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

Plant Biotechnol J. 2017 May;15(5):605-613. doi: 10.1111/pbi.12658. Epub 2016 Dec 18.


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 (CGPase) and results in the release of β-aspartic acid (Asp)-Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB 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 CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant-expressed CGP fed in combination with plant-made CGPase 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 CGPase 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.

Keywords: Nicotiana benthamiana; arginine; cyanophycin; cyanophycinase; digestion; dipeptide; protein stability.

MeSH terms

  • Animals
  • Arginine / pharmacokinetics
  • Bacterial Proteins / metabolism*
  • Biological Availability
  • Chloroplasts / genetics
  • Chloroplasts / metabolism
  • Cytosol / metabolism
  • Dietary Supplements
  • Dipeptides / pharmacokinetics
  • Hydrolysis
  • Intestinal Mucosa / metabolism*
  • Male
  • Mice
  • Nicotiana / genetics
  • Nicotiana / metabolism*
  • Peptide Hydrolases / genetics*
  • Peptide Hydrolases / metabolism*
  • Plant Extracts / chemistry
  • Plants, Genetically Modified


  • Bacterial Proteins
  • Dipeptides
  • Plant Extracts
  • cyanophycin
  • Arginine
  • Peptide Hydrolases
  • cyanophycinase