Dual-opto-CRISPRi-mediated dynamic regulation of fructose metabolism for high-efficiency D-allulose biosynthesis in E. coli

D-allulose, a low-caloric sweetener, represents a promising alternative to sucrose across diverse applications. In contrast to enzymatic production, fermentation-based approaches coupled with metabolic engineering strategies present a more economically viable and operationally streamlined production route. Nevertheless, traditional regulatory tools, including gene deletion and isopropyl β-D-thiogalactoside (IPTG)-mediated induction, exhibit limitations in achieving precise temporal control over metabolic processes. Herein, two mutually orthogonal optogenetic systems were combined with clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology to enable the spatiotemporal control of cellular behavior and dynamically reallocate metabolic resources. Specifically, the YF1/FixJ and iLight-ho1 systems were employed in the CRISPRi/dCpf1 system, with protein production triggered by blue light and dark conditions, respectively, to redirect metabolic pathways towards D-allulose production, enables precise balancing without the metabolic fragility caused by gene knockout. Consequently, D-allulose production achieved a titer of 5.924 g/L, indicating a 3.26-fold increase compared to the unregulated control, with a productivity of 0.197 g/L/h and an in vitro conversion yield (the intracellular D-allulose to residual D-fructose ratio) of 75.8 %. These represented the highest titer, productivity and in vitro conversion yield of D-allulose from D-fructose by fermentation reported to date. The findings validate the dual-opto-CRISPRi system as an efficient metabolic engineering strategy for enhancing product yields, highlighting its potential for broader industrial applications.