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

Abstract

SummaryFood 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.

Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Induces Immune Responses in Mice

“…Fusion of mZP3-1 to the GFP coding region, resulting in GFP-mZP3-1, led to a more than 25-fold higher accumulation of mZP3. GFP has already been used successfully to improve protein production and stability under various conditions [ 70 , 71 , 72 ]. This stabilization seems to depend not only on the GFP sequence, but also on the protein size since mZP3-3 showed a similar effect on the stability and accumulation of the protein.…”

Direct Delivery of Health Promoting β-Asp-Arg Dipeptides via Stable Co-expression of Cyanophycin and the Cyanophycinase CphE241 in Tobacco Plants

“…Nevertheless, in case the plants are feed without prior extraction, the enzyme has to be stable and effective in the digestive tract, and the persistence of the feed should be sufficient to provide high amounts the CGP. Ponndorf et al (2016) could show that CphB Te, is stable enough to degrade CGP in the digestive tract of mice (Ponndorf et al, 2016). We could also demonstrate that there are no detectable differences in protein stability between CphB Te and CphE241 and that CphE241 showed a drastically higher enzyme activity, both as purified proteins under in vitro conditions and in crude leaf extracts (Ponndorf et al, 2017).…”

“…Complete CGP degradation was only observed for CphE241, but not for CphB Te , which was assigned to a drastically higher enzyme activity of CphE241 (Ponndorf et al, 2017). In a feeding study with mice, in which purified plantproduced CGP and CphB Te were added to the diet, it could be shown that the CGPase is capable to degrade CGP in the digestive tract and that the β-Asp-Arg DPs were transferred to the blood (Ponndorf et al, 2016). This should also be true for CphE al , since this CGPase type naturally occurs in the colon (Sallam and Steinbuchel, 2009a) and since it has been demonstrated that CphE al is active in a pH range from 5 to 9 (Sallam and Steinbuchel, 2011).…”

“…To prevent premature CGP degradation and subsequent metabolizing of the resulting DPs, CGP, and CGPase need to be spatially separated. The production of both a CphBtype CGPase from T. elongatus BP-1 (CphB Te , 30.7 kDa) and a mutant of the CphE-type CGPase CphE al , termed as CphE241 (44.7 kDa), in the cytosol of plants was already shown via transient expression in tobacco (Ponndorf et al, 2016;Nausch and Broer, 2017). In addition, transient expression of both cytosolic enzymes in plastidic CGP-producing tobacco demonstrated that CGP was not degraded until the plant material was homogenized.…”