Metabolite-Centric Flux Analysis for Rational Design and Engineering of Microbes

The development of various constraint-based model-driven computational algorithms has immensely aided the rational redesign of metabolic networks for the targeted metabolite overproduction in microbial cell factories. While most such methods rely on a reaction-centric approach whereby multiple reactions are suggested to be overexpressed/knocked, we have recently presented a method which is metabolite-centric. In this work, we demonstrated how the previously developed concept of flux-sum, i.e. the turnover of metabolites in constraints-based flux analysis, can be utilized for identifying metabolic engineering targets by applying it to Escherichia coli, as a case study for enhancing ethanol and succinate production. Remarkably, while several metabolite targets identified well corresponded to the gene targets which have been identified by reaction-centric algorithms, certain unique targets such as the pyruvate attenuation for enhancing succinate production were also unraveled. Similarly, we also show how the concept of flux-sum can be utilized to analyze the intracellular redox balancing by estimating overall NAD(H) and NADP(H) turnover. For this purpose, we examine the NADPH regeneration potentials in E. coli, Saccharomyces cerevisiae, Bacillus subtilis, and Pichia pastoris across multiple environmental conditions and uncovered E. coli and glycerol as the best microbial host and most suitable carbon source, respectively, for producing NADP-dependent products. Moreover, we also identified the optimal cofactor specificity engineering (CSE) enzyme targets using our previously developed cofactor modification analysis (CMA), whose cofactors when switched from NAD(H) to NADP(H) augmented the overall NADP(H) flux-sum significantly. Overall, the metabolite-centric approach offer significant potential for rational design and engineering of industrial microbes in the field of systems metabolic engineering.