biostudies-literatureSheng YNational Outstanding Youth Science Fund Project of National Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaFoundation for Innovative Research Groups of the National Natural Science Foundation of ChinaJiangsu Basic Research Center for Synthetic BiologyNational Science Fund for Excellent Young ScholarsNatural Science Foundation of Jiangsu Provincee70145https://www.ebi.ac.uk/biostudies/studies/S-EPMC12093341biostudies-literatureUnknown18(5)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 leMicrobial biotechnologyReconstitution of Methionine Cycle With ATP Regeneration for Whole-Cell Catalysis of Creatine Production in Engineered Escherichia coli.PMC12093341BK202330033217234932021005BK2020200232222069Liu YSheng YLi JLiu LChen JLv XDu GZhang LWu YfalseReconstitution 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 le2025-01-01T00:00:00Z2025 May2026-07-15T05:03:42.722Z2026-06-30T03:08:25.259ZS-EPMC120933414039656610.1111/1751-7915.70145