Reconstitution of Methionine Cycle With ATP Regeneration for Whole-Cell Catalysis of Creatine Production in Engineered Escherichia coli

Creatine (CR) is a naturally occurring amino acid derivative that plays a key role in cellular energy homeostasis, which has wide-ranging applications in food and medicine. Currently, the lack of green and sustainable CR biomanufacturing methods has led to reliance on chemical methods for industrial CR synthesis. This study presents a biological approach to synthesising CR using whole-cell catalysis by engineered Escherichia coli. First, through screening of critical enzymes from different sources and dual-enzyme co-expression strategies, arginine: glycine amidinotransferase (AGAT) from Amycolatopsis kentuckyensis and guanidinoacetate N-methyltransferase (GAMT) from Mus caroli were introduced to construct the CR biosynthesis pathway, yielding 0.83 g/L CR production. Then, the expression level of GAMT, the critical rate-limiting enzyme, was optimised by screening the ribosome binding site and N-terminal coding sequences, resulting in a 92% enhancement of CR production, reaching 1.59 g/L. Next, the endogenous ornithine and methionine cycles were further engineered to boost the synthesis of the precursor guanidinoacetate (GAA) and methyl donor S-adenosylmethionine (SAM), leading to a 68% increase in CR production, reaching 2.67 g/L. Finally, considering adenosine triphosphate (ATP) is required as a cofactor for SAM biosynthesis, we integrated the reconstitution methionine cycle with a polyphosphate kinase-based ATP regeneration system, achieving a CR titre of 5.27 g/L with a productivity of 0.22 g/L/h, and the molar conversion of substrate arginine was 71 mol% over 24 h following the engineering process. This study is the first report achieving whole-cell catalysis of CR production in engineered E. coli with a dual enzyme cascade using arginine as substrate, providing a new platform for CR production and insights into the biosynthesis of high-value metabolites that rely on ATP consumption.