Systematizing the different notions of growth-coupled product synthesis and a single framework for computing...

A widely used design principle for metabolic engineering of microorganisms aims to introduce interventions that enforce growth-coupled product synthesis such that the product of interest becomes a (mandatory) by-product of growth. However, different variants and partially contradicting notions of growth-coupled production (GCP) exist. Herein, we propose an ontology for the different degrees of GCP and clarify their relationships. Ordered by coupling degree, we distinguish four major classes: potentially, weakly, and directionally growth-coupled production (pGCP, wGCP, dGCP) as well as substrate-uptake coupled production (SUCP). We then extend the framework of Minimal Cut Sets (MCS), previously used to compute dGCP and SUCP strain designs, to allow inclusion of implicit optimality constraints, a feature required to compute pGCP and wGCP designs. This extension closes the gap between MCS-based and bilevel-based strain design approaches and enables computation (and comparison) of designs for all GCP classes within a single framework. By computing GCP strain designs for a range of products, we illustrate the hierarchical relationships between the different coupling degrees. We find that feasibility of coupling is not affected by the chosen GCP degree and that strongest coupling (SUCP) requires often only one or two more interventions than wGCP and dGCP. Finally, we show that the principle of coupling can be generalized to couple product synthesis with other cellular functions than growth, for example, with net ATP formation. This work provides important theoretical results and algorithmic developments and a unified terminology for computational strain design based on GCP.