Metabolic engineering-driven precursor accumulation for enhanced biosynthesis of O-succinyl-L-homoserine via L-aspartate and L-homoserine pathway optimization

Abstract

O-succinyl-L-homoserine (OSH) is a critical intermediate in L-homoserine synthesis and serves as a precursor for L-methionine, which is essential for various biosynthetic processes. This study employs Escherichia coli W3110 as the starting strain to enhance OSH production by eliminating competing and degrading pathways, while simultaneously promoting the accumulation of precursor substances. Initially, we optimized the phosphoenolpyruvate-pyruvate-oxaloacetate pathway through the overexpression of the genes ppc, aspC, aspA, and gdhA*, which significantly increased the supply of L-aspartate. In addition, we adjusted the promoters of the key enzyme genes, metA11 and yjeH, involved in the primary OSH synthesis pathway, resulting in the creation of strain OSH23. The engineered strain demonstrated notable improvements in OSH titer, yield, and productivity in a 5 L fermenter, achieving values of 131.99 g/L, 0.49 g/g, and 1.14 g/L/h, respectively. Further overexpression of thrA^fbr enhanced carbon flux, yielding strain OSH24 which produced 120.32 g/L OSH in a 5-L bioreactor with a glucose conversion of 0.51 g/g and productivity of 1.18 g/L/h. When the fermentation scale was scaled up to 50 liters, OSH24 achieved a final OSH concentration of 131.06 g/L, while maintaining a sugar acid conversion rate of 0.51 g/g. This study not only illustrates the successful application of advanced metabolic engineering for high-yield OSH production but also establishes a robust foundation for its industrial application in L-methionine biosynthesis.