Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine

Summary Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin ( CGP ), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase ( CGP ase) and results in the release of β‐aspartic acid (Asp)‐Arg dipeptides. The simultaneous production of CGP and CGP ase in plants could be a convenient source of Arg dipeptides. Different variants of the cph B coding region from Thermosynechococcus elongatus BP ‐1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGP ase. Protein stability was increased by the translational fusion of CGP ase to the green fluorescent protein ( GFP ) or to the transit peptide of the small subunit of RuBis CO for peptide production in the chloroplasts. Studies in mice showed that plant‐expressed CGP fed in combination with plant‐made CGP ase was hydrolysed in the intestine, and high levels of ß‐Asp‐Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGP ase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes.