Discovering a novel d-xylonate-responsive promoter: the PyjhI-driven genetic switch towards better 1,2,4-butanetriol production

Abstract

The capability of Escherichia coli to catabolize d-xylonate is a crucial component for building and optimizing the Dahms pathway. It relies on the inherent dehydratase and keto-acid aldolase activities of E. coli. Although the biochemical characteristics of these enzymes are known, their inherent expression regulation remains unclear. This knowledge is vital for the optimization of d-xylonate assimilation, especially in addressing the problem of d-xylonate accumulation, which hampers both cell growth and target product formation. In this report, molecular biology techniques and synthetic biology tools were combined to build a simple genetic switch controller for d-xylonate. First, quantitative and relative expression analysis of the gene clusters involved in d-xylonate catabolism were performed, revealing two d-xylonate-inducible operons, yagEF and yjhIHG. The 5′-flanking DNA sequence of these operons were then subjected to reporter gene assays which showed PyjhI to have low background activity and wide response range to d-xylonate. A PyjhI-driven synthetic genetic switch was then constructed containing feedback control to autoregulate d-xylonate accumulation and to activate the expression of the genes for 1,2,4-butanetriol (BTO) production. The genetic switch effectively reduced d-xylonate accumulation, which led to 31% BTO molar yield, the highest for direct microbial fermentation systems thus far. This genetic switch can be further modified and employed in the production of other compounds from d-xylose through the xylose oxidative pathway.