Enhanced Production of Leucine-Leucine Dipeptide by Whole-Cell Catalysis

The Leucine-Leucine dipeptide (Leu-Leu) is recognized for its ability to enhance muscle growth, promote recovery, and reduce fatigue, demonstrating therapeutic potential for conditions such as sarcopenia and metabolic disorders. Recent research in the biosynthesis of the Leu-Leu has gained increasing attention; however, its production is hampered by low catalytic efficiency, hydrolysis of the peptide bond as side reaction, and the generation of tripeptides as byproducts. In this study, a novel L-amino acid ligase (Lal) from Pseudomonas sessilinigenes was identified through data mining. This ligase effectively catalyzes the conversion of l-Leu to the Leu-Leu, exhibiting a k_cat/K_m value of 14.6 s^-1 M^-1 and a melting temperature of 57 °C. Furthermore, the disruption of four endogenous peptidases in Escherichia coli (E. coli) led to the accumulation of Leu-Leu dipeptide via whole-cell catalysis. The regeneration of adenosine 5'-triphosphate (ATP) is facilitated by the simultaneous action of a polyphosphate kinase derived from Erysipelotrichaceaebacterium. The engineered E. coli chassis, which coexpresses the L-amino acid ligase and polyphosphate kinase, achieves a space-time yield of 23.85 g/L/d in an ATP self-sufficient manner under optimized conditions. Our study enhances the capabilities of Lals in the synthesis of dipeptides and highlights their potential application for the overproduction of the Leu-Leu dipeptide.