Comparison of pathway analysis and constraint-based methods for cell factory design

Background: Computational strain optimisation methods (CSOMs) have been successfully used to exploit genome-scale metabolic models, yielding strategies useful for allowing compound overproduction in metabolic cell factories. Minimal cut sets are particularly interesting since their definition allows searching for intervention strategies that impose strong growth-coupling phenotypes, and are not subject to optimality bias when compared with simulation-based CSOMs. However, since both types of methods have different underlying principles, they also imply different ways to formulate metabolic engineering problems, posing an obstacle when comparing their outputs. Results: In this work, we perform an in-depth analysis of potential strategies that can be obtained with both methods, providing a critical comparison of performance, robustness, predicted phenotypes as well as strategy structure and size. To this end, we devised a pipeline including enumeration of strategies from evolutionary algorithms (EA) and minimal cut sets (MCS), filtering and flux analysis of predicted mutants to optimize the production of succinic acid in Saccharomyces cerevisiae. We additionally attempt to generalize problem formulations for MCS enumeration within the context of growth-coupled product synthesis. Strategies from evolutionary algorithms show the best compromise between acceptable growth rates and compound overproduction. However, constrained MCSs lead to a larger variety of phenotypes with several degrees of growth-coupling with production flux. The latter have proven useful in revealing the importance, in silico, of the gamma-aminobutyric acid shunt and manipulation of cofactor pools in growth-coupled designs for succinate production, mechanisms which have also been touted as potentially useful for metabolic engineering. Conclusions: The two main groups of CSOMs are valuable for finding growth-coupled mutants. Despite the limitations in maximum growth rates and large strategy sizes, MCSs help uncover novel mechanisms for compound overproduction and thus, analyzing outputs from both methods provides a richer overview on strategies that can be potentially carried over in vivo.