Developing a Redox Imbalance Forces Drive (RIFD) strategy and its application in L-threonine production

Abstract

The design of synthetic driving forces for biosynthetic pathway is crucial for directing carbon flux toward the target product. Optimizing cellular redox status is one of the key strategies for constructing microbial cell factories. In this study, we attempt to create a novel redox imbalance force-driven (RIFD) strategy to direct carbon flow toward the target synthetic pathway. Initially, we increased the NADPH pool through a strategy of "open source and reduce expenditure" employing four approaches to achieve excessive NADPH levels and growth inhibition: (I) the expression of cofactor-converting enzymes, (II) the expression of heterologous cofactor-dependent enzymes, (III) the expression of enzymes involved in the NADPH synthesis pathway, and (IV) reduced NADPH wastage by knocking down non-essential genes that consume NADPH in vivo. Next, multiple automated genome engineering (MAGE) techniques were employed to evolve redox-imbalanced engineered strains and drive the metabolic flux to L-threonine production. Finally, we developed a NADPH and L-threonine dual-sensing biosensor, combined it with Fluorescence-Activated Cell Sorting (FACS), and a high-yield (0.65 g/g) L-threonine-producing strain with a titer of 117.65 g L^-1 was obtained. This research presents a general approach to increasing the production of cofactor-related products. Utilizing redox imbalance forces to drive metabolic flow toward the target product, it is possible to increase production while simultaneously restoring cell growth.

Keywords: Driving force; Escherichia coli; High-throughput screening; L-threonine; MAGE; Metabolic engineering; NADPH.