Heterologous Expression of Cyanophycin Synthetase and Cyanophycin Synthesis in the Industrial Relevant Bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida

Aboulmagd, Elsayed; Voß, Ingo; Oppermann‐Sanio, Fred Bernd; Steinbüchel, Alexander

doi:10.1021/bm010075a

Cited by 71 publications

(69 citation statements)

References 39 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: Discussionsupporting

confidence: 77%

“…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). Through slight modifications of the cultivation conditions, the CDM average was increased 1.5-fold for strains harboring pYEX-BX::cphA 6308 , and interestingly, soluble CGP was no longer produced when the CGP constituents aspartate and arginine were added to the medium.…”

Section: Discussionmentioning

confidence: 81%

“…For the production of CGP at a technical scale, cyanobacteria were shown to be unsuitable due to their low cell densities and polymer contents (3.5% [wt/wt]) and slow growth and circumstantial growth conditions in a photobioreactor (18,19 Also, in the industrially relevant bacteria Pseudomonas putida, Ralstonia eutropha, and Corynebacterium glutamicum, considerable amounts of CGP could be produced after heterologous expression of cphA (3,13,48,49). Recently, CGP production was also achieved for the first time in eukaryotic organisms.…”

mentioning

confidence: 99%

“…In contrast, much higher amounts of the polymer were produced with the heterotrophic bacterium Acinetobacter baylyi strain ADP1 (46% [wt/wt]) (14) and with recombinant strains of E. coli (24% [wt/wt] [15] and 34.5% [wt/wt] [21], respectively). Also, in the industrially relevant bacteria Pseudomonas putida, Ralstonia eutropha, and Corynebacterium glutamicum, considerable amounts of CGP could be produced after heterologous expression of cphA (3,13,48,49). Recently, CGP production was also achieved for the first time in eukaryotic organisms.…”

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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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: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 81%

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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“…By the same mechanism, the C-terminal active site connects the b-carboxyl group of the C-terminal aspartic acid residue to the a-amino group of arginine, thereby forming an isopeptide bond [cyanophycin : L-arginine ligase (ADP-forming; EC 6.3.2.30)] (Berg, 2003;Berg et al, 2000). In accordance with the proposed mechanism, two ATP per b-aspartylarginine, the formal building block of cyanophycin, are converted to ADP and phosphate (Aboulmagd et al, 2001). Genes for CphA1 are present in the genomes of most cyanobacterial species and some heterotrophic bacteria (Füser & Steinbüchel, 2007).…”

Section: Cyanophycinmentioning

confidence: 84%

CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria

et al. 2016

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Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N 2 -fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide b-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N 2 -fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.

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Cyanophycin

Oppermann‐Sanio¹,

Steinbüchel²

2002

Biopolymers Online

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Introduction Historical Outline Chemical Structure Chemical Analysis and Detection Solubility Properties of CGP and Isolation Molecular Weight and Structure of CGP Quantification of CGP Variations in Composition and CGP‐like Polymers Occurrence Occurrence in Cyanobacteria Occurrence in Chemotrophic Bacteria Functions Physiology Biochemistry Primary Structures and Motifs Occurring in CGP Synthetases Assay of CGP Synthetase Activity and Substrate Specificity Mechanism of Catalysis Molecular Genetics Biodegradation Intracellular Mobilization Extracellular Breakdown Biotechnological Production of CGP Fermentative Production after Heterologous Expression of cphA in E. coli Isolation of CGP Fermentative Production after Heterologous Expression of cphA in other Bacteria In‐Vitro Biosynthesis of CGP Outlook and Perspectives Patents

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Heterologous Expression of Cyanophycin Synthetase and Cyanophycin Synthesis in the Industrial Relevant Bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida

Cited by 71 publications

References 39 publications

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria

Cyanophycin

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