Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases

Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-l-arginyl-poly-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 (cphA₆₃₀₈) 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 cphA₆₃₀₈ and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA₆₃₀₈Δ1, which was truncated by one amino acid at the C terminus; point mutated CphA₆₃₀₈C595S; and the combined double-mutant CphA₆₃₀₈Δ1C595S 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 CphA₆₃₀₈ (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA₆₃₀₈Δ2) or three (CphA₆₃₀₈Δ3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA₆₃₀₈. In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA₆₃₀₈Δ1 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 CphA₆₃₀₈ and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA₆₃₀₈Δ1.Since the first isolation of a methylotrophic yeast, Kloeckera sp. strain 2201, in 1969 (43), the two methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have become the most popular methylotrophs in industry and academia (9, 23, 24). The main benefits of these organisms for the production of recombinant proteins are their growth to cell densities as high as 130 g cell dry matter per liter (50, 57) and the availability of strong and tightly regulated promoters that result in a high product yield (13). Viral hepatitis B surface antigen, S. cerevisiae mating factor α, and S. cerevisiae invertase are only a few examples of compounds produced by recombinant P. pastoris (reviewed in reference 9).A variety of strains were optimized for the expression of recombinant proteins (9). Protease-deficient strains such as strain KM71(H) were generated to circumvent the proteolytic degradation of recombinant proteins (17). Three different phenotypes exist that differ in the ability to utilize methanol (reviewed in reference 37). (i) Mut⁺ strains grow on methanol as the sole carbon and energy source at the wild-type rate. (ii) Mut^s strains possess a disrupted alcohol oxidase 1 (AOX1) gene and therefore rely on the weaker AOX2 gene, leading to decreased methanol utilization rates in comparison to those exhibited by Mut⁺ strains. (iii) Mut⁻ strains are not able to utilize methanol as a carbon and energy source; consequently, such strains use the compound as an inducer only and are dependent on the concomitant addition of carbon sources that do not repress the AOX1 promoter (30, 31). Depending on the required product, any of these phenotypes can be optimal (37). The AOX1 promoter is totally repressed during growth on, e.g., glycerol, whereas it is strongly expressed after methanol is supplied (11). Therefore, P. pastoris fermentations are divided into two phases. (i) During growth on glycerol, high cell densities are reached; (ii) subsequent growth on methanol leads to induction of heterologous protein synthesis, resulting in a high product yield (14). Besides glycerol, several other carbon sources, such as, e.g., glucose, acetate, ethanol, or sorbitol, were used for the production of foreign proteins (30, 31). Several fermentation strategies that allow optimal cell and product yields have been established (8, 25, 28).Besides the AOX1 promoter, several other suitable promoters are available (10), e.g., the copper-inducible CUP1 promoter from S. cerevisiae (33, 38), the inducible ICL1 promoter from the isocitrate lyase gene (8), or the constitutive GAP promoter from glyceraldehydes-3-phosphate dehydrogenase (56).Synthesis of cyanophycin (multi-l-arginyl-poly-l-aspartic acid CGP]) was only recently established in the yeast S. cerevisiae. Recombinant strains harboring cphA from Synechocystis sp. strain PCC 6308 but otherwise with a wild-type background accumulated CGP up to 6.9% (wt/wt) (52), whereas recombinant strains with a mutation in arginine metabolism accumulated CGP even up to 15.3% (wt/wt) of the cell dry mass (CDM) (54). All of the strains synthesized the polymer in soluble and insoluble forms, which was also observed in transgenic plants (29, 42); the soluble type of CGP was first observed in Escherichia coli expressing the cphA gene from Desulfitobacterium hafniense (59). Several cyanobacterial and heterotrophic CGP synthetase genes were expressed heterologously in the past (16, 26, 29, 52, 59). To unravel structurally or catalytically relevant residues of the enzyme, a few site-directed mutations were generated in cyanobacterial cphA genes (26, 27, 35, 53). In addition, several variations in the amino acid composition of the polymer were recently obtained; while cyanobacterial CGP or CGP synthesized by specific CphA proteins exhibiting a narrow substrate range contained aspartate and arginine only (18, 51); lysine was observed as a component replacing arginine at up to 18 mol% in recombinant strains of E. coli and S. cerevisiae harboring CphA with a broader substrate range (34, 54). Moreover, citrulline and ornithine were also detected as constituents replacing arginine in mutants of S. cerevisiae expressing CphA from Synechocystis sp. strain PCC 6308 (54). The soluble CGP contained up to 20 mol% citrulline or up to 8 mol% ornithine instead of arginine. The latter enzyme also revealed a wide substrate range in vitro comprising agmatine and canavanine besides arginine, lysine, citrulline, and ornithine (2, 58).A multitude of technical or pharmaceutical applications are known for degradation products of CGP (44, 48, 49). Dipeptides obtained after α cleavage of the polymer by cyanophycinases are employed as high-value pharmaceuticals (45, 46). Through β cleavage of the polymer, polyaspartic acid can be obtained, which serves as a biodegradable alternative to the persistent polyacrylic acid (9). Finally, research on the synthesis of bulk chemicals such as urea or acrylonitrile from CGP has become of special interest (40, 48, 49).In this study, the methylotrophic yeast P. pastoris was, for the first time, employed for synthesis of the polyamide CGP to analyze if this organism provides a perspective for the production of the polymer. For further optimization of polymer yields, mutated CphA proteins were generated by site-directed mutagenesis and characterized and optimal growth parameters were determined in parallel fermentations.