Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants

Neumann, Katrin; Stephan, D.; Ziegler, K.; Hühns, Maja; Broer, Inge; Lockau, Wolfgang; Pistorius, Elfriede K.

doi:10.1111/j.1467-7652.2005.00122.x

Cited by 82 publications

(71 citation statements)

References 44 publications

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“…Nevertheless, the antibodies generated provide a suitable tool for the detection of insoluble CGP from different organisms. The molecular mass distributions of CGP was between 20 and 35 kDa for the soluble CGP and between 26 and 45 kDa for the insoluble CGP and was similar to distributions observed with recombinant bacteria and plants (3,15,37). In contrast, in cyanobacteria, the apparent molecular masses were much higher, ranging up to 130 kDa (18).…”

Section: Discussionmentioning

confidence: 64%

“…Determination of the cell densities of transgenic yeasts harboring cphA 6308 yielded lower values than the negative controls without cphA, thereby indicating that the synthesis and accumulation of CGP resulted in slower growth. Such an inhibition due to CGP biosynthesis was recently reported in transgenic plants, too (37). However, a total CGP content of almost 7%, which was obtained without having varied the cultivation conditions, is still high in comparison to that of CGP-producing plants or some recombinant strains of C. glutamicum and P. putida (3,37).…”

Section: Discussionmentioning

confidence: 91%

“…This study describes for the first time the synthesis of CGP in recombinant yeast and, besides the recently described synthesis of CGP in plants (37), the only investigation of the production of this polymer in eukaryotic organisms. It was shown that both of the investigated S. cerevisiae strains were able to produce the polymer in considerable amounts.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, CGP production was also achieved for the first time in eukaryotic organisms. Transgenic tobacco plants accumulated up to 1.14% (wt/wt) and transgenic potato plants up to 0.24% (wt/wt) of watersoluble and water-insoluble CGP (37).…”

mentioning

confidence: 99%

“…CGP is a polydispersed polymer; the molecular size distribution of CGP varies with the producing host strains (1,15,18,20,30,37,52). Due to its branched structure, CGP is not degradable by a wide range of proteinases (45).…”

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confidence: 99%

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Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Steinle

Oppermann‐Sanio

Reichelt

et al. 2008

Appl Environ Microbiol

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Cyanophycin [multi-L-arginyl-poly(L-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae. The episomal vector systems pESC (with the galactoseinducible promoter GAL1) and pYEX-BX (with the copper ion-inducible promoter CUP1) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308 ) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA 6308 reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.Cyanophycin, also referred to as multi-L-arginyl-poly(L-aspartic acid) or cyanophycin granule polypeptide (CGP), is a nonribosomally synthesized polypeptide consisting of a poly-(aspartic acid) backbone with arginine residues linked to the ␤-carboxyl group of each aspartate by the ␣-amino group (45). CGP is a polydispersed polymer; the molecular size distribution of CGP varies with the producing host strains (1,15,18,20,30,37,52). Due to its branched structure, CGP is not degradable by a wide range of proteinases (45). Biosynthesis of CGP from aspartate and arginine requires only one enzyme, cyanophycin synthetase, which is encoded by cphA (51). CGP is insoluble at neutral pH and under physiological ionic strength, but it is soluble at low (Ͼ3) or high (Ͻ9) pH. Ziegler et al. were the first to observe a water-soluble form of CGP after the heterologous expression of cphA from Desulfitobacterium hafniense strain DSM 10664 in Escherichia coli (52). A detailed study of the solubility behavior of CGP isolated from recombinant E. coli in inorganic salts has been carried out by Füser and Steinbüchel (16). It was shown that the occurrence of the soluble form was not dependent on the origin of cphA or on the host.Recently, several putative applications for CGP and its derivatives have become available, indicating there is a need for its efficient biotechnological production (36,42,43). For the production of CGP at a technical scale, cyanobacteria were shown to be unsuitable due to their low cell...

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Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Steinle

Oppermann‐Sanio

Reichelt

et al. 2008

Appl Environ Microbiol

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Renewable Polymers in Transgenic Crop Plants

Hausmann

Broer

2011

Renewable Polymers

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Polymerases for Biosynthesis of Storage Compounds

Bröker

Steinbüchel

2010

Biocatalysis in Polymer Chemistry

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enzyme catalyzing the polymerization of two peptidoglycan precursors in bacterial cell wall biogenesis.1. The best investigated mechanism, distributed ubiquitously in living matter, is the template -dependent ribosomal synthesis of proteins. Here the amino acids are activated by adenylation catalyzed by aminoacyl -tRNA synthetases [1] .2. The second mechanism is performed by nonribosomal peptide synthetases ( NRPS ) which are multienzyme complexes consisting of four domains [2, 3] . The adenylation domain required for activation of the substrate at the expense of ATP, via the formation of an enzyme -stabilized aminoacyl adenylate. As the formed adenylate is not stable, the energy is further conserved by transfer of the peptide to: the thiolation domain giving rise to a thioester bond formed between the amino acid and a cysteine of the enzyme complex.The condensation domain is required for formation of peptide bonds between the itemized monomers, and the thioesterase domain catalyzes the release of the fi nal product from the NRPS by cyclization to an amide or ester, or by hydrolysis to the free acid [3] . The transfer of the intermediates is mediated by the cofactor pantetheine. Numerous peptide antibiotics such as penicillin, bacitracin, and actinomycin are synthesized by NRPS (reviewed by [2] ). These compounds are known to contain non -proteinogenic amino acids, d -amino acids, hydroxy acids, methylated or cyclic forms, and other unusual constituents; these modifi cations are catalyzed by the NRPS. As the enzyme complex itself functions as matrix, the resulting peptides have a strictly defi ned length which is in contrast to poly(amino acids).3. The third mechanism is represented by nonmodular one -step peptide synthesis. Enzymes belonging to this group catalyze the biosynthesis of poly(amino acids). Naturally occurring poly(amino acids) comprise cyanophycin [multil -arginyl -poly -( l -aspartic acid); cyanophycin granule polypeptide , ( CGP )], ( poly -( ε -lysine) ( PL ), and poly -( γ -glutamate) ( PGA ). As a consequence of non -ribosomal biosynthesis these peptides reveal a polydisperse mass distribution.Further aspects distinguishing poly(amino acids) from proteins are the following:proteins consist of a mixture of 22 amino acids, whereas poly(amino acids) consist of one amino acid in the polymer backbone;their biosynthesis is not constrained by translational inhibitors such as chloramphenicol [4] ;the amide bonds formed between the monomers are not exclusively linked between the α -carboxylic and α -amino groups, as for proteins, but also between β -or γ -carboxylic groups or ε -amino groups. Polyhydroxyalkanoate Synthases 249

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Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants

Cited by 82 publications

References 44 publications

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Renewable Polymers in Transgenic Crop Plants

Polymerases for Biosynthesis of Storage Compounds

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