Construction and optimization of a multienzyme cascade system centered on carbamoyl phosphate synthesis module for efficient guanidinoacetic acid production

Abstract

Guanidinoacetic acid (GAA) production is limited by insufficient arginine supply and the accumulation of the byproduct ornithine. Here, we constructed a multi-enzyme cascade centered on carbamoyl phosphate synthesis to enable efficient GAA production through arginine regeneration and byproduct recycling. A structurally guided screen of arginine: glycine amidinotransferase mutants from Arabidopsis thaliana identified the AtE31K/G351N variant, which exhibited a 2.5-fold increase in enzyme activity (7.4 U/mg) and elevated GAA titer from 11.1 mM in wild-type to 15.2 mM. Metabolic flux analysis revealed carbamoyl phosphate as rate-limiting; introducing glutamine synthetase and carbamate kinase enhanced carbamoyl phosphate synthesis and cycle throughput. The eight-enzyme cascade was functionally co-expressed in Escherichia coli BL21 (DE3) through dual-plasmid systems (pRSFDuet-1 and pETDuet-1). After process optimization, 18.35 g/L GAA (156.75 mM) was produced via 60-hour bioconversion with the molar conversion rate of arginine reaching 261.12 %. This work establishes a robust platform for industrial-scale GAA production, and presents a broadly applicable strategy for the rational design and coordinated expression of complex multi-enzyme biosynthetic systems.