Metabolic engineering of Escherichia coli for highly efficient N-acetylneuraminic acid production

The overflow of carbon to acetate is often encountered in Escherichia coli cell factories, especially when the target products require PEP as their precursor. The accumulation of acetate exerts a pronounced inhibitory effect on both cell growth and biosynthesis efficiency, which significantly constrains the application of E. coli cell factories. The biosynthesis of N-acetylneuraminic acid (Neu5Ac) represents such a challenging process that requires PEP as a precursor, which has become an attractive value-added chemical with growing applications in various areas. In this study, we employ the biosynthesis of Neu5Ac as a model to systematically explore strategies for optimizing PEP distribution in the synthesis of the target product, carbon source assimilation, and central carbon metabolism pathways. Additionally, we aim to reduce acetate accumulation and achieve highly efficient production of the target product. Through the combined implementation of metabolic engineering strategies targeting the acetate synthesis pathway, along with the optimization of fermentation temperature and ATP supply, we successfully achieved a significant reduction and delay in acetate accumulation. The final strain NEA-27 produced 77.12 g/L Neu5Ac at 56 h in a 5 L bioreactor at a fermentation temperature of 30 ℃, with a yield and productivity of 0.217 g/g glucose and 1.38 g/L/h, respectively. This is the most efficient de novo production of Neu5Ac ever reported and shows great potential in large-scale application. These strategies offer critical mechanistic insights into harnessing PEP as a metabolic precursor for heterologous biosynthesis of value-added chemicals.