Synthetic Biology and Systems Metabolic Engineering of Aminoglycoside Antibiotics: From Biosynthetic Pathways to Resistance-Evading and Low-Toxicity Design

Aminoglycoside antibiotics are important therapeutic agents in the clinical treatment of Gram-negative bacterial infections; however, the spread of antimicrobial resistance, together with dose-limiting ototoxicity and nephrotoxicity side effects, substantially restricts their clinical utility. Recent advances in synthetic biology and systems metabolic engineering have provided powerful approaches for elucidating and engineering the biosynthetic pathways of these antibiotics. This review systematically summarizes the conserved biosynthetic logic of aminoglycosides, with particular emphasis on the 2-deoxystreptamine and streptidine core scaffold assembly and on the catalytic features and substrate selectivity of four major enzyme families involved in aminoglycoside biosynthesis: glycosyltransferases, pyridoxal phosphate-dependent aminotransferases, S-adenosyl-L-methionine-dependent methyltransferases, and phosphorylation/deamination enzymes. On this basis, the core strategies for synthetic biology and systems metabolic engineering of aminoglycoside antibiotics are reviewed from five perspectives: genetic circuit design, chassis cell optimization, cell-free synthesis, heterologous de novo biosynthesis, and systems biology-driven global optimization. Using gentamicin, kanamycin, tobramycin, apramycin, neomycin, and streptomycin as representative examples, we highlight the distinctive features of their respective biosynthetic pathways and discuss their potential for synthetic biology-based exploitation. Particular attention is given to the development of novel derivatives, including resistance-evading derivatives such as plazomicin, low-toxicity designs based on 4-monosubstituted apramycin structures, and advances in combinatorial biosynthesis for generating hybrid antibiotics. Finally, key challenges are examined, including heterologous expression compatibility, bottlenecks in product yield, and an incomplete understanding of structure–activity relationships. Emerging strategies for overcoming these challenges are also discussed, with a focus on cutting-edge technologies such as artificial intelligence-driven dynamic regulation, genome-scale metabolic model-guided design, and cell-free synthesis systems.