(281g) 13c-Metabolic Flux Analysis of Microbial Cell Factories for Rational Strain Development

Metabolic flux analysis via ¹³C labeling is a powerful tool to delineate metabolic pathways and to determine novel enzyme functions in microbial hosts. Three core techniques are used during ¹³C-metabolic flux analysis: 1) steady-state or dynamic labeling of cell cultures; 2) precise measurements of the labeling pattern of targeted metabolites using GC-MS and LC-MS; and 3) model based interpretation of culture kinetics, spectrometry data sets, and other omics results to reveal the flux network and metabolic regulations. ¹³C-based metabolic analysis has diverse applications for studying environmental microorganisms that are important for bioremediation and bio-manufacturing. This presentation will give three examples that apply ¹³C-based analysis to characterize microbial factory metabolisms and identify unintuitive engineering targets: 1) ¹³C-Metabolic flux analysis of free fatty acid E.coli producers to elucidate metabolic burdens and strain improvement guidelines; 2) isotopically nonstationary metabolic flux analysis of fast-growing phototrophic metabolisms, metabolite channeling, and flux network rigidity in cyanobacterial workhorses; 3) ¹³C-metabolic flux analysis can be simplified to ¹³C-fingerprinting, ¹³C-pulse-tracing, and ¹³C-metabolomics to offer fast screening of novel pathways, metabolic shifts, co-utilization of complex nutrients, and biosynthesis bottlenecks during Yarrowia terpenoid fermentations. These ¹³C-tools can play important role in design-build-test-learn based strain development.