Cyanophycin (multi-L-arginyl-poly-L-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a putative precursor for numerous biodegradable technically used chemicals. Therefore, the biosynthesis and production of the polymer in recombinant organisms is of special interest. The synthesis of cyanophycin derivatives consisting of a wider range of constituents would broaden the applications of this polymer. We applied recombinant Saccharomyces cerevisiae strains defective in arginine metabolism and expressing the cyanophycin synthetase of Synechocystis sp. strain PCC 6308 in order to synthesize CGP with citrulline and ornithine as constituents. Strains defective in arginine degradation (Car1 and Car2) accumulated up to 4% (wt/wt) CGP, whereas strains defective in arginine synthesis (Arg1, Arg3, and Arg4) accumulated up to 15.3% (wt/wt) of CGP, which is more than twofold higher than the previously content reported in yeast and the highest content ever reported in eukaryotes. Characterization of the isolated polymers by different analytical methods indicated that CGP synthesized by strain Arg1 (with argininosuccinate synthetase deleted) consisted of up to 20 mol% of citrulline, whereas CGP from strain Arg3 (with ornithine carbamoyltransferase deleted) consisted of up to 8 mol% of ornithine, and CGP isolated from strain Arg4 (with argininosuccinate lyase deleted) consisted of up to 16 mol% lysine. Cultivation experiments indicated that the incorporation of citrulline or ornithine is enhanced by the addition of low amounts of arginine (2 mM) and also by the addition of ornithine or citrulline (10 to 40 mM), respectively, to the medium.Cyanophycin (multi-L-arginyl-poly-[L-aspartic acid]), also referred to as cyanophycin grana peptide (CGP), represents a polydisperse nonribosomally synthesized polypeptide consisting of poly(aspartic acid) as backbone and arginine residues bound to each aspartate (49) (Fig. 1). One enzyme only, referred to as cyanophycin synthetase (CphA), catalyzes the synthesis of the polymer from amino acids (55). Several CphAs originating from different bacteria exhibit specific features (2,7,5,32,50,51). CphAs from the cyanobacteria Synechocystis sp. strain PCC 6308 and Anabaena variabilis ATCC 29413, respectively, exhibit a wide substrate range in vitro (2, 7), whereas CphA from Acinetobacter baylyi or Nostoc ellipsosporum incorporates only aspartate and arginine (23,24,32). CphA from Thermosynechococcus elongatus catalyzes the synthesis of CGP primer independently (5); CphA from Synechococcus sp. strain MA19 exhibits high thermostability (22). Furthermore, two types of CGP were observed concerning its solubility behavior: (i) a water-insoluble type that becomes soluble at high or low pH (34, 48) and (ii) a water-soluble type that was only recently observed in recombinant organisms (19,26,42,50,56). In the past, bacteria were mainly applied for the synthesis of CGP (3,14,18,53), whereas recently there has been greater interest in synthesis in eukaryotes (26,42,50). CGP was accumulated to al...

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...

Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-L-arginylpoly-L-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308 ) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA 6308 and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA 6308 ⌬1, which was truncated by one amino acid at the C terminus; point mutated CphA 6308 C595S; and the combined double-mutant CphA 6308 ⌬1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA 6308 (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA 6308 ⌬2) or three (CphA 6308 ⌬3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA 6308 . In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA 6308 ⌬1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26°C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA 6308 and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA 6308 ⌬1.

No abstract

An efficient, time-saving, and cost-effective method for isolation of the polyamide cyanophycin from recombinant Saccharomyces cerevisiae was established. Due to its simple procedure, this isolation method may be also applicable at industrial scale and also to other intracellular compounds in this yeast. Production of cyanophycin gained preferential interest in the past, as degradation products thereof are of pharmaceutical and technical interest. Recently, it was shown that Saccharomyces cerevisiae represents a putative candidate for cyanophycin synthesis at industrial scale. For identification of optimal isolation procedures, several parameters such as heat stress, freeze drying, and freeze/thaw cycles of transgenic yeast cells were compared for their effectiveness of cyanophycin isolation. Additionally, optimal resuspension solutions for the applied cells and minimal required materials or chemicals were determined to make the process most environmentally and economically friendly. Maximal cyanophycin granule polypeptide yields of 21% (w/w) were obtained after incubation of dry cells at 70 degrees C or 80 degrees C and precipitation of the polymer with two volumes of ethanol.