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Fig. 3 | Microbial Cell Factories

Fig. 3

From: GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways

Fig. 3

Experimental design for 13C metabolic flux analysis of photomixotrophic Synechocystis 6803. Different setups using different combinations of 13C tracer substrates and 13C labelling data were analyzed for achievable flux precision and accuracy, assuming a flux scenario with zero flux through the ED and the PK pathway. Here, the key fluxes of upper and lower carbon metabolism, i.e., through the ED, OPP, EMP, and PK pathways, the CBB cycle, and the TCA cycle, are shown. Each setup was evaluated by a Monte-Carlo approach that mimicked 100 repetitions of the corresponding flux study while taking experimental errors into account. The sensitive substrates [1-13C], [3-13C], [6-13C], and [13C6] glucose seemed useful for the following reasons. The combination of [1-13C] glucose and [6-13C] glucose well discriminated the fluxes through the EMP, the PP, and the ED pathway in glucose-grown pseudomonads, revealing a similarly cyclic pathway architecture as cyanobacteria [9]. Metabolization of [3-13C] glucose (based on the underlying carbon transitions) via the ED route should selectively lead to 13C label enrichment at the C1 of pyruvate (and amino acids derived therefrom), providing a sensitive readout, should this pathway be active. The use of [13C6] glucose appeared beneficial, likely because it helped to estimate the relative uptake of 13C sugar versus (non-labelled) CO2, as previously demonstrated for Basfia succiniciproducens, grown on sucrose under high rates of CO2 assimilation [22]. The color indicates flux determinability: green, < 0.1%, yellow < 1%, orange < 10%; and red, > 10%

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