Tobacco as platform for a commercial production of cyanophycin

Nausch, Henrik; Hausmann, Tina; Ponndorf, Daniel; Hühns, Maja; Hoedtke, Sandra; Wolf, Petra; Zeyner, Annette; Broer, Inge

doi:10.1016/j.nbt.2016.08.001

Cited by 17 publications

(23 citation statements)

References 31 publications

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“…For super-transformation with the CphE241 encoding T-DNA, the T2 individual BG 176-4-3 of the one-copy event BG 176 (Nausch et al, 2016) was used. Tobacco seeds were surface sterilized in a saturated calcium hypochlorite solution, germinated on Linsmaier and Skoog (LS) medium (4.4 g/L of LS medium including vitamins (Duchefa, Haarlem, Netherlands), 30 g/L sucrose, 6.5 g/L plant agar (Duchefa), [pH 5.7].…”

Section: Plant Materials and Transformationmentioning

confidence: 99%

“…Cyanophycin was quantified as described by Nausch et al (2016) which bases upon the method of Simon (1973). Three samples per individual plant of 30-35 mg of freeze-dried leaf material were homogenized with ceramic pills in a Precellys 24 homogenisator (VWR International GmbH, Erlangen, Germany) and incubated in 1 mL 50 mM Tris [pH 8.0] for 30 min.…”

Section: Cgp Quantificationmentioning

confidence: 99%

“…In order to make the AA stored in CGP bioavailable, we aimed to co-express a CGPase. For coexpression of the CGPase CphE241 in tobacco with plastidic CGP-production, we selected T2 plants of BG transformant (event) 176, described in our previous study (Nausch et al, 2016), because (1) this line carries one-copy of CGP-synthetase encoding transgene, (2) was bred to homozygosity, and (3) has a homogenous and reliable CGP-accumulation of ∼2.5% CGP/leaf DW. These T2 plants were super-transformed with the gene for CphE241syn under the control of the constitutive CaMV 35s (pLH7000-35s-CphE241syn, Figure 1), using the bar gene and the herbicide phosphinothricin as selection system.…”

Section: Cphe241 Can Be Co-expressed In High Amounts In Cgp-producing Plantsmentioning

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%

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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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Section: Plant Materials and Transformationmentioning

confidence: 99%

Section: Cgp Quantificationmentioning

confidence: 99%

Section: Cphe241 Can Be Co-expressed In High Amounts In Cgp-producing Plantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…No entanto, o gene AccA possui um sítio próprio de início da transcrição (SIT) identificado em várias linhagens cianobacterianas, semelhante à separação transcricional de Aar/Ado e possivelmente indicando um regulamento independente (KLAHN et al, 2014). Entanto, a enzima desidrogenase de cadeia curta pertenceu à família SDR de oxidoredutases dependentes de NAD ou NADP, similar a 3-oxoacil-ACP redutase (EC NAUSCH et al, 2016;BROER, 2017;ARAHAL et al, 2018; LI; ZHOU, 2019a). Cianoficina (ácido multi-l-arginil-poli-l-aspártico) é uma poliamida de reserva rica em nitrogênio/ carbono, distribuida em várias linhagens de cianobactérias e algumas bactérias heterotróficas (WATZER; FORCHHAMMER, 2018), e estratégicamente armazenada durante a noite MONTI;LUBENSKY;WOLDE, 2018).…”

Section: Hidrocarbonetos Derivados De áCidos Graxosunclassified

Taxonomia, genômica e potencial funcional da cianobactéria >i<Oxynema>/i< sp. CENA135

