Numerous secondary metabolites from plants are important for their medicinal, nutraceutical or sensory properties. Recently, significant progress has been made in the identification of the genes and enzymes of plant secondary metabolic pathways. Hence, there is interest in using synthetic biology to enhance the production of targeted valuable metabolites in plants. In this article, we examine the contribution that metabolic flux analysis will have on informing the rational selection of metabolic engineering targets as well as analysis of carbon and energy efficiency. Compared to microbes, plants have more complex tissue, cellular and subcellular organization, making precise metabolite concentration measurements more challenging. We review different techniques involved in quantifying flux and provide examples illustrating the application of the techniques. For linear and branched pathways that lead to end products with low turnover, flux quantification is straightforward and doesn't require isotopic labeling. However, for metabolites synthesized via parallel pathways, there is a requirement for isotopic labeling experiments. If the fed isotopically labeled carbons don't scramble, one needs to apply transient label balancing methods. In the transient case, it is also necessary to measure metabolite concentrations. While flux analysis is not able to directly identify mechanisms of regulation, it is a powerful tool to examine flux distribution at key metabolic nodes in intermediary metabolism, detect flux to wasteful side pathways, and show how parallel pathways handle flux in wild-type and engineered plants under a variety of physiological conditions.
Keywords: 13C MFA, Steady state isotopically labeled metabolic flux analysis; BA, Benzoic acid; DMAPP, Dimethylallyl diphosphate; GC, Gas chromatography; INST-MFA, Isotopically non-steady state metabolic flux analysis; IP, Isopentenyl phosphate; IPP, Isopentenyl diphosphate; LC, Liquid chromatography; MEP, Methylerythritol 4-phosphate; MFA, Metabolic flux analysis; MS, Mass spectrometry; MVA, Mevalonic acid; MVAP, Mevalonate 5-phosphate; MVAPP, Mevalonate 5-diphosphate; Metabolic channeling; Metabolic flux analysis; NMR, Nuclear magnetic resonance; Phe, Phenylalanine; Plant secondary metabolites; Stable isotopic labeling; Subcellular compartmentation.
© 2020 The Authors.