Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Abstract

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...

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

“…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 almost 7% (wt/wt) of dry matter in recombinant Nicotiana tabacum and Saccharomyces cerevisiae (26,50).In S. cerevisiae the arginine metabolism is well understood and has been investigated (30) (see Fig. 2).…”

“…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 almost 7% (wt/wt) of dry matter in recombinant Nicotiana tabacum and Saccharomyces cerevisiae (26,50).…”

“…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.…”

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

“…Experiments with yeasts maintained the idea of a water-soluble form and a modified composition of CGP synthesized by CphA 6308 . In 2008, Steinle et al (11) showed that in Saccharomyces cerevisiae expressing cphA 6308 , two forms of CGP are available. The major part accounts for a water-soluble form, and a minor part accounts for an insoluble form.…”

“…Heterologously synthesized CGP, e.g., from bacterial hosts like Escherichia coli, Pseudomonas putida, and Ralstonia eutropha or from yeasts, is quite different (9,10,11). The water-insoluble cyanophycin-like material isolated from recombinant E. coli expressing cphA from Synechocystis sp.…”