Metabolic Engineering of <i>Escherichia coli</i> for High-Yield Production of <scp>l</scp> -Homoserine Using Glycerol as a Sole Carbon Source

l-Homoserine, a nonproteinic amino acid with extensive applications, holds significant potential for sustainable production via a cell factory, particularly with the nongrain feedstock glycerol. Here, <i>Escherichia coli</i> was systematically engineered to enhance l-homoserine production using glycerol as an alternative carbon source. Initially, we truncated homoserine kinase (ThrB) to avoid l-homoserine degradation and support cell growth. Subsequently, the main synthesis pathway was strengthened by limited-step gene overexpression and protein scaffold strategies. With further reprogramming of the glycerol assimilation pathway and heterologous introduction of the Entner-Doudoroff pathway to balance cofactor supply, an l-homoserine hyperproducer HZ23/p99A_<i>rhtB</i> could achieve a titer of 90.21 g/L, with a yield of 0.49 g/g glycerol and a productivity of 1.50 g/L/h. Our study demonstrated the potential of glycerol as a sustainable feedstock for high-yield production of l-homoserine and provides a robust framework for the industrial-scale production of this important precursor.