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

Feed supplementation with ß-arginine-aspartate dipeptides (ß-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 ß-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 ß-Asp-Arg DP in field-grown plants.

Tobacco as platform for a commercial production of cyanophycin.

Cyanophycin (CP) is a proteinogenic polymer that can be substituted for petroleum in the production of plastic compounds and can also serve as a source of valuable dietary supplements. However, because there is no economically feasible system for large-scale industrial production, its application is limited. In order to develop a low-input system, CP-synthesis was established in the two commercial Nicotiana tabacum (N. tabacum) cultivars 'Badischer Geudertheimer' (BG) and 'Virginia Golta' (VG), by introducing the cyanophycin-synthetase gene from Thermosynecchococcus elongatus BP-1 (CphATe) either via crossbreeding with transgenic N. tabacum cv. Petit Havana SR1 (PH) T2 individual 51-3-2 or by agrobacterium-mediated transformation. Both in F1 hybrids (max. 9.4% CP/DW) and T0 transformants (max. 8.8% CP/DW), a substantial increase in CP content was achieved in leaf tissue, compared to a maximum of 1.7% CP/DW in PH T0 transformants of Hühns et al. (2008). In BG CP, yields were homogenous and there was no substantial difference in the variation of the CP content between primary transformants (T0), clones of T0 individuals, T1 siblings and F1 siblings of hybrids. Therefore, BG meets the requirements for establishing a master seed bank for continuous and reliable CP-production. In addition, it was shown that the polymer is not only stable in planta but also during silage, which simplifies storage of the harvest prior to isolation of CP.

Effect of ensiling treatment on secondary compounds and amino acid profile of tropical forage legumes, and implications for their pig feeding potential.

BACKGROUND: Smallholders in the tropics depend on local protein supplements to balance pig diets. Thus, various tropical forage legumes are a potential feeding option. Ensiling allows converting forages into a ready-to-feed-out choice, but the lactic acid fermentation may influence various (anti)nutritional components. The purpose of this study was to evaluate the effects of sucrose (SU) and a tropical Lactobacillus plantarum strain (LAB) as ensiling treatments (control, SU, LAB, LAB + SU) on the potential nutrient availability of 10 forage legume species. RESULTS: Ensiling commonly reduced antinutritional compounds such as tannins (by 49-84%) and trypsin inhibitory activity (by 74-78%), as well as oxalic acid (by 51-100%). An improved potential absorbability of protein and minerals for pigs is thus inferred. There was no major loss in total amino acids. In general, the species effect was stronger than the treatment effect. A clear effect of the treatments SU, LAB and LAB + SU over all 10 forage species was only observed for oxalic acid, single amino acids and trypsin inhibitory activity. CONCLUSION: Ensiling is a viable option to enhance nutrient utilization of tropical forages for pigs. Species-specific treatment of forage legumes is recommended.

Comparative evaluation of laboratory-scale silages using standard glass jar silages or vacuum-packed model silages.

BACKGROUND: The objective of this study was to compare the fermentation variables of laboratory-scale silages made in glass preserving jars (GLASS) and vacuum-packed plastic bags (Rostock model silages, ROMOS). Silages were prepared from perennial ryegrass (fresh and wilted, 151 g kg(-1) and 286 g kg(-1) dry matter (DM), respectively) and remoistened coarsely ground rye grain (650 g kg(-1) DM) either with or without the addition of a lactic acid bacteria inoculant (3×10(5) colony forming units (cfu) g(-1) , LAB). Quintuplicate silos were opened on days 2, 4, 8, 49 and 90. RESULTS: Silage pH (P=0.073), acetic acid content (P=0.608) and ethanol content (P=0.223) were not influenced by the ensiling method. The contents of DM (P<0.001) and propionic acid (P=0.008) were affected by the ensiling method, but mean differences were only marginal. In ROMOS the concentration of lactic acid was increased (P=0.007) whereas butyric acid was produced less (P=0.001) when compared to GLASS. This suggested slightly better ensiling conditions for ROMOS. CONCLUSIONS: ROMOS represents a reasonable alternative to glass jar silages and opens the possibility for further investigations, e.g. studying the impact of packing density as well as the quantitative and qualitative analysis of fermentation gases.