Abstract
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 Bacillus subtilis. First, we introduced a heterologous arginine: glycine amidinotransferase (AgaT) from Amycolatopsis kentuckyensis into B. subtilis 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 B. subtilis. 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.
Highlights
To convert glycine and arginine to GAA, agaT was first introduced into B. subtilis
The plasmid was transformed into wild-type B. subtilis of168, a strong promoter
The results showed that these eight single colonies had completely different mutations in both ribosome binding site (RBS) and N-terminal coding sequence (NCS) of agaT (Table 3)
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Guanidinoacetate (GAA) has the physiological functions of promoting insulin secretion, influencing neuromodulation, changing arginine metabolism, etc. It is widely used as an intermediate in pharmaceutical organic synthesis; GAA can promote energy metabolism and improve antioxidant capacity, so it is often used in feed additives and food additives [1,2]. Creatine has an important role in the intracellular energy transport process, assisting in providing energy to muscle and nerve cells [3]
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