Castillo¹,

Angélica²

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e em especial a Fernanda Rios Jacinavicius pela guia, ensinamentos e disponibilidade na realização de análises. Às funcionarias do laboratório Ana Paula Dini Andreote e Renata Beatriz Cruz, pelas distintas atividades que realizam no laboratório e ainda assim me forneceram apoio. Aos colegas do laboratório, amigos, com todo carinho e gratidão para Bruno Evangelista de Souza, Taiane Fernanda Barradas, Endrews Delbaje, Caroline Rosa de Carvalho e Rafael Barty Dextro. Em especial para Juliana dos Santos Costa e Thierry Alexandre Pellegrinetti pelo companherismo e o suporte em momentos cinzentos. A nossa Pós-doutoranda Simone Cotta e às estagiárias Julia Polezi Formagio e Lara Losovoi pela juda recebida. Ao pesquisador Danillo Alvarenga pelos conselhos fornecidos A Guilherme K. Hosaka, pelo amor, amizade, companhia, ensinamentos, apoio inestimável durante minha formação, e pelo Pisco. A sua familia pelas atenções recebidas e as sempre interessantes conversas culturais. A Rodolfo Silva e Fernando Matias, pela amizade, apoio e aventuras neste lindo país. Ao Programa de Pós-Graduação em Microbiologia Agrícola e ao Serviço da Pós-Graduação, em especial a Maria Solizete Granziol Silva e Alexandre Joviniano dos Santos, por todo o auxílio e orientação fornecida. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Código de Financiamento 001 ABSTRACT Taxonomy, genomics and functional potential of the cyanobacteria Oxynema sp. CENA135 Oxynema is a derivated genus from the polyphyletic Phormidium and with few registered species, which were found in mangroves, marine or high salinity environments. The taxonomic and genomic informations are almost nonexisting for this genus, with a few exceptions of 16S rRNA and 16S-23S ITS sequences available at databases. Previous studies performed with Oxynema sp. CENA135 isolated from a Brazilian mangrove showed its potential to synthesize siderophores, aliphatic hydrocarbons, anticancer and antibiotic substances, besides its effective degradation of some types of textile dyes. In this work, the strain Oxynema sp. CENA135 was analyzed under a polyphasic approach (morphology, habitat, phylogenomics, phylogeny of the 16S rRNA gene and secondary structure of 16S-23S ITS), including the search for genes or gene clusters related to the biosynthesis of natural products and the detection of associated bacteria. After morphological characterization using an optic and a scanning microscopes, the genomic sequencing was performed from a mate pair library at the HiSeq (Illumina) platform. This is the first genome of the genus Oxynema and, therefore, for its assembly it was used a de novo method resulting in 11 scaffolds in a total size of 6.2 Mbp, with N50 of 3,591,108 pb, GC content of 51.6% and average coverage of 190x. The checkM software estimated a high completeness of the genome (99.29%) and few contaminations (1.22%). Genome mining revealed a biorremediation potential due to the presence of genes related to resistance of copper,...

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Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts

Nausch

Broer

2016

Appl Microbiol Biotechnol

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One of the major constraints in pig and poultry farming is the supply of protein-rich forage, containing sufficient amounts of key amino acids such as arginine (Ufaz and Galili 2008). Since these are underrepresented in plant proteins, the usage of plants as feed is limited. The heterologous production of the cyanobacterial storage polymer cyanophycin granule polypeptide (CGP) in plastids increases the amount of arginine substantially (Huhns et al. 2008; Huhns et al. 2009; Nausch et al. 2016a). CGP degradation releases arginine-aspartate dipeptides. CGP is stable in plants because its degradation is exclusively restricted to bacterial cyanophycinases (CGPases; Law et al. 2009). Since animals are also unable to digest CGP, CGPases need to be co-delivered with CGP-containing plant feed in order to release the dipeptides in the gastrointestinal tract of animals during digestion. Therefore, an extracellular CGPase, CphE from Pseudomonas alcaligenes DIP-1, was targeted to the cytosol, ER, and apoplasm of Nicotiana benthamiana. Translocation to the chloroplast was not successful. Although CphE accumulated in high amounts in the cytosol, only moderate levels were present in the ER, while the enzyme was nearly undetectable in the apoplasm. This correlates with the higher instability of post-translationally modified CphE in crude plant extracts. In addition, the production in the ER led to an increased number and size of necroses compared with cytosolic expression and might therefore interfere with the endogenous metabolism in the ER. Due to the high and robust enzyme activity, even moderate CphE concentrations were sufficient to degrade CGP in plant extracts.

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Tobacco as platform for a commercial production of cyanophycin

Cited by 17 publications

References 31 publications

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

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

Taxonomia, genômica e potencial funcional da cianobactéria >i<Oxynema>/i< sp. CENA135

Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts

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