Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression

Neubauer, Katja; Hühns, Maja; Hausmann, Tina; Klemke, Friederike; Lockau, Wolfgang; Kahmann, Uwe; Pistorius, Elfriede K.; Kragl, Udo; Broer, Inge

doi:10.1016/j.jbiotec.2011.12.008

Cited by 18 publications

(7 citation statements)

References 47 publications

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“…CGP-containing tobacco leaves can be stored in a dried form or as silage without loss of CGP ( Nausch et al, 2016 ). Isolation of plant-made CGP on a small scale has been reported for tobacco and potato, achieving purities of 58–90%, respectively ( Neubauer et al, 2012 ). This method is based on acid extraction on lyophilized starting material.…”

Section: Introductionmentioning

confidence: 99%

Sustainable Production of the Cyanophycin Biopolymer in Tobacco in the Greenhouse and Field

Huckauf

Brandt

Dezar³

et al. 2022

Front. Bioeng. Biotechnol.

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The production of biodegradable polymers as coproducts of other commercially relevant plant components can be a sustainable strategy to decrease the carbon footprint and increase the commercial value of a plant. The biodegradable polymer cyanophycin granular polypeptide (CGP) was expressed in the leaves of a commercial tobacco variety, whose seeds can serve as a source for biofuel and feed. In T0 generation in the greenhouse, up to 11% of the leaf dry weight corresponded to the CGP. In T1 generation, the maximum content decreased to approximately 4% dw, both in the greenhouse and first field trial. In the field, a maximum harvest of 4 g CGP/plant could be obtained. Independent of the CGP content, most transgenic plants exhibited a slight yield penalty in the leaf biomass, especially under stress conditions in greenhouse and field trials. After the harvest, the leaves were either Sun dried or ensiled. The resulting material was used to evaluate the extraction of CGP compared to that in the laboratory protocol. The farm-level analysis indicates that the extraction of CGP from tobacco plants can provide alternative income opportunities for tobacco farmers. The CGP yield/ha indicates that the CGP production in plants can be economically feasible depending on the cultivation and extraction costs. Moreover, we analyzed the consumer acceptance of potential applications associated with GM tobacco in four European countries (Germany, Finland, Italy and the Netherlands) and found unexpectedly high acceptance.

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Section: Introductionmentioning

confidence: 99%

Sustainable Production of the Cyanophycin Biopolymer in Tobacco in the Greenhouse and Field

Huckauf

Brandt

Dezar³

et al. 2022

Front. Bioeng. Biotechnol.

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“…CGP was isolated from Solanum tuberosum tubers (PsbY‐cphA TE ‐12) as described by Neubauer et al . (), and 10 mg was dissolved in 1 mL 0.1 m HCL pH < 2. CGPase was isolated from E. coli BL21 cells carrying pET22b‐ cph B‐his and N. benthamiana leaves using Ni 2+ NTA affinity chromatography as described in detail in the supplementary information (Data S1).…”

Section: Methodsmentioning

confidence: 99%

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

Ponndorf

Ehmke

Walliser

et al. 2016

Plant Biotechnology Journal

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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.

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“…A calibration curve was prepared with purified CGP from potato tubers, isolated as described by Neubauer et al (2012). OD values of leaf samples from non-transgenic plants were subtracted from OD values of samples of non-transgenic nearisogenic control plants.…”

Section: Cgp Quantificationmentioning

confidence: 99%

“…Studies estimated that the production costs are on average less than a tenth compared to conventional microbial and mammalian cell cultures (Broz et al, 2013;Tusé et al, 2014;Chen, 2015;Gecchele et al, 2015;Walwyn et al, 2015;Dirisala et al, 2016;Nandi et al, 2016;Buyel et al, 2017). The synthesis of CGP in plants was established via expression of the CGP-synthetase gene from Thermosynechococcus elongatus BP-1 (cphA Te ) in plastids of stably transformed tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants, yielding up to 9.4% leaf DW in commercial tobacco cultivars (Neumann et al, 2005;Hühns et al, 2008Hühns et al, , 2009Neubauer et al, 2012;Nausch et al, 2016). Plastid targeting was achieved via fusion of the coding region of cphA Te to the transit peptide of the integral protein of photosystem II PsbY (Hühns et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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 fieldgrown 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.

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Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression

Cited by 18 publications

References 47 publications

Sustainable Production of the Cyanophycin Biopolymer in Tobacco in the Greenhouse and Field

Sustainable Production of the Cyanophycin Biopolymer in Tobacco in the Greenhouse and Field

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

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

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