| Summary: | Guanidinoacetate (GAA) is a naturally occurring amino acid derivative and the direct precursor of creatine, which is widely used in feed additives and the pharmaceutical industry. The current industrial synthesis of GAA is based on chemical methods, which limits the application of GAA. Here, a biological approach is developed for food safety GAA production via whole-cell biocatalysis by the generally regarded as safe (GRAS) bacterium <i>Bacillus subtilis</i>. First, we introduced a heterologous arginine: glycine amidinotransferase (AgaT) from <i>Amycolatopsis kentuckyensis</i> into <i>B. subtilis</i> and optimized its expression level using strategies including: promoter optimization, ribosome binding site (RBS) and N-terminal coding sequence (NCS) screening. In order to alleviate the waste of arginine and the inhibition of AgaT by ornithine, we optimized the natural ornithine cycle in <i>B. subtilis</i>. At the same time, the first gene in the glycine degradation pathway was knocked out. After optimization using these strategies, the titer of GAA was 4.26 g/L with a productivity of 0.21 g/L/h in 20 h, which provides a new method for the biosynthesis of GAA.
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