Directed evolution and metabolic engineering generate an Escherichia coli cell factory for de novo production of 4-hydroxymandelate.

ABSTRACT

4-hydroxymandelate is a high-value aromatic compound used in the medicine, cosmetics, food, and chemical industry. However, existing natural extraction and chemical synthesis methods are costly and lead to environmental pollution. This study employed metabolic engineering and directed evolution strategies for de novo 4-hydroxymandelate biosynthesis. Two key challenges were addressed insufficient precursor supply and limited activity of crucial enzymes. Through gene overexpression and multi-level gene interference using CRISPRi, An Escherichia coli chassis capable of producing the key precursor 4-hydroxyphenylpyruvate and the titer reached 5.05 mM (0.91 g/L). A mutant clone was obtained, HmaSV152G, which showed a 5.13-fold improvement in the catalytic rate. During fermentation, a high production of 194.87 mM (32.768 g/L) 4-hydroxymandelate was achieved in 76 h with a batch supply of glucose in a 5-L bioreactor. This study demonstrated the great potential of biosensors in protein engineering and provides a reference for large-scale production of other high-value aromatic compounds.