Metabolic Engineering of Saccharomyces cerevisiae for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids

Abstract

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...

Increased Lysine Content Is the Main Characteristic of the Soluble Form of the Polyamide Cyanophycin Synthesized by Recombinant Escherichia coli

“…The major part accounts for a water-soluble form, and a minor part accounts for an insoluble form. Metabolic engineering of S. cerevisiae confirmed the results of Krehenbrink et al (6) concerning the incorporation of lysine (16 mol%), citrulline (20 mol%), and ornithine (8 mol%) in CGP synthesized by CphA 6308 (13). Wiefel et al (14) also demonstrated that in recombinant P. putida ATCC 4359 expressing cphA 6308 , a soluble polymer could be isolated consisting of 8 mol% lysine and 8 mol% citrulline instead of arginine.…”

“…Amino acid constituents of isolated CGP were determined by high-performance liquid chromatography (HPLC) with a Waters B801 column (300 by 4 mm) as described by Aboulmagd et al (25) and Steinle et al (13). Precolumn o-phthalaldehyde derivatization was carried out in a Smartline Autosampler 3900 (Knauer GmbH, Berlin, Germany) as described in the manual.…”

Solubility Behavior of Cyanophycin Depending on Lysine Content

“…The amino acid constituents of isolated CGP and CGP dipeptides were determined by high-performance liquid chromatography (HPLC) using a Waters B801 (300-by 4-mm) column as described by Aboulmagd et al (17) and Steinle et al (7). Precolumn OPA derivatization was done with a Smartline autosampler 3900 as described in the manual (Knauer GmbH, Berlin, Germany).…”

“…CGP is interesting due to its easy and lowcost purification using alternating steps of solubilization in 0.1 M HCl and precipitation by neutralization to pH 7 (5). Also of interest is the possibility of obtaining CGP with different side chains in which L-arginine is replaced by other compounds, like lysine (6), ornithine (7), or citrulline (7,8). Synthesis of these forms of CGP became possible because the amino acids mentioned also possess a certain affinity for the cyanophycin synthetase CphA (EC 6.3.2.29 and EC 6.3.2.30).…”

“…Steinle et al (7), for example, synthesized soluble CGP with a citrulline content of over 20 mol%, while the corresponding insoluble form contained citrulline only at a fraction of about 5 mol%. Unlike the normal insoluble CGP, which can only be solubilized in weak acids, like 0.1 M HCl, and which is insoluble at neutral pH, the soluble CGP is still soluble at pH 7.0 and can only be isolated by precipitation using ethanol (EtOH) or acetone (4).…”