Economical Production of Creatine in Corynebacterium glutamicum via a Multisubstrate Self-Cycling System

Creatine (CR) is an essential energy substance that is widely used as a dietary supplement. Currently, industrial-scale biosynthesis of CR remains challenged by its low efficiency and high costs. Here, a cost-effective platform was developed in Corynebacterium glutamicum for high-yield CR production, achieving a remarkable 92.2% reduction in production costs. Through combinatorial enzyme screening and expression regulation, a heterologous pathway consisting of l-arginine: glycine amidinotransferase (AGAT) from Cylindrospermopsis raciborskii and guanidinoacetate methyltransferase (GAMT) from Homo sapiens was established and optimized. Next, implementation of the l-ornithine cycle coordinately enhanced l-arginine carbon fluxes from l-ornithine and alleviated feedback inhibition of l-ornithine for AGAT. Furthermore, an S-adenosyl-l-methionine (SAM) self-supply system was established, achieving a 66.2-fold increase in SAM levels by enhancing the SAM synthetic flux, establishing an adenosine-mediated adenosine-5′-triphosphate (ATP) regeneration cycle, and optimizing the recycling pathways of the byproduct S-adenosyl-homocysteine (SAH). The final strain demonstrated a CR titer of 16.8 g/L in a 5 L bioreactor with a productivity of 0.23 g/(L h), which represented the highest levels reported to date. This study presents a novel cost-effective strategy for CR synthesis through eliminating the reliance on costly and unsustainable substrates.