Reconstruction of a Cofactor Self-Sufficient Whole-Cell Biocatalyst System for Efficient Biosynthesis of Allitol from d-Glucose.

Abstract

The combined catalysis of glucose isomerase (GI), d-psicose 3-epimerase (DPEase), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) provides a convenient route for the biosynthesis of allitol from d-glucose; however, the low catalytic efficiency restricts its industrial applications. Here, the supplementation of 0.32 g/L NAD+ significantly promoted the cell catalytic activity by 1.18-fold, suggesting that the insufficient intracellular NAD(H) content was a limiting factor in allitol production. Glucose dehydrogenase (GDH) with 18.13-fold higher activity than FDH was used for reconstructing a cofactor self-sufficient system, which was combined with the overexpression of the rate-limiting genes involved in NAD+ salvage metabolic flow to expand the available intracellular NAD(H) pool. Then, the multienzyme self-assembly system with SpyTag and SpyCatcher effectively channeled intermediates, leading to an 81.1% increase in allitol titer to 15.03 g/L from 25 g/L d-glucose. This study provided a facilitated strategy for large-scale and efficient biosynthesis of allitol from a low-cost substrate.