Design of a Continuous GAA-Producing Probiotic as a Potential Mitigator of the Effects of Sleep Deprivation

Creatine is a popular athletic supplement that has also been shown to improve cognitive performance upon sleep deprivation. However, it is rapidly cleared from the gastrointestinal tract a few hours after consumption. Toward providing a persistent creatine dose, we engineered the human probiotic <i><i>Escherichia coli</i></i> Nissle (EcN) to produce guanidinoacetic acid (GAA), which is converted to creatine in the liver. We find GAA-producing enzymes present in the human microbiome and compare their activities to known enzymes. Three copies of arginine:glycine amidinotransferase (AGAT) from <i>Actinokineospora terrae</i> are expressed from the genome, and native <i>gcvP</i>, <i>argR</i>, and <i>argA</i> are edited or deleted to improve substrate availability without negatively impacting cell viability. A standard EcN dose (10¹² cells) produces 41 ± 7 mg GAA per hour under laboratory conditions. This work demonstrates that a probiotic bacterium can be engineered to produce sustained GAA titers known to impact cognitive performance.