Synthetic Cofactor-Linked Metabolic Circuits for Selective Energy Transfer

Abstract

A cellular energy-transfer process can be analogized as the running of an energy carrier (EC)-linked metabolic circuit between an energy-supplying module (ESM) and an energy-utilizing module (EUM). Because natural EC such as the reduced nicotinamide adenosine dinucleotide (NAD) links multiple energy-transfer modules and metabolic circuits, and the formation of natural EC is routinely coupled with the transformation of endogenous substances, it is challenging to transfer energy selectively. Here we devise synthetic cofactor-linked circuits for pathway-selective energy transfer. We engineer phosphite dehydrogenase as ESM to use the synthetic cofactor nicotinamide cytosine dinucleotide (NCD). We construct diverse circuits in vitro by combining different ESM, EUM, and EC, and we demonstrate that an energy-transfer process is controllable by tuning the feature of each component of the circuit. More specifically, we show that it is possible to drive the NCD-linked subsystem while leaving the NAD-linked reaction virtually unaffected. When armed with such circuits, Escherichia coli cells used phosphite as the electron source to generate reduced NCD that drove reductive carboxylation of pyruvate for improved malate production from glucose. Together, this study provides an opportunity to establish an orthogonal energy-transfer system for engineering cell factories and may be used to set an additional layer of a control mechanism for life.