Deriving metabolic engineering strategies from genome-scale modeling with flux ratio constraints

Optimized production of bio‐based fuels and chemicals from microbial cell factories is a central goal of systems metabolic engineering. To achieve this goal, a new computational method of using flux balance analysis with flux ratios (FBrAtio) was further developed in this research and applied to five case studies to evaluate and design metabolic engineering strategies. The approach was implemented using publicly available genome‐scale metabolic flux models. Synthetic pathways were added to these models along with flux ratio constraints by FBrAtio to achieve increased (i) cellulose production from Arabidopsis thaliana; (ii) isobutanol production from Saccharomyces cerevisiae; (iii) acetone production from Synechocystis sp. PCC6803; (iv) H2 production from Escherichia coli MG1655; and (v) isopropanol, butanol, and ethanol (IBE) production from engineered Clostridium acetobutylicum. The FBrAtio approach was applied to each case to simulate a metabolic engineering strategy already implemented experimentally, and flux ratios were continually adjusted to find (i) the end‐limit of increased production using the existing strategy, (ii) new potential strategies to increase production, and (iii) the impact of these metabolic engineering strategies on product yield and culture growth. The FBrAtio approach has the potential to design “fine‐tuned” metabolic engineering strategies in silico that can be implemented directly with available genomic tools. Constraining the distribution of a single metabolite among competing pathways can massively reorder global metabolism. In this study, authors show a new method – Flux Balance Analysis with Flux Ratios (FBrAtio) – along with genome‐scale modeling, which can be used to determine the global outcome of metabolic engineering strategies. The authors apply this technique to five case studies to evaluate and improve existing metabolic engineering strategies in the production of biofuels and commodity chemicals.