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Free keywords:
GRAM-NEGATIVE BACTERIA; CYANOPHYCIN SYNTHETASE; ALCALIGENES-EUTROPHUS;
MOLECULAR CHARACTERIZATION; RECOMBINANT STRAINS; ANABAENA-CYLINDRICA;
PSEUDOMONAS-PUTIDA; ESCHERICHIA-COLI; CO2 ASSIMILATION; GENE-EXPRESSION
Abstract:
With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong P-cbbL promoter of the R. eutropha H16 cbb(c) operon (coding for autotrophic CO2 fixation). In batch cultures growing on H-2 and CO2 as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with (CO2)-C-13 and (NH4Cl)-N-15, and the C-13/N-15-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [C-13/N-15] arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.
