Orthogonal Design of Metabolic Pathways

Traditional metabolic engineering strives to achieve overproduction of target metabolites through growth coupling strategies that alter these structures and functions in such a way that neither cell growth nor metabolite production is optimal due to extensive network interactions. Hence, we are motivated to explore an alternative philosophy that prioritizes optimal network structures for metabolite production over cell growth. Our antithetical approach aims to achieve mutually exclusive operation of pathways responsible for cell growth and metabolite production by minimizing interactions between these pathways.

 We show that it is possible to obtain optimal metabolite production through engineered orthogonal pathways that are not optimal for cell growth by reducing the number of shared enzymatic steps between pathways producing biomass precursors and those that produce the metabolite. We found succinate production through the Embden-Meyerhoff-Parnas, the Entner-Doudoroff and the methylglyoxal bypass to have orthogonality values of 0.42, 0.46 and 0.40 respectively compared to production through the synthetic pathway that had an orthogonality of 0.52. Consequentially, we establish that growth coupling, which aims to improve connectivity between the production and growth components of a metabolic network, lowers the orthogonality of the resulting network, characterized by increased interactions between growth and metabolite production.

 We present an algorithmic approach to engineering orthogonal pathways using a cut sets based method. To facilitate switching between growth-independent production and production-independent growth phases, a metabolic valve reaction is connected to a node that acts as a branching point between pathways that serve as growth precursors and those that produce the target metabolite. Turning off the metabolic valve would result in true orthogonal pathways and could be an attractive target for two phase fermentation processes and dynamic metabolic engineering. We found isocitrate dehydrogenase to be this valve reaction for succinate production from glucose.

 Constraining cellular pathways by the chosen substrate-product pair also constraints the quantity and quality of interactions witnessed within the production network. By making orthogonal pathways dependent on the chosen substrate-product pairing, we have enabled de novo engineering of pathways for growth independent optimal production from many non-conventional substrates that are not glucose. We show that using xylose instead of glucose, to produce succinic acid minimizes the interactions prevalent in natural pathways to obtain orthogonal production pathways. The orthogonality of the xylose-based production pathway (0.59) supports this statement.

 Hence, our proposed orthogonal pathways could bring about a reduction in the number of interactions within the network without necessitating an equivalent number of gene deletions while also delivering greater independence between the production and growth components of metabolism. In doing so, it enables substrate-dependent design of cell factories to leverage the use of several substrates previously thought be unsuitable. Through our process, we have also developed a qualitative assessment of the ability of a given metabolic network to support the two different objectives of cell growth and metabolite production as a measure of connectivity within the metabolic network