (13)C flux analysis provide actionable information for metabolic engineers to identify knockout, overexpression, and/or media optimization targets. ILEs have been used in both academic and industrial labs to increase product formation, discover novel metabolic functions in previously uncharacterized organisms, and enhance the metabolic efficiency of host cell factories. This review highlights specific examples of how ILEs have been used in conjunction with enzyme or metabolic engineering to elucidate host cell metabolism and improve product titer, rate, or yield in a directed manner.

Isotopically nonstationary 13C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation.

Improving plant productivity is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe leaf metabolism, although such information would be valuable for increasing photosynthetic capacity, enhancing biomass production, and rerouting carbon flux toward desirable end products. Isotopically nonstationary metabolic flux analysis (INST-MFA) has been previously applied to map carbon fluxes in photoautotrophic bacteria, which involves model-based regression of transient (13)C-labeling patterns of intracellular metabolites.

13 C flux analysis of cyanobacterial metabolism.

(13)C metabolic flux analysis (MFA) has made important contributions to our understanding of the physiology of model strains of E. coli and yeast, and it has been widely used to guide metabolic engineering efforts in these microorganisms. Recent advancements in (13)C MFA methodology combined with publicly available software tools are creating new opportunities to extend this approach to examine less characterized microbes.

Isotopically nonstationary MFA (INST-MFA) of autotrophic metabolism.

Metabolic flux analysis (MFA) is a powerful approach for quantifying plant central carbon metabolism based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstationary (13)C MFA (INST-MFA), which is applicable to autotrophic systems that are at metabolic steady state but are sampled during the transient period prior to achieving isotopic steady state following the introduction of (13)CO2. We describe protocols for performing the necessary isotope labeling experiments, sample collection and quenching, nonaqueous fractionation and extraction of intracellular metabolites, and mass spectrometry (MS) analysis of metabolite labeling.

Isotopically nonstationary 13C metabolic flux analysis.

(13)C metabolic flux analysis (MFA) is a powerful approach for quantifying cell physiology based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstationary (13)C MFA (INST-MFA), which is applicable to systems that are at metabolic steady state, but are sampled during the transient period prior to achieving isotopic steady state following the introduction of a (13)C tracer. We describe protocols for performing the necessary isotope labeling experiments, for quenching and extraction of intracellular metabolites, for mass spectrometry (MS) analysis of metabolite labeling, and for computational flux estimation using INST-MFA.