Abstract
2-Pyrrolidone is a valuable bulk chemical with myriad applications as a solvent, polymer precursor and active pharmaceutical intermediate. A novel 2-pyrrolidone synthase, ORF27, from Streptomyces aizunensis was identified to catalyze the ring closing dehydration of γ-aminobutyrate. ORF27's tendency to aggregate was resolved by expression at low temperature and fusion to the maltose binding protein (MBP). Recombinant Escherichia coli was metabolically engineered for the production of 2-pyrrolidone from glutamate by expressing both the genes encoding GadB, a glutamate decarboxylase, and ORF27. Incorporation of a GadB mutant lacking H465 and T466, GadB_ΔHT, improved the efficiency of one-pot 2-pyrrolidone biosynthesis in vivo. When the recombinant E. coli strain expressing the E. coli GadB_ΔHT mutant and the ORF27-MBP fusion was cultured in ZYM-5052 medium containing 9 g/L of l-glutamate, 7.7 g/L of l-glutamate was converted to 1.1 g/L of 2-pyrrolidone within 31 h, achieving 25% molar yield from the consumed substrate.