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

Henrik Nausch; Mandy Dorn; Andrej Frolov; Sandra Hoedtke; Petra Wolf; Inge Broer

doi:10.3389/fpls.2020.00842

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

Front Plant Sci. 2020 Jun 19:11:842. doi: 10.3389/fpls.2020.00842. eCollection 2020.

Authors

Henrik Nausch¹, Mandy Dorn², Andrej Frolov^{2

3}, Sandra Hoedtke⁴, Petra Wolf⁴, Inge Broer¹

Affiliations

¹ Department of Agrobiotechnology and Risk Assessment for Bio- und Gene Technology, Faculty of Agricultural and Environmental Sciences, University of Rostock, Rostock, Germany.
² Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
³ Department of Biochemistry, Saint Petersburg State University, Saint Petersburg, Russia.
⁴ Department of Nutrition Physiology and Animal Nutrition, Faculty of Agricultural and Environmental Sciences, University of Rostock, Rostock, Germany.

Abstract

Feed supplementation with β-arginine-aspartate dipeptides (β-Asp-Arg DP) shows growth promoting effects in feeding trials with fish and might also be beneficial for pig and poultry farming. Currently, these DPs are generated from purified cyanophycin (CGP), with the help of the CGP-degrading enzyme cyanophycinase (CGPase). As alternative to an in vitro production, the DPs might be directly produced in feed crops. We already demonstrated that CGP can be produced in plastids of tobacco and potato, yielding up to 9.4% of the dry weight (DW). We also transiently co-expressed CGPases in the cytosol without degrading CGP in intact cells, while degradation occurs in the homogenized plant tissue. However, transient co-expression is not feasible for field-grown CGP plants, which is necessary for bulk production. In the present study, we proved that stable co-expression of the CGPase CphE241 in CGP-producing tobacco is sufficient to degrade 2.0% CGP/DW nearly completely within 3 h after homogenization of the leaves. In intact senescing leaves, CGP is partially released to the cytosol and degraded into DPs which limits the overall accumulation of CGP but not the level of the stable DPs. Even after 48 h, 54 μmol β-Asp-Arg DP/g DW could be detected in the extract, which correspond to 1.5% DP/DW and represents 84% of the expected amount. Thus, we developed a system for the production of β-Asp-Arg DP in field-grown plants.

Keywords: Nicotiana tabacum; co-expression; cyanophycin; cyanophycinase CphE; β-Asp-Arg dipeptides.