Multienzyme cascades for non-grain carbon feedstocks valorization: Recent advances and perspectives

Biomanufacturing is increasingly transitioning toward sustainable non-grain carbon feedstocks, including gaseous C1 compounds, liquid C1 substrates, and solid non-grain biomass. However, the efficient utilization of these feedstocks remains hindered by mass-transfer limitations, thermodynamic constraints, substrate and intermediate cytotoxicity, and the inherent physical limitations of macromolecular catalysts. Intracellular multienzyme cascade catalysis offers a promising solution by organizing highly coordinated catalytic networks within living cells, thereby enhancing metabolic efficiency and overcoming multiple bioconversion barriers. In this review, we systematically analyze the mechanisms and engineering strategies underlying whole-cell cascade systems from the integrated perspectives of material, energy, and information flows. Based on these principles, we propose a universal diagnostic and design framework for the bioconversion of non-grain carbon feedstocks. We further illustrate how these general design rules can be tailored and implemented at the systems level for the efficient conversion of specific substrate classes. In addition, we highlight the emerging role of artificial intelligence in enabling predictive system reconstruction through generative design, global pathway optimization, and automated design-build-test-learn (DBTL) cycles. Collectively, this review establishes a unified theoretical and practical framework for the rational design of next-generation biomanufacturing platforms based on intracellular multienzyme cascade catalysis